Arabidopsis CME appears to follow the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal-basket type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the plasma membrane but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mechanism during the autonomous rise of multicellularity in eukaryotes.In plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques.  Clathrin mediated endocytosis (CME) is the key cellular progress of internalising a cargo, either on the plasma membrane (PM) or outside the cell, into the cell. The cargos are packaged inside a small vesicle, which is covered by a coat made of the protein clathrin. CME represents a major endocytic route in eukaryotes . In planCME in plants is poorly characterized and its hypothesized mechanism is largely inferred from studies in mammalian and yeast systems, where CME components are highly conserved; with at least 60 homologous endocytosis accessory proteins (EAPs) well characterized in both these model systems . Plants Despite a high degree of conservation between mammalian and yeast EAPs, there are key differences in the actual CME mechanism; chiefly related to the requirement of actin cytoskeleton. While actin co-localises with single sites of CME in both systems, interference with actin results in a total arrest of CME in yeast  whereas in vivo. For example, electron microscopy, the key technology credited for the first CME characterisation  risation , can be risation  but the risation .in vivo, and combined this with live quantitative TIRF-M imaging at a high spatial and temporal resolution. Contrary to expectations, actin is not present or required during CCV formation on the PM, but is critical for the early post-endocytic traffic supporting an unsuspected active role of dynamic actin and moving endosomes in organizing CME events at the PM. Imaging of CCVs deeper inside the cell revealed strongly delayed and gradual uncoating on route to the endosomes. Thus, we provide an extensive mechanistic framework for CME in plants and identify multiple unexpected, evolutionary non-conserved aspects of this fundamental cellular process.Here we provide a detailed characterisation of plant CME, starting with the formation of clathrin-coated pits (CCP) at the PM and up until the later endocytic events in the cell interior. We established new electron microscopy sample preparation protocols, which produced an abundance of clathrin-coated structures at an unprecedented level in planta, largely due to the lack of efficient ultra-structure analysis of the key events of CME. Therefore, we established a method for using Scanning Electron Microscopy (SEM) on metal replicas of unroofed Arabidopsis root protoplast cells, which allowed the direct examination of clathrin coated structures (CCSs). Availability of such a technique to study their size, shape and invagination enabled us to investigate the mechanism of CCV formation.Endocytic events have not yet been well characterized Arabidopsis root protoplast cells provided a detailed view of well-preserved CCPs at various stages of invagination at the PM and fully formed CCVs deeper inside the cell, which are often attached to cytoskeletal and intracellular membrane structures  a flat lattice, or (ii) dome-shaped and developing attached to PM, or (iii) spherical and mature  stays the same along the dome-shaped and the spherical CCSs, it is indicative that the membrane bending follows the constant curvature model  in our protoplast cell replica SEM images into flat, dome-shaped and spherical CCPs . We founHowever, we could not entirely dismiss the existence of other mechanism of membrane bending, given that there was a small percentage of flat CCSs that could potentially develop and bend to form a spherical CCV, characteristic of constant area model. In addition, due to the irregular ordering of the clathrin in the irregular basket-type CCVs , it is iInterestingly, the diameters of the CCVs measured in our unroofed protoplast cells is similar to CCVs purified from the entire seedlings (where the CCV size ranges between 60–80 nm ). TherefArabidopsis roots.In summary, ultrastructural and live imaging analysis of clathrin at the PM did not detect any larger clathrin lattices that could develop into vesicles. Moreover, the ultrastructure observations of developmental stages of CCPs in root protoplasts suggest that the membrane bending follows ‘constant curvature model’ supported also by live imaging-based clathrin intensity profiles in While TIRF-M has been used in plants , a detaibone fide clathrin endocytic population, we used an approach based on a mixture model distribution fitting (A large data set of lifetimes was generated (12 cells and 149162 tracks) by imaging plant root epidermis expressing CLC2-GFP. CLC2 was used as a marker for CME, as it is one of the key components of the clathrin coat, comprised of both clathrin light and heavy chains. Additionally, we found a high degree of co-localization (~62%) between CLC2 and CHC1, indicating that the CLC2 is present at the majority of total CME events . The mea fitting . Further fitting . There w fitting .bona fide CME, we used dual channel TIRF-M on root cells expressing CLC2-GFP and an additional endocytosis marker TPLATE, which has been proposed to function as an endocytic adaptor  and CLC2-mKO, using TIRF-M. We were able to observe the intact cortical actin filaments of hypocotyl epidermal cells , which fThus, in contrast to expectations based on observations in yeast and some mammalian systems, where CCP invagination is facilitated by actin polymerization, plant cortical actin only forms filaments and does not accumulate around the invaginating CCPs as concrete foci.The absence of actin foci at the endocytic spots is unexpected given that actin polymerization energy had been proposed to be a critical factor for plant CCP development.In order to test the importance of actin in CCV formation, we perturbed actin by either stabilization of actin filaments treating with Jasplakinolide (Jasp)  or depolbona fide endocytic sub-population of CCPs with the lifetime of 44 s  fused with EE/TGN compartments; which is specific to plant systems . The EE/Next, we examined the actin dynamics in relation to the endocytic events at the PM in detail. Using TIRF-M, in hypocotyl epidermis, we observed the dynamics of actin subsidiary filaments (marked by ABD2-GFP) and the CCVs (marked by CLC2-mKO). We observed a spatial and temporal correlation between the appearance of a small branch of an actin subsidiary filament and the disappearance of CCVs from the PM . This coin vivo imaging of endocytotic events at the PM suggest that actin filaments traffic the CCVs directly from the site of endocytosis. Also, actin guides the EE/TGN compartments movement to ‘collect’ CCVs as they move. This is not only a first observation how EE in plants are formed by directional traffic and fusion of CCVs, but it also suggests a more active role of endocytic compartments in organizing the early post-endocytic events  after scission at the endocytic spot, but the clathrin coat is retained for a prolonged time, shedding its components gradually  on its way to the EE .Arabidopsis tissues. We found that while there is some evolutionary conservation of endocytic molecular components between plants and other model systems .Arabidopsis thaliana genes studied and their corresponding accession numbers: Fim1 – AT4G26700, CHC1 - AT3G11130, CLC2 - AT2G40060, Drp1C – AT1G14830, Tplate- AT3G01780, VHA-a1 - At2g28520, PIN2 - AT5G57090, FLS2 – AT5G46330, Axl2 - AT4G12770, AP2A1 –AT5G22770. All lines used are from Arabidopsis thaliana:The 35 s::GFP-fABD2 ; p35S::ST-RFP x pCLC2::CLC2-GFP  sucrose, stratified for 2–3 days and then grown at 21°C in a 16 hr/8 h day/night cycle. Seedlings to examine roots were grown for 3 or 7 days, seedlings to examine the hypocotyl seedlings were grown for 3 days in dark conditions.Arabidopsis (Col-0) root-derived suspension was obtained from Eva Kondorosi . The suspension culture is maintained in growth medium (GM) of pH5.7 containing 4.25 g/l of MS salts (Sigma M5524), 30 g/l of sucrose, 0.250 mg of 2,4D, 0.015 mg of kinetin and 2 ml Vitamin B5 stock .LatrunculinB, β-estradiol, MS powder and all chemicals used for metal replicas and resin embedding were from Sigma Aldrich; with the exceptions of EM grade glutaraldehyde and pioloform , Osmium tetroxide , Acetone and polyethylene glycol , picric acid , Uranyl acetate . Jasplakinolide was from Santa Cruz Biotechnology, Cellulase from Serva, Macerozyme from Yakult pharmaceuticals, Aclar foil from Ted Pella Inc , Aluminium planchettes and sapphire disks from Wohlwend , Cryo-vials from Biozym GmbH , Glass cover slips from Roth , Maxtaform H15 finder grids from Science Services GmbH  and Platinum and carbon rods were from Leica Microsystems .Arabidopsis suspension cultured cells were used. Protoplast isolation from the cells was carried out as described by 3 day old 2, pH 6.9) plus 1% high-MW polyethylene glycol  and 2 µM phalloidin ±2 µM taxol) was applied for 4 mins. Samples were then washed three times in PEM plus 1% PEG for 1 min and then fixed in 2% Glutaraldehyde in phosphate buffer (PB) for 20 mins. After washing in distilled water, samples were treated with 0.1% tannic acid in water (w/v) for 20 mins at room temperature and 0.2% uranyl acetate in water (w/v) for 20 mins at room temperature. Samples were then ‘critical point dried’ as described by The plated protoplasts were washed in PBS equilibrated to room temperature and the extraction solution , the protoplasts were incubated in one of the following solutions for 30 mins at room temperature: GM buffer or hyperosmolar buffer (75 mM Mannitol in GM buffer). Protoplasts were then fixed in 2% formaldehyde plus 2.5% glutaraldehyde and 15% of a saturated solution of picric acid in phosphate buffer  for 30 mins at room temperature. The protoplasts were then washed with PB buffer and then incubated with 0.5% tannic acid in PB (w/v) for 1 hr at 4°C, then 1% osmium tetroxide in PB (w/v) for 30 mins at 4°C and then 1% uranyl-acetate in water (w/v) overnight at 4°C. Protoplasts were then contrast-enhanced with Walton’s lead aspartate for 30 mins at 60°C, dehydrated in graded ethanols and anhydrous acetone, and embedded in epoxy resin (Durcupan ACM). Serial ultrathin sections of 40 nm were cut with an ultramicrotome UC7 (Leica Microsystems), collected onto Formvar-coated copper slot grids and stained with 1% aqueous uranyl acetate and 0.3% lead citrate.XVE >>Axl2 x pPIN2::PIN2-Dendra were induced by transferring them to solid ½ AM with 1% sucrose plate supplemented with 2 µM β-estradiol. The seedlings were continuously under chemical induction during subsequent imaging.All the treatments were carried out at room temperature in ½ MS medium containing 1% (w/v) sucrose. Jasp and LatB treatments of the seedlings were carried out by dissolving the compounds in DMSO, and then diluting to the working concentration with liquid 1/2 MS and 1% sucrose solution. Specific concentrations and duration of treatments are defined in the relevant figure legends. Throughout imaging experiments, the seedlings were kept in the mock or treatment solutions, with the exception of FM4-64 uptake experiments. To induce the overexpression of Auxilin2, 2 day old seedlings expressing To examine the uptake of FM4-64, following the actin/mock treatment, 3 day old seedlings were incubated with 2 µM FM4-64 dissolved in liquid ½ MS with 1% sucrose medium for 2 mins. The seedlings were then washed twice before being imaged. To examine Flagellin uptake, 3-day-old etiolated seedlings were incubated in 10 µM flg22 in liquid ½ MS with 1% sucrose medium for 30 mins. Flagellin treatment was continuously present during imaging.PIN2, photo conversion and imaging of photo converted PIN2-Dendra at the PM was conducted using a Zeiss LSM700 vertical confocal microscope equipped with a Plan-Apochromat 20x/NA 0.8 air objective; as previously described (To determine the endocytic rate of escribed . The whoescribed  and the Roots of 7-day-old seedlings or hypocotyls of 3-day-old etiolated seedlings were imaged with a Olympus IX83 inverted microscope equipped with a Cell^TIRF module using a OLYMPUS Uapo N 100x/1.49 Oil TIRF objective. A quad line beam splitter emission filter (Chroma) in combination with an EM-CCD camera (Hamamatsu) was used to collect images. Excitation wavelengths were 488 for GFP tags and 561 nm for RFP tags. Time-lapse imaging in the hypocotyl was done on the epidermal cells that are closet to the root-hypocotyl junction, after cutting the cotyledon. Single channel images were collected at two frames per second. Dual channel imaging was done sequentially at the frame rate as specified in the figures/movies.Roots were prepared for imaging as previously described by To examine the metal replicas of unroofed protoplasts a Zeiss FE-SEM Merlin VP Compact was used. An accelerating voltage of 0.5–5 kV was applied under high vacuum condition and In-lens duo detector was used for image recording. For transmission electron microscopy of resin embedded samples, the protoplasts were examined and imaged at an accelerating voltage of 80kV in a Thermo Ficher TEM-Tecnai10 equipped with EMSIS Megaview G3 camera.en face surface-view analysis, only the CCSs with the entire view of the basket were considered; and the baskets that were partially visible or lying on the side were dismissed. To study the invagination of CCSs at the PM, TEM images of CCSs after resin embedding and sectioning of protoplasts were visually examined and classified using Fiji (NIH).The classification of the SEM images of the CCSs after metal replica was done manually using Fiji (NIH) and plotted using R. For all the Time series movies were subjected to just the particle detection and tracking programs of the cmeAnalysis package, as previously described . The frahttps://imagej.net/Spots_colocalization_(ComDet)) for Fiji was used to calculate a co-localization value.For co-localization, 10 frames of the detections made by the cmeAnalysis package were used to make a median z projection. Then the comDet plugin (The intensity time-course of the endocytic foci marked by CLC2-GFP was processed as described in h(t), where t denotes different possible lifetimes. As the only tracks with a lifetime of at least five frames were considered for analysis, the lifetimes have a specific lower bound tlow = 5*frame rate. The lifetimes also have an upper bound thigh, corresponding to the total imaging time. Hence, we have h(t)=0, for t < tlow and t > thigh. We choose a specific bin size, Δt, equal to the inverse of the frame rate, and obtain h(t) as a discrete density function over a set of N lifetimes (Adamowski and Friml), 1 ≤ i ≤ N, uniformly separated by Δt, between t1 = tlow and tN = thigh. Hence, h(ti)Δt denotes the probability that the lifetime is in the interval As previously indicated, there is consensus related to the great interest of your results and the thoroughness of this study. In particular, all reviewers agreed on the importance of the experiments showing that actin is dispensable for endocytic uptake but rather plays a role in post-internalization events, as well as on the evolutionary divergence regarding the endocytic protein dynamics and the mechanism of clathrin assembly and un-coating in plants, as compared to mammalian cells or yeast. The reviewers agree now that the manuscript reads better than the first version and some important issues have been addressed. However, they still have some concerns that you need to address in the text:You still classify the clathrin coated structures to "hexagonal basket-type" and "pentagonal basket-type". It remains unclear why you think this is a useful classification. All clathrin coated vesicles are expected to have both hexagons and pentagons, and therefore, observing one or the other on the vesicle surface would not be informative. You need to indicate how the lattice is built in the text . In keeping with this, your data indicate that the "pentagonal pits" are smaller in diameter, likely because they have fewer hexagons. This needs to be clarified also.The data showing that CLC2 labels 60% of CHC1 coated pits is a good addition, but now, acknowledging that there are two types of CHC's further complicates interpretation of the data. It would be more accurate if you can clearly state that the live cell imaging data can technically be applicable to only one type of clathrin. Or maybe CHC1 represents most of the clathrin in these cells? Further clarification of whether CLC2 can associate with both CHCs or just one should be explicit in the text so it is apparent how much of the clathrin in plant cells is represented in the live cell imaging.The issue of different CHC's doesn't change the conclusions about the actin association with pits, but it does change the interpretation of the uncoating results. Looking at CLC2 vs adaptors means that you could be looking at more than one type of clathrin (CHC1 or CHC2 with CLC2 bound to either CHC). Maybe CHC1 clathrin dissociates faster than CHC2 clathrin, so the residual CLC2 represents a different clathrin uncoating by a different mechanism. Again, the fact that the uncoating dynamics in plants appear different to yeast and mammals is still valid, but the possibilities of more than one type of clathrin with potentially different behaviors needs to be acknowledged and discussed in full to ensure that the reader understands the implications. Essential revisions:1) The characterization of the "hexagon" versus "pentagon" basket is puzzling. All coats will have 12 pentagons . Size is increased by adding hexagons. It does not make sense to talk about a pentagon basket, as they are all pentagon baskets. If the authors are characterizing baskets with different numbers of hexagons, that would be a better way to describe their data. Given that the two types of baskets identified by the authors are the same size, could these not be the same basket from different angles? If not, better to categorize by numbers of hexagons or even by numbers of clathrin molecules in the basket.We thank the reviewers for highlighting the potential for confusion caused by the terminology we have used to characterize the whole clathrin basket. We have altered the manuscript to make our classifications clearer to the readers. We have re-written this section and the added a graphical illusion in Figure 1. Briefly, the whole clathrin basket is characterized into ‘pentagonal’, ‘hexagonal’ or ‘irregular’ baskets based on the surface-view, where the number of rings around a central ring is used to make this determination. For example, if 5 rings are counted around a central ring, it is a pentagonal CCP appearing to form a pentagon shaped.During this quantification, care was taken to exclude any CCS which appeared to be lying on its side or appearing at an angle. But to alleviate the reviewer’s concerns that the basket shape  recorded could be due to the viewing angle, we have included details about this point in the Materials and methods section of the text to highlight this concern.The reason we have not quantified the individual clathrin hexagonal/pentagonal rings or the number of clathrin molecules themselves is that they cannot clearly be identified in our SEM replica images. Therefore, we chose to look at the size and shape of the entire basket as this was a much more reliable component to accurately measure.2) The authors make a very strong conclusion about the plant cells using the constant curvature model to assemble their endocytic vesicles. However, the arguments about constant curvature versus re-arrangement need to be supported by measurements of the theta angle and TEM, as was done in Avinoam et al., 2015. Looking at Figure 1—figure supplement 1D, the theta angle looks like it's changing, which is not expected in the constant curvature model. Looking at pentagons and hexagons does not provide this information (see point 1). The authors should also be aware that recent studies suggest intermediate models, where the coat area keeps growing during vesicle budding, but the coat curvature still changes .We agree with the reviewers that approach used by Avinoam et al., is a good way to determine the ‘radius of curvature’ during CCP development. However, during current existing plant TEM protocols, the preservation of invaginations on the PM is incredibly rare. Over decades of TEM work on endocytosis in plants, there is only limited number to events recorded and this also applies to our own TEM efforts. Therefore, it is impossible to make a real quantitative analysis such as ‘the radius of curvature’, and any conclusion from our very few TEM section images are hard to justify.To overcome this issue, we therefore have to rely on a different approach to address the question of membrane re-modeling during CCV formation using the replica protocol; as this is the only EM method in plant cells which produces intact CCSs with any reliability and quantity. Therefore, we relied on the characterization of the whole basket view . Using this surface-view approach, we can make an approximation of the ‘radius of curvature’ of all the CCPs, which we found to be predominantly constant, thus pointing to constant curvature model. We have explained our methodology more clearly in the text and Figure 1 and Figure 1—figure supplement 1.Avinoam et al., measured theta, the angle between PM and the invaginating vesicle, as a proxy to stages of CCP development which changes the same way for both the models. To try and analyze our CCSs in a similar fashion to the theta angle approach demonstrated by Avinoam et al., we assume theta to represent the degree of invagination. Therefore, by assessing the side-view of each CCP, we were able to determine an approximation of the degree of invagination. Using both the side-view analysis of degree of invagination, and the surface-view analysis of the baskets, we have estimated the ‘radius of curvature’ of different stages of CCPs. We have presented the quantifications in Table 1 in Supplementary file 1 and explanations in the main text.In the intermediate models highlighted by the reviewer, clathrin is required to first assemble into flat lattices on the PM and then invaginate inwards, with the addition of more clathrin. Because of this requirement, we loosely classified the constant area and intermediate models together . In our analysis, there were <4% CCSs that were flat, which could potentially develop and form CCVs following either ‘intermediate’ or ‘constant area’ model (as the reviewer pointed out); which we have now discuss in the text.3) The lack of large clathrin plaques in EM is interesting. However, the fluorescence studies show some apparently large areas of clathrin accumulation . Can the authors somehow verify that those large structures are not flat lattices?The reviewers are correct to point out that there are large clathrin structures in the live plant samples. However, they do not represent static lattices on the PM as when looking at the dynamic movies of the live plant samples , these are highly mobile elements bellow the PM. They are mobile EE/TGN structures and are not PM associated, like those seen in the EM work of Figure 1F.To clarify this, in the text we have referred to the movies showing the motility of these structures in the main text. For example, in Figure 2 we have referred the supplemental videos.4) The EM is done in protoplasts, whereas the live cell imaging is done in intact cells. These are quite different growth condition and endocytosis could also behave differently, which would complicate the interpretation of the data. Could the authors do live cell imaging in protoplasts or EM in tissues?Arabidopsis root culture cells (as used in the replica EM experiments) are much smaller and rounder. When we tried to image them, we were unable to successful establish a protocol which allowed reproducible TIRF imaging and analysis to be conducted, which was most likely due to their small surface area on the coverslip and the roundness of their overall shape.We agree that there indeed could be difference in endocytic processes in protoplasts and intact plant tissues. Unfortunately, the metal replica approach used here has been developed and optimized for the use of single cells and remains a technical challenge to successfully work with intact plant tissues. While TIRF-M has been used on some types of plant protoplasts , the While there may be potential differences in the vesicle formation in protoplast and plants, it is important to note that the size of the CCVs quantified in our EM work closely matches the size of CCVs purified from whole plant tissue extracts , thus suggesting that the same mechanisms are functional in both tissues and single cells. We have now included this sentiment in the main text.5) Providing background on Arabidopsis clathrin would be helpful to understand whether the dynamic labeling using the clathrin light chain 2 (CLC2) is representative of all clathrin or just a subset of coated pits. There is the concern, particularly for uncoating, that measuring clathrin dynamics with a single tagged subunit could influence this process. Could an additional clathrin marker (eg CHC) be used to support the results? If the GFP tagged CHC does not complement, the authors should at least present co-staining of the CHC and the GFP-tagged CLC2.Arabidopsis clathrin in the Introduction of the paper. We also included new data. Using plants co-expressing CLC2-FP (1 of the 3 Arabidopsis clathrin light chain isoforms) and CHC1-FP (1 of the 2 clathrin heavy chain isoforms), we have determined the co-localization to be around 60% and included this new data in the text and as a Figure 2—figure supplement 1.We have included some more information about 6) The authors need to demonstrate that the used latB concentration lead to depolymerisation of the actin structures in their experimental system.We agree that this is a critical point, and as such, we have highlighted the depolymerisation effect for both roots and hypocotyl after LatB in Figure 3C and Video 2. We show that the concentrations used were effective in the experimental systems.As previously indicated, there is consensus related to the great interest of your results and the thoroughness of this study. In particular, all reviewers agreed on the importance of the experiments showing that actin is dispensable for endocytic uptake but rather plays a role in post-internalization events, as well as on the evolutionary divergence regarding the endocytic protein dynamics and the mechanism of clathrin assembly and un-coating in plants, as compared to mammalian cells or yeast. The reviewers agree now that the manuscript reads better than the first version and some important issues have been addressed. However, they still have some concerns that you need to address in the text:You still classify the clathrin coated structures to "hexagonal basket-type" and "pentagonal basket-type". It remains unclear why you think this is a useful classification. All clathrin coated vesicles are expected to have both hexagons and pentagons, and therefore, observing one or the other on the vesicle surface would not be informative. You need to indicate how the lattice is built in the text . In keeping with this, your data indicate that the "pentagonal pits" are smaller in diameter, likely because they have fewer hexagons. This needs to be clarified also.We believe that the classification of clathrin coated structures (CCSs) is useful as it provides an insight into the type of arrangements of clathrin used in plant cells, something which has never been examined before. We constantly found that these distinct basket formations exist. However, the exact physiological significance of this remains unclear and highlights the need for further work to define the precise structure of plant CCVs. We have added a few sentences to make these points clearer, Results paragraph three and Discussion subsection “Key characteristics of plant CME”.In detail, the main reason we focused on the overall surface view shape of the basket , and not the arrangements of individual polygons, is because with current plant EM protocols we are unable to produce EM images where we can clearly visualize all the clathrin polygons within single fully formed CCVs. Further to this, the replica approach allows the classification of the basket arrangements/formations in situ which has the benefit of examining CCVs in a somewhat physiological environment. This is an advantage as it is well documented that CCVs in cells or tissues are substantially more heterogeneous in size and shape than the coats assembled for cryoEM studies . This is an important consideration, as the vesicle size and shape are affected by both membrane tension and specific adaptor proteins ) and AP2 only have a 50% rate of co-localization ) . During the examination of the plant cell replicas, we consistently found that the basket of the CCVs were actually quite similar, however it was apparent that they exist in different populations definable by their basket arrangement and size. Therefore, the basket classification is useful to show that different populations of clearly identifiable assembles exist in the plant cells and it provides a way to examine the membrane bending using the only plant ultrastructural method which produced large numbers of CCVs.We have also added a few lines to include information about the lattice is made from 12 pentagons and how numbers of hexagons effect the size of the CCV in Results paragraph four.The data showing that CLC2 labels 60% of CHC1 coated pits is a good addition, but now, acknowledging that there are two types of CHC's further complicates interpretation of the data. It would be more accurate if you can clearly state that the live cell imaging data can technically be applicable to only one type of clathrin. Or maybe CHC1 represents most of the clathrin in these cells? Further clarification of whether CLC2 can associate with both CHCs or just one should be explicit in the text so it is apparent how much of the clathrin in plant cells is represented in the live cell imaging.The issue of different CHC's doesn't change the conclusions about the actin association with pits, but it does change the interpretation of the uncoating results. Looking at CLC2 vs adaptors means that you could be looking at more than one type of clathrin (CHC1 or CHC2 with CLC2 bound to either CHC). Maybe CHC1 clathrin dissociates faster than CHC2 clathrin, so the residual CLC2 represents a different clathrin uncoating by a different mechanism. Again, the fact that the uncoating dynamics in plants appear different to yeast and mammals is still valid, but the possibilities of more than one type of clathrin with potentially different behaviors needs to be acknowledged and discussed in full to ensure that the reader understands the implications.We agree that the rate of co-localization of CLC2 and the CHCs is indeed interesting. And as such, it is an area of ongoing research within our lab. Currently, there has been no detailed assessment of the rate of co-localization between all the different clathrin isoforms in plant cells and it has been not an ambition of this report. However, it has been shown that the two CHC isoforms are functionally redundant for each other  and mass spectroscopy data show that the CHCs can interact with each other and all the CLCs , so from a genetical and biochemical point of view, there is not a reason to assume that different isoforms can have distinct mechanistic roles but this needs to be examined in detail. We have added a few sentences to address this issue.


Risk communication is a core aspect of a physician’s work and a fundamental prerequisite for successful shared decision-making. However, many physicians are not able to adequately communicate risks to patients due to a lack of understanding of statistics as well as inadequate management of conflicts of interest (COI).To evaluate the effects of an integrated curriculum encompassing COI and shared decision-making on the participants’ risk communication competence, that is, their competence to advise patients on the benefits and harms of diagnostic or therapeutic interventions.A rater-blind randomized controlled trial with a 30 (± 1)-week follow-up conducted from October 2016 to June 2017 at two German academic medical centers.Sixty-three medical students in their fourth or fifth year.Participants received either a newly developed 15-h curriculum or a course manual adapted from teaching as usual.Primary outcome: change in risk communication performance in a video-observed structured clinical examination (VOSCE).d of 2.35  1.62 to 3.01, p < 0.01) and of 1.83  30 (± 1) weeks later. Secondary outcomes with the exception of frequency of interactions with the pharmaceutical industry also showed relevant improvements in the intervention as compared with the control group (d between 0.91 and 2.04 (p < 0.001)).Participants were 25.7 years old on average (SD 3.6); 73% (46/63) were female. Increase in risk communication performance was significantly higher in the intervention group with post-intervention Cohen’s Our results show that an integrated curriculum encompassing COI and risk communication leads to a large and sustainable increase in risk communication performance. We interpret the large effect sizes to be a result of the integration of topics that are usually taught separately, leading to a more effective organization of knowledge.Trial Registration: The trial is registered in the International Clinical Trials Registry with the trial number DRKS00010890.The online version of this article (10.1007/s11606-019-05420-w) contains supplementary material, which is available to authorized users. Studies show that many physicians are not up to the challenge.8Risk communication is a core aspect of a physician’s work and a fundamental prerequisite for successful shared decision-making.7 For example, many physicians rely on the wrong statistical parameters to judge the effect of screenings for common cancers6 and are not able to explain basic statistic concepts such as absolute or relative risk reduction.7 Conflicts of interest (COI) resulting from interactions with the pharmaceutical industry further impede the interpretation of risks: they lead to a biased view on risk information as well as a biased presentation of scientific data in information brochures and scientific publications.11 Physicians thus need to avoid such conflicts or manage them professionally to avoid bias. Yet data from the Physician Payments Sunshine Act as well as surveys among physicians show that interactions with pharmaceutical companies continue to be pervasive.14For one, studies suggest that many physicians cannot accurately interpret statistics commonly used to describe effects of screening and therapeutic interventions; some have called this “statistical illiteracy.”15 Some curricula on the topics have been published,18 but no integrated curriculum encompassing all aspects important for risk communication exists in the literature. We thus hypothesize that an integration of the relevant topics—basic statistics, bias detection, professional management of COI, and communication skills—is necessary to improve physicians’ risk communication competence by improving knowledge organization, facilitating knowledge transfer into practice, and emphasizing the importance of basic statistics and professionalism for the treatment quality of patients. We developed an integrated curriculum covering these topics and choosing didactic methods primarily based on adult learning theory.19 The objective of this study was to evaluate the effects of this curriculum on the participants’ risk communication performance.According to our literature search and knowledge from a curricular mapping project, most aspects of risk communication are being taught in medical school—however, they are taught separately.This study received a positive ethics review from the two responsible ethics committees for Mainz and Heidelberg ; and Ethikkommission der Medizinischen Fakultät Heidelberg, case number S-314/2016)This was a rater-blind randomized controlled trial conducted from October 2016 to June 2017 at two German academic medical centers (Mainz and Heidelberg). Eligible for participation were all medical students in their fourth and fifth year at the two universities. All participants had had training in statistics and basic communication skills. Each student who completed the entire study received a cash incentive of 100 €.It was calculated that a sample size of 60 participants (30 per group) was necessary to guarantee a power of 0.85 to detect an effect size of 0.8 standard deviations regarding the primary outcome , see below).Participating students provided informed consent before completing a baseline assessment consisting of a VOSCE as well as a questionnaire (details see below). The students were then randomized 1:1 to the intervention or control group. After receiving the respective intervention, participants completed a similar assessment twice more: once up to 2 weeks after the intervention (post-test) and once 30 (± 1) weeks later (follow-up).20 The randomization sequence was generated by the IMBEI in Mainz. Participants could not be blinded to the allocation because of the nature of the intervention. However, raters and standardized patients (SP) were blinded to the allocation of the participants.Randomization to the two groups was stratified by knowledge  at baseline.21 For the choice of methods, we used principles of adult learning theory and integrated activities in the active mode of learning according to the “ICAP hypothesis” and used the concept of deliberate practice to design practice sessions.23The intervention group received an integrated curriculum developed based on a literature review and expert opinions , i.e., generic communication skills, and risk communication content (RCC), i.e., the key risk information being communicated. Students could score a maximum possible of 120 points for the two consultations, 72 for risk communication process, and 48 for risk communication content . For the final analysis, means of the two ratings were used.Secondary outcomes were changes in knowledge, attitudes, professionalism regarding COI, frequency of interactions with the pharmaceutical industry, and self-assessed interest, knowledge, and competence.26 from which a skepticism score was calculated where higher scores indicate higher skepticism.25 Professionalism regarding the management of COI was assessed using a situational judgment test (SJT) encompassing five scenarios involving a COI and five possible ways to behave in each. Students could score a maximum of 125 points on the SJT . A p < 0.05 was regarded to be significant for the primary outcome. Results are reported as Cohen’s d with 95% confidence interval. The McNemar test was used to test for a bias blind spot. To assess the success of the rater training and the inter-rater reliability for the VOSCE within the study, the average measures, absolute agreement, and two-way random effects ICC were calculated.Data were analyzed using SPSS 23. To test for group differences between the control and intervention groups regarding continuous outcome measures, two-tailed Figure d of 2.35  and 1.83 , respectively. The difference between the two groups thus persisted until follow-up, though it narrowed slightly  at post-test and 0.91  at follow-up). Assuming the abovementioned passing score of 60%, at post-test, 27/29 (93.1%) of the intervention group and 16/27 (59.3%) of the control group would have passed. At follow-up, 23/28 (82.1%) of the intervention group and 17/24 (70.8%) of the control group would have passed.Increase in knowledge was larger in the intervention than in the control group  at post-test and 0.96  at follow-up). Looking at the items individually, the intervention group showed a trend toward more skeptical attitudes that persisted until follow-up in all attitude items. Table Skepticism increase in the intervention group was larger than in the control group  and 1.09 , respectively. These results were similarly found for each scenario separately  of all participants indicated that they had not interacted with pharmaceutical companies within the last 6 months. This percentage increased to 39.2% (20/51) at follow-up; however, there was no relevant difference between the intervention and control groups (intervention group 40.7% (11/27), control group 37.5% (9/24)). Further data on gift acceptance can be found in online Appendix Table 2 of all The integrated curriculum on conflicts of interest and risk communication we developed led to a large, statistically significant increase in risk communication performance of the participants compared with the control group at both post-test and follow-up 30 weeks later. In addition, the intervention group showed increased knowledge regarding both risk communication and COI and a change in attitudes toward more skepticism toward interactions with pharmaceutical companies as well as improved intentions of managing COI in a way that reduces their risk of bias. However, there was no change in acceptance of gifts or attendance of sponsored events. At follow-up, differences between the two groups decreased slightly for all measures. This was especially pronounced for the MC knowledge exam, where the intervention group score declined while the control group score increased slightly. Risk communication performance and attitude items, however, were relatively stable.18 Regarding the baseline skepticism, students in our study were comparable with previous German and international studies, albeit slightly more skeptical.26 In studies on curricula regarding COI, similar changes in attitude were found as in this study. However, none of these had a comparable follow-up and effects seen were usually somewhat smaller or less consistent than in this study.16 To our knowledge, no study has assessed the management of COI by students using an SJT. Previous studies assessing the effect of curricula on accepting gifts have had contradictory results.16The change in risk communication competence is generally in line with the results of one other study evaluating a curriculum on the topic. In this single blind controlled trial, the intervention group also had significantly better scores for objective risk communication competence (content as well as process) than the control group.29 When topics are taught separately, students organize knowledge around separate categories and may fail to make connections for tasks that require incorporating different areas of expertise. Teaching the topics together with a clear structure denoting how the topics are relevant to the tasks may thus lead to a larger learning gain. Additionally, following adult learning theory, the practical exercises based on situations encountered during clinical practice underscore the relevance of topics to the participants’ daily lives, leading to a larger motivation to learn.19 Despite the large learning gains, only 40% of students in the intervention group would have passed the VOSCE when applying a criterion-based passing score of 60%. We think this may be due to a combination of factors: our rating scale is not externally validated, so it is unclear which score corresponds to a sufficient mastering of the task; students may not have prepared for the formative assessment within our study as intensively as they would have for a grade-relevant exam; and finally, the students may have needed more time to master this highly complex task.We suspect that the large effects we found are due to the integration of topics that are usually taught separately. This is supported by the fact that all the students had had courses on almost all of the topics covered in our curriculum and still showed a large learning gain. In our view, this cannot be exclusively due to the coverage of novel content, because only very little content, mostly pertaining to conflicts of interest, was taught to the intervention but not the control group. One of the reasons why integrated curricula may lead to a larger learning effect is that they support a knowledge organization by students that is better matched to the tasks that are required of them.23In addition, the gain in risk communication competence was enduring over the 30-week follow-up, unlike the knowledge gain. Possibly, risk communication is a form of procedural knowledge less prone to decline. In addition, the intense (“deliberate”) practice of this skill during the curriculum using both role play and training with SPs could explain that this competence was more enduring than the declarative knowledge needed for the MC exam.30 Furthermore, the prediction of behavior from attitudes is generally difficult.32 Lastly, our study was only powered to detect a difference in the primary outcome, possibly explaining why we did not find a difference regarding gift acceptance.It is unclear why the marked change in attitudes as well as behavioral intent did not translate to a change in behavior regarding accepted gifts or attended sponsored events. This could be due to environmental factors, conforming with the social cognitive theory of behavior.16 The long follow-up period is also an advantage compared with other trials of curricula covering similar topics. A weakness of our study is that the evaluation instruments were not formally validated. However, they had been piloted and the rating scale for the VOSCE showed a good internal consistency and high inter-rater reliability.This study had several strengths. One was the meticulous development process for the integrated curriculum, which resulted in a well-crafted course with a high acceptance by the participants. Methodically, the randomized controlled design is a major strength of this study. It is the first study to investigate the effects of a curriculum on COI or risk communication with such a design.The fact that most of the individual topics necessary for risk communication had already been taught to participants previously supports our hypothesis that the large learning gains we found are mostly due to our method of integration of topics rather than due to the teaching of previously unknown subject matter. Consequently, an entrustable professional activity testing clinical decision-making under the consideration of COI has been introduced as a requirement for the licensing examinations in Germany following the results of this study. However, our study was not designed to test which elements of the curriculum were responsible for the large effects. Further studies are needed to identify whether our hypothesis that integration is the reason for the large effects holds true and to demonstrate the implementation at other universities is feasible and leads to comparably strong effects as seen in our study.ESM 1(DOCX 41.5 kb)


Background: Pneumococcal conjugate vaccines (PCVs) have prevented deaths due to pneumonia among children. The effect may differ between higher- and lower-income populations due to various factors, such as differences in the distribution of pneumococcal serotypes, healthcare access, and PCV uptake. This study aims to evaluate an association between increasing PCV coverage and population-level declines in death due to pneumonia and its variation by socioeconomic status of subnational regions.Methods: We analyzed municipality-level mortality data from 2005 and 2015 for children aged 2-23 months in Brazil, Colombia, and Peru. We fit Poisson regression models to estimate the relationship between changes in PCV uptake and deaths due to all-cause pneumonia among subnational regions with different income levels. We controlled for changes unrelated to PCV by using data on non-respiratory deaths over time.Results: Uptake of the third dose of PCV varied across subnational regions and was higher in high-income regions. Higher uptake of PCV was associated with larger declines in pneumonia mortality. This association did not differ by income level of the region in Brazil and Colombia. In Peru, low-income regions observed larger declines in pneumonia deaths, but there was large uncertainty in the difference between the low- and high-income regions. We estimated that, with 90% coverage, there would be 4-38% declines in all-cause pneumonia mortality across income levels and countries.Conclusions: Regions with higher PCV coverage experienced larger declines in pneumonia deaths, regardless of the income level. Having more reliable data on mortality records and vaccine uptake would improve the reliability of vaccine impact estimates. For instance, national-level analyses found that estimated declines in pneumonia mortality among children 2–11 months of age were 8% : 2 to 14%) in Brazil, 14% (-5 to 35%) in Colombia, and 36% (17 to 44%) in Peru7. The impact of PCVs on preventing deaths due to pneumonia could differ between higher- and lower-income populations. Differences in the etiology of pneumonia, the distribution of pneumococcal serotypes, and access to healthcare can influence the proportion of deaths that are preventable. Variations in vaccine uptake can also influence the overall reduction.Vaccination programs targeting pneumococcus are now established globally. Pneumococcal conjugate vaccines (PCVs) have effectively reduced the burden of disease and death due to pneumonia and other severe pneumococcal infections The countries in the Latin America and Caribbean region are ideal for conducting evaluations of the impact of PCVs. Countries in this region were among the first lower- and middle-income countries to introduce PCVs. The strong health and mortality data systems in these countries make it possible to perform detailed vaccine evaluation studies at a subnational level and to evaluate how variations in vaccine uptake relate to changes in rates of pneumonia deaths. The countries also have a wide variation in socioeconomic status at a subnational level, which provides a unique opportunity to compare the impact of PCV by income level. Therefore, this study aims to evaluate how the association between increasing vaccine uptake and population-level declines in death due to pneumonia varies by socioeconomic status in Brazil, Colombia, and Peru.7. Among the 10 countries included in the national-level study, Brazil, Colombia, and Peru had subnational data on mortality, socioeconomic status, and vaccine coverage with a high spatial resolution, which enabled us to conduct this analysis. Mortality data were obtained from the National Mortality Information Systems. Both primary and non-primary causes of deaths were recorded for Brazil and Peru, while only primary causes were available for Colombia. The cause of death was classified using the International Statistical Classification of Diseases and Related Health Problems tenth revision (ICD-10) codes. Each country conducted standardized data cleaning and quality control8. We extracted mortality data for children at the municipality level that were available during the study period (2005-2015). The quality of mortality data was assessed and reported in the previous study7. Durations of pre- and post-PCV periods for each country are described inIn a previous study, we evaluated the population-level impact of PCVs against pneumonia mortality at the national level in ten Latin American and Caribbean countriesAn outcome of our analysis was all-cause pneumonia deaths, defined as having an ICD-10 code in the range of J12-J18. For Brazil and Peru, we used J12-J18 recorded as either primary or non-primary causes of death as the outcome in the main analysis, and used J12-J18 recorded as the primary cause of deaths as the outcome in the sensitivity analysis. For Colombia, we used J12-J18 recorded as the primary cause of death as the outcome, as they did not have data on non-primary causes of death.7. Children under two months of age were not included in the analysis for both biological and practical reasons7.We analyzed data for children 2–23 months of age, as our previous national-level analysis did not detect any changes in pneumonia mortality among older children (24–59 months of age) following the introduction of PCV in any of the ten countries we analyzed10 These income categories were defined for each country separately and cannot be directly compared across countries. We then created subnational regions by grouping municipalities based on these three income-level categories in each of the departments (n=32 in Colombia and n=25 in Peru) or states (n=27 in Brazil). For example, Peru had 75 possible combinations of departments and income levels; however, there were only 68 subnational regions simply because some strata did not exist . Furthermore, we excluded subnational regions from the analysis if there were less than ten all-cause pneumonia mortality per year on average. After this exclusion, we had 49 subnational regions in Brazil , 16 in Colombia (12 medium- and four high-income regions), and 23 in Peru  in the final analysis.For Brazil and Colombia, we classified municipalities into three income levels  using the Gross Domestic Product (GDP) per capita for the year of PCV introduction. For Peru, these three categories of the income level were created based on a five-level income level indicator, reflecting the per capita income in the year of PCV introduction. This indicator is obtained from household surveys and is based on quintiles of household per capita expenditure as it relates to the costs of a “basic consumption basket.”Brazil introduced the 10-valent PCV with a 3+1 schedule  in March 2010. Colombia introduced the 10-valent PCV in November 2011 and used a 2+1 schedule (2 and 4 months + 12 months of age). Peru introduced the 7-valent PCV with the 2+1 schedule in August 2009 (2 and 4 months + 12 months of age), and switched to the 10-valent PCV with the 2+1 schedule in December 2011.Data on the PCV third dose coverage  by year were obtained from the National Immunization Programs at the municipality level in all three countries. For Colombia and Peru, we first conducted linear interpolation for missing coverage data, if there were any, in each municipality. We then calculated a weighted average of the coverage in each subnational region. We used the population size of each municipality as a weight for Brazil and Colombia. For Peru, a total number of deaths during the study period in each municipality was used as a weight, as municipality-level population data were not available.To estimate changes in pneumonia mortality among children after the introduction of PCVs, we fit the following Poisson regression model to the data from each country separately such thati,tY, is the number of all-cause pneumonia deaths in subnational regioni in yeart. To control for unmeasured temporal factors affecting pneumonia mortality, we included all-cause deaths other than those caused by respiratory illness as a covariate in the model  whereβi1 represents the region-specific regression parameter that describes the association between this covariate and deaths andβi0 is the region-specific intercept. To account for potential overdispersion and unexplained correlation in deaths across time, we included an observation-level random effect,ϕi,t, that follows a first order autoregressive process. The weighted average of the PCV third dose coverage in subnational regioni in yeart is given as vi,t and the region-specific vaccine effect,iθ, is modeled as a function of region-level socioeconomic status such thatThe outcome,iθ that is not explained by the effect of income level is captured byiη which are modeled using independent Gaussian distributions with zero mean and shared variance parameter.where11. The aggregated time series data and code can be found in the following GitHub repository:https://github.com/weinbergerlab/PAHO_subnational. A stable copy of the repository is available fromhttps://zenodo.org/badge/latestdoi/291341482. More details on the model, such as the information on prior distributions, can be found in theExtended data, Supplementary methods12.Posterior medians were used as point estimates and the 95% highest density CrIs were calculated to quantify uncertainty. All analyses were performed in R Extended data, Supplementary methods12.We evaluated how the population-level impact of PCV changes by income level in a few different ways. First, we quantified declines in all-cause pneumonia deaths based on the actual coverage in the last year of the study period in each subnational region. Next, to compare the effect of PCVs between regions while holding vaccine uptake constant, we estimated declines in all-cause pneumonia deaths that would be expected with 90% uptake of the third dose of PCV. More details on the calculations are described in theExtended data, Figure S1)13. For example, although the average uptake of the third dose was 82% at the national level in the last year of the study period in Peru 13. For example, in Peru, the median uptake was 67%, 88%, and 90% in low, medium, and high-income regions, respectively, in 2014.The uptake of PCV increased rapidly within a few years after introduction in all countries, but there was variation by region 12. The discrepancy in Peru was due to changes over time in the proportion of pneumonia deaths recorded as the primary cause vs contributing cause of death 13, and this shift exaggerated the estimated impact of PCV when analyzing the primary cause of death only.For Brazil and Peru, we repeated the analysis but only included deaths where pneumonia was recorded as the primary cause of death as the outcome variable. This modification did not appreciably change the estimated declines in Brazil, while it increased the estimated impact of PCV in Peru 2. On the other hand, a multi-country study conducted time series analysis and did not find declines in Guyana and Honduras due to large uncertainty7.To date, seven post-licensure studies have evaluated the impact of PCV on pneumonia mortality, all of which were conducted in Latin American and Caribbean regions6, while we did not see differences across income levels. That study used three indicators , while we used GDP per capita in the year of PCV introduction. Among children 2–23 months of age, the previous study found the largest decline in low-income regions only when municipalities were grouped based on maternal primary education. Our income classification based on GDP is most closely related to their HDI-based classification. With this HDI category, the previous study found that medium-level regions observed slightly larger declines than low- and high-level regions, which is consistent with our findings for Brazil , both our outcome (J12–18) and controls (other ICD-10 chapters) had small counts per unit time at subnational level. To reduce the noise introduced by sparse counts, we aggregated data by year. When aggregating time series data by a larger time interval, models will be fit to fewer data points, which may also cause an issue, such as increased uncertainty in estimates.7. To minimize the influence of data quality, each country collected and cleaned the data using standardized methodology in collaboration with the Pan American Health Organization (PAHO). We also controlled for changes in data quality by including the control cause of death in the model, assuming that changes in data quality affect both the outcome and the control. Our results, however, should be interpreted carefully. Regarding the data on PCV uptake, some municipalities in Brazil had unreliable data exceeding 100% with abrupt increases and decreases. We defined the income level of each municipality using the data in the year of PCV introduction, although the income level could change over time. For trend adjustment, we were only able to include one control cause of death  due to data sparsity. In Peru, even common control causes of death only had 0–2 deaths per year in many regions. However, we believe that all-cause deaths were able to successfully adjust for some key trends because its estimated coefficient was different from zero in many regions.Our study has some limitations. The quality of mortality data varied by country and changed over time, as discussed in detail in the national-level studyOne challenge with the type of analysis conducted here is that vaccine uptake data tend to be less reliable at local scales. Estimating both the numerator (number of children vaccinated) and denominator (number of children eligible for the vaccine) becomes more challenging at small spatial scales. Because the vaccine uptake data were measured with error, the estimates for the association with pneumonia deaths could be biased towards zero. This trend was clear especially among small low-income regions in Peru which were filtered out from the final analysis. Having higher quality data on vaccine uptake at a local level could facilitate robust estimates of vaccine impact in other countries and regions.In conclusion, in three middle-income countries in South America, regions with higher PCV coverage experienced larger declines in pneumonia deaths, regardless of the income level. Focusing public health efforts on increasing the vaccine coverage could prevent additional childhood deaths.http://doi.org/10.5281/zenodo.401109714.Zenodo: AHO_subnational 0.2;https://github.com/weinbergerlab/PAHO_subnational/tree/master/Data.Subfolder ‘Data’ contains the aggregated time series data used in this study. Data are also available atbr.meso.v1.csv, co.meso.v1.csv, and pr.dept.inc.v1.csv include time series for subnational-level mortality data for Brazil, Colombia, and Peru, respectively. pr.cov.by.dept.inc.csv includes the PCV coverage data for Peru.https://doi.org/10.6084/m9.figshare.12934901.v112.Figshare: OnlineSupplement.pdf;This file contains the following extended data:Supplementary methodsTable S1. Average declines in all-cause pneumonia mortality among children aged 2–23 months expected with the 90% coverage of PCV by income level in Brazil and Peru using J12-18 coded as the primary cause of death as an outcome.https://doi.org/10.6084/m9.figshare.12957887.v113.Figshare: Supplementary Figures;This file contains the following extended data:Figure S1. Uptake of the third dose of pneumococcal conjugate vaccines by subnational region in Brazil, Colombia, and Peru.Figure S2. Coverage of PCV third dose in the last year of the study period by income level in Brazil, Colombia, and Peru.Figure S3. Proportion of J12-J18 recorded as the primary cause of deaths among J12-J18 recorded as any causes of deaths in Brazil and Peru.https://github.com/weinbergerlab/PAHO_subnational.Analysis code available from:http://doi.org/10.5281/zenodo.401109714.Archived analysis code at time of publication:Creative Commons Attribution 4.0 International license (CC-BY 4.0).All data and code are available under the terms of the This is a very timely and appropriate piece of research.  In the past some (including this reviewer) have argued that PCVs have the potential to increase inequity in child survival unless high coverage is reached in the poorest communities.  This research addresses this issue in 3 countries of the Americas where quality mortality data are available at subnational level. In each country departments or states are categorized into 3 levels based on income data from other sources.  Then the pneumonia mortality rates were evaluated by income level and time.  To account for important differences in PCV coverage by income, the authors extrapolated to a hypothetical PCV coverage level of 90%.  This seems particularly important in Peru where PCV coverage ranged from 67% to 94% in the 3 strata.  Exactly how this extrapolation was done is unclear to this reviewer.  Logically the relationship between PCV coverage and mortality is non-linear as the unvaccinated will be at higher risk, regardless of income level, and this effect may be more acute at lower income levels. Figure 2 seems to show that this adjustment had little effect on the conclusions. Figure 3 which shows estimated mortality declines with PCV coverage is a bunch of straight lines with various data points on it.  What does this mean? On Page 5, “In Peru, this association was especially strong in low-income regions, while it was modest among high-income regions and not clear among medium-income regions.” This seems a polite way to put this.  The graph seems to show increasing mortality among most of the middle income regions. This probably reflects issues with data, but it is difficult to conclude that.  At least it should be discussed. As a reader it is a little difficult to really understand the paper without the basic pneumonia mortality data in the 3 strata against which the trends are being analysed.  All data are presented relative to the year of vaccine introduction, 2009, 2010 and 2008 for Brazil, Colombia and Peru respectively. The 3 countries have all seen substantial falls in child mortality over the period which predated PCV introduction, so the key question seems to be what was the impact of PCV, over-and-above the declines that were taking place. That would appear to be presented in Figure 1, which seems to show that there was no significant decline in any of the 3 countries, once non-pneumonia mortality trends were accounted for. Figure S3 is difficult to follow, probably because of the legend. In conclusion, as a reader I was reassured that PCV is associated with reduced child pneumonia mortality in most communities in the 3 countries, but this is quite in line with overall changes in child mortality in those countries so it is difficult to attribute the decline to PCV.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Infectious disease epidemiology; vaccinology, paediatricsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. This study sets out to evaluate how the association between increasing vaccine uptake and population-level declines in death due to pneumonia in children 2-23 months varies by socioeconomic status in Brazil, Colombia, and Peru. The authors have recently published an evaluation of the population-level impact of PCVs against death due to pneumonia in children under 5 years of age. The value of such national level analyses is size but the datasets from Peru and Colombia are modest. The analysis is confounded by heterogeneity of vaccine programmes and falling pneumonia death rates before vaccine introduction. These data nonetheless would be a valuable addition to the literature. I have some suggestions that could improve the quality of the manuscript:TITLE: The authors have specifically focused on children 2-23 months of age – this should be reflected in the title.INTRODUCTION: Although it may seem obvious, the authors should introduce what is already known and why differences in pneumonia deaths related to socioeconomic status between and within countries may be expected.METHODS:This analysis is highly dependent on the coding of pneumonia episodes in each country, uniformity of practice between countries and over time. The reader is pointed to a WHO document on standardized data cleaning and quality control which is in Spanish. Some further detail should be provided. Have any checks been done to ascertain miscoding? Do the authors have any data to demonstrate the fidelity of the coding.The authors state that income level was ascertain in “household surveys”. Were these undertaken by the national governments?To provide a context for the data, the authors should state the approximate birth cohorts for each country and the total number of deaths during the same period.To account for overdispersion, an observation-level random effect was included in the analysis. Is there evidence that this was successful?Were permissions required to use this national data?RESULTS:Table 1 shows that the total number of J12-18 among children 2–23 months in Brazil coded as primary or non-primary causes of death is 16,303 4700 and the total number of J12-18 among children 2–23 months of age in the study period coded as primary cause of death is 35,259. Is this the correct way around?The authors provide the weighted average of PCV third dose coverage in the last year of the study period in the main text but the rapidly of vaccine uptake and the difference between and within countries are best illustrated by Figure S1 which should be included in the main manuscript.To understand trends, all-cause pneumonia has been adjusted for all-cause mortality other than respiratory illness but the underlying trend could be in infectious diseases only. What is the overall trend in non-pneumonia infectious diseases? Has this been used to adjust all-cause pneumonia?Figure 3 is referred to in the statement “Higher uptake of the third dose of PCV was associated with additional declines in pneumonia deaths in most of the regions in all three countries”. An analysis should be provided to confirm what seems to be apparent visually.DISCUSSION:The statement, “The estimated decline in all-cause pneumonia deaths associated with increasing PCV uptake was similar across income levels in Brazil and Colombia, while there was a larger decline among low-income regions in Peru”, should include a statement about the uncertainty.Limitations should include: the number of pneumonia deaths were relatively modest in Peru and Columbia; heterogeneity in schedules and vaccine valency which may be particularly relevant for younger ages; and the estimates of vaccine coverage.The main conclusion is that “in three middle-income countries in South America, regions with higher PCV coverage experienced larger declines in pneumonia deaths”. This needs to be formally substantiated in the results (see above).Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Microbial Pathogenesis; Mucosal Immunity; Vaccine Preventable DiseasesI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. This is an important study contributing to the body of literature documenting PCV impact on mortality. The authors apply complex analytic methods to evaluate association between increasing vaccine uptake and pneumonia-related deaths by socioeconomic factor. The paper is well written and methods are clearly explained.Declines in mortality are reported; however, for all estimates wide CrIs and all estimates cross the null value. Is the conclusion about the declines observed in all countries in the last year of study warranted given high level of uncertainty around the estimates? Perhaps the conclusion and corresponding statements in the results could be softened.How do the authors explain increases in mid-income regions in Peru and mid- and low-income regions in Brazil? Several lines for corresponding regions in Fig 3. suggest there were increases in deaths and the lines are above the dotted “null” line as vaccine coverage increases.Changes in coding practices over time could have contributed to observed trends. This should be included as a limitation.Unreliable PCV coverage data is mentioned in limitations without explanation of how this was resolved in the analysis (e.g. >100% coverage or unstable coverage estimates).Have any control “exposures” been evaluated, in addition to adjusting for “control cause of deaths”? If an association is found between control “exposures” (e.g. vaccine uptake other than PCV) that should not be associated with pneumonia-related deaths, this could indicate that the model is not adjusting for time-dependent confounders. Specific comments:Table 1. Number of deaths for Brazil appear to be flipped - numbers are higher for pneumonia as primary-only cause of death than for pneumonia as primary or secondary cause of death.Figure 1B interpretation in the text suggests that after adjusting for non-respiratory causes of death, the trends became “unclear”. From the figure, it appears that for most strata there was no longer a downward trend before or after PCV introduction. A couple of minor comments:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


This report describes a case of an electrical storm of Torsades De Pointes in a structurally normal heart, following an H1N1 infection in the presence of a genetic variant of unknown significance. The patient was successfully treated with isoproterenol. This case highlights the dilemma of evaluating novel genetic testing results in a clinical setting. A 60-year-old male with an implantable cardioverter-defibrillator (ICD) presented to the emergency room (ER) for being shocked multiple times. He was hemodynamically stable with a blood pressure of 115/72 and a heart rate of 93. He reported fever, muscle aches, runny nose, and a productive cough with white sputum for the last 2 days. The physical exam was unremarkable except for mild erythema in the oropharynx with no exudate.His past medical history was significant for recurrent Torsades De Pointes (TdP) in association with aspiration pneumonia in 2014 status post-ICD placement; heart catheterization at that time showed completely normal coronary arteries. He denies any family history of cardiac disease in his parents or grandparents.Differential diagnoses at this point included emergent interrogation of the ICD device which confirmed the diagnosis of TdP. Thus, the differential was for the eliciting factor which was broadly similar to the data found in Upon further investigations and after interrogating his pacemaker, eleven shocks for ventricular tachycardia (V-Tach) and TdP were confirmed. Electrocardiogram (ECG) showed sinus rhythm , first-dIn ER, an amiodarone drip and a bolus were started by the ER physician. Oseltamivir was started, 2 grams of magnesium was given, and he was admitted to the intensive care unit. Overnight, the patient was shocked for multiple runs of TdP and V-Tach as shown in The definition of cardiac electrical storm (ES) is electrical instability of the heart manifesting as recurrent ventricular arrhythmia in a short period of time (>3 in 24 hr). Despite the significant improvement of survival with ICD placement in these patients, ES remains to hold high mortality and morbidity and has a negative impact on long-term outcomes . It is iα-subunits  of these channels are detected [Recognizing the genetic substrate underlying the inherited arrhythmia syndromes has remarkably improved the field of the molecular basis of cardiac electrophysiology, including arrhythmia mechanism and the role of the different ion channels , 3. Genodetected . Currentdetected . DespiteSince the ES is an emergency, it is initially managed with the advanced cardiac life support (ACLS) protocol , regardlBut what if the inducing factor is H1N1 infection without the genetic predisposition or myocarditis? Or the upper respiratory infection with H1N1 in the setting of KCNH2, p.Leu586Val variant is the combination inducing this TdP ES?In our case, the patient had TdP in 2014 that was believed to be induced by his aspiration pneumonia, and since then, the patient was symptoms free until the recent H1N1 that caused his ES. Oseltamivir was given, which has a safety profile far outweighs the side effects that includes 5-10% nausea, vomiting, and no known arrhythmic side effects that might complicate the patient's situation . While mAlso, it has been reported that H1N1 could induce ES in a patient with LQTS which was treated with perfusing magnesium sulfate, increasing the resting pacing rate from 40 to 85 bpm, and increasing propranolol dosage . In contWhile more studies are required to understand the effect of H1N1 in patients with genetic variants of uncertain significance, it is worthwhile to consider the potential effect of H1N1 influenza infection in triggering an electrical storm in this population. This may have a great impact as ventricular arrhythmias are a cause of sudden death.