Herpesviridae protein kinases (CHPK) are conserved among all members of the Herpesviridae. Herpesviruses lacking CHPK propagate in cell culture at varying degrees, depending on the virus and cell culture system. CHPK is dispensable for Marek’s disease herpesvirus (MDV) replication in cell culture and experimental infection in chickens; however, CHPK—particularly its kinase activity—is essential for horizontal transmission in chickens, also known as natural infection. To address the importance of CHPK during natural infection in chickens, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomics of samples collected from live chickens. Comparing modification of viral proteins in feather follicle epithelial (FFE) cells infected with wildtype or a CHPK-null virus, we identified the US10 protein (pUS10) as a potential target for CHPK in vivo. When expression of pUS10 was evaluated in cell culture and in FFE skin cells during in vivo infection, pUS10 was severely reduced or abrogated in cells infected with CHPK mutant or CHPK-null viruses, respectively, indicating a potential role for pUS10 in transmission. To test this hypothesis, US10 was deleted from the MDV genome, and the reconstituted virus was tested for replication, horizontal transmission, and disease induction. Our results showed that removal of US10 had no effect on the ability of MDV to transmit in experimentally infected chickens, but disease induction in naturally infected chickens was significantly reduced. These results show CHPK is necessary for pUS10 expression both in cell culture and in the host, and pUS10 is important for disease induction during natural infection.Conserved  Herpesviridae protein kinase (CHPK) of MDV is essential for the initiation of natural infection, and we identified the virion protein US10 as a potential target for CHPK during natural infection. Our results showed that MDV CHPK was required for expression of pUS10 in both cell culture and in chickens. Although MDV lacking pUS10 replicated and spread in experimentally infected chickens, it was less virulent implicating an important role of pUS10 in natural infection of the host. These results emphasize the importance of studying specific viral proteins and their functions during infection of the natural host.Marek’s disease herpesvirus (MDV) is an important pathogen in the poultry industry. Current vaccines reduce disease but do not protect chickens from infection. Understanding natural infection provides vital information for developing potential therapies to protect against Marek’s disease, while also providing a robust natural virus-host model to study herpesvirus pathogenesis. The conserved  Herpesviridae protein kinase (CHPK) that is common to all members of the Herpesviridae and produces serine/threonine kinases  belonging to pUS10 that was consistently phosphorylated on Serine 20 (S20) in vUL47eGFP and vCHPKwt-infected cells but not in vCHPKmut-infected FFE cells. Furthermore, the sequence containing the phosphorylation site of interest (SPKE) was identified as a casein kinase 2 (CKII) (SxxE/D) and cyclin-dependent kinase (CDK) (SPxK) motifs using MyHits ) from our preliminary study but identified additional and small peptides, most likely due to the implementation of an optimized tryptic digestion protocol. Now, four phosphorylated peptides were identified, exclusively in samples from vCHPKwt/10HA-infected FFE cells that were not detected in vΔCHPK/10HA-infected chickens.To validate our preliminary data concerning S20 phosphorylation on pUS10 during MDV infection, we examined the global proteome and phosphoproteome of infected FFE cells. This two-pronged approach is summarized in Combining the data from the vCHPKwt/10HA and vΔCHPK/10HA-infected samples, pUS10 was confidently identified by 20 peptide sequences; 19 of these peptides were phosphopeptides . InteresAlthough we did detect multiple pUS10 peptides in vΔCHPK-infected samples, pUS10 expression appeared to be completely abrogated in the absence of pCHPK expression in CECs  and FFE Next, the ability of vCHPKwt/Δ10HA to replicate in experimentally infected chickens and to infect naïve contact chickens was tested. qPCR assays for MDV genomic copies in the blood of experimentally infected chickens showed significant differences (p <0.05) in virus replication at 10 and 35 days pi but no other differences at 4, 7, 14, 21, and 28 days pi between the two groups . MonitorThere was no significant difference (P>0.05) between vCHPKwt/10HA and vCHPKwt/Δ10HA for MD induction in experimentally infected chickens with both inducing 88 and 75% MD (tumor) incidence, respectively . HoweverExpression of pUS10 was evident in FFs  and FFE When pUS10 expression was evaluated, two forms of pUS10 were also seen in vCHPKwt/10HA-infected cells, while only one band was observed in vCHPKmut/10HA-infected FFE cells . DephospIt has been firmly established that MDV pCHPK is required for horizontal transmission in chickens , 40, 42.E. coli two-hybrid screening system and Ly6E was found to be associated with the length of survival and incidence of tumors following MDV challenge  A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Robert F. KalejtaAssociate EditorPLOS PathogensBlossom DamaniaSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens <span style="font-family: "Noto Sans", sans-serif; font-size: 12px;"> orcid.org/0000-0002-7699-2064 </span> ***********************The reviewers considered your manuscript as interesting and well-written, but raised several issues that would need to be addressed in any resubmission. While all reviewer comments would need to be addressed, particularly important ones include additional controls for IP, co-localization, and phosphorylation experiments, excluding secondary mutations as a source of the phenotype of mutant viruses, exploring autophagy as a possibility for the decrease in Us10 protein levels, and eliminating the use of "data not shown".Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: Ponnuraj et al., pursue the very interesting observation that the MDV conserved herpesvirus protein kinase (CHPK) is necessary for natural transmission of the virus and explore the possibility that this function is mediated by phosphorylation of one the virus tegument proteins, pUS10. They present proteomic evidence that pUS10 is phosphorylated at several specific sites in the presence of CHPK catalytic activity, but not in its absence and go on to explore the significance of CHPK and pUS10 in production of infectious virus, virus transmission and development of disease in experimentally and naturally infected chickens.There are many interesting findings, including that the MDV CHPK catalytic activity is required for improving the specific infectivity of MDV virions, that CHPK strongly affects the expression level of pUS10 at some post-transcriptional level and that CHPK and pUS10 strongly affect each other’s localization in the infected cell. The authors also provide suggestive, but not conclusive evidence (see specific comments below), that these two proteins interact physically. The principal finding regarding the functional consequence of pUS10 phosphorylation is negative – deletion of the US10 gene has no significant effect on natural transmission but may possibly promote development of Marek’s disease following natural transmission.1. The co-localization of pUS10 and CHPK looks very convincing in the pair of cells shown in Figure 3E, but it is not clear from the text or legend how many cells were sampled for the quantitative analysis of colocalization. This number should be at least 10 and information should be provided about how cells were selected for analysis to minimize investigator bias. This same comment applies to the colocalization results shown in Figure 5 and 6.2. The Co-IP shown in Figure 3F is not well-controlled. Showing that VP5 is not co-precipitated goes some way toward demonstrating specificity, but important negative controls are not shown. Ideally, the results of co-IPs in which pUS10 and CHPK are not tagged should be shown.3. The evidence that the K170M mutation changes CHPK expression is not convincing since the diminished expression of the mutant shown in Figure 3D is not at all evident in the no-inhibitor controls shown in Figure 4B. Indeed, in Figure 4B, it looks like expression of the mutant CHPK is enhanced compared to the wild-type. Since there is evidently variability in the relative expression levels, this effect would need to be carefully quantitated to be convincing. In this light, the molecular dynamics simulation of the effect of the K170M mutation in CHPK is a distraction from the rest of the experimental findings in the manuscript and should be removed.4. The title for the legend of Figure 5 states that pCHPK and pUS10 copurify in cells, but the figure contains no copurification results.5. The authors state that the results show that “. . . tagging MDV pUS10 with 2×HA at the C-terminus did not affect the ability of MDV to reach the FFs, induce MD, and horizontally transmit in chickens . . .” There is no condition in these experiments in which US10 is not tagged. Thus, the authors can conclude that tagging MDV pUS10 does not prevent MDV from reaching the FFs and so on, but the data leave open the possibility that tagging US10 may delay these events.6. The authors would like to conclude that the two pUS10 bands observed in immunoblotting represent differently phosphorylated forms of the protein, since the lower band is absent in cells infected with the CHPKmut virus. This is reasonable, but not conclusive. Loss of the lower band upon phosphatase treatment is needed to demonstrate this conclusively.7. The first and second paragraphs of the discussion repeat almost the same information.8. The discussion does not address the large difference in specific infectivity in virions made by CHPK and CHPKmut viruses. It would be nice to know what the authors think about this and whether there might be a role for the tegument protein pUS10 in this function of CHPK.Reviewer #2: In this clearly written article by Ponnuraj et al from the Jarolinski Group, the role of the Mareks disease virus conserved herpesvirus protein kinase (CHPK) and one of its potential targets, the US10 gene, are examined in the context of infected cells and in spread of the virus in chickens. The authors go through a number of experiments to show that the protein kinase has an influence on the amount of US10 protein in cell culture and in host animals. They have previously partially detailed the role of the CHPK previously in a publication using a virus with point mutant inactivated protein kinase (ref 23), showing that it has such a role. Here they make a new mutant deleted for a large part of the protein kinase. The identify US10 as a substrate by phosphoproteomics, and show that its phosphorylation is dependent on the viral protein kinase. It interacts with the protein kinase. Virus lacking the US10 protein shows viability and some attenuation in animal models but is surprisingly not that severe. An interesting observation is that lacking US10 changes the cellular distribution of the protein kinase. While overall the work appears to be well done, controlled and has some very intriguing observations, there are some outstanding and unresolved issues that do not outline with clarity the mechanisms going on.Reviewer #3: In this manuscript, the authors investigated the targets of the conserved herpesvirius protein kinases (CHPK) of Marek’s disease virus (MDV) using LC-MS/MS. They identified the US10 capsid/tegument-associated phosphoprotein as a target of CHPK in MDV infection in vivo. Expression of US10 was shown to be dependent on the CHPK activity. In addition, they examined the role of US10 in this model of virus induced tumor formation and could demonstrate that the protein is dispensable for transmission but plays an important role in the disease induction in naturally infected chickens. The manuscript is very well written and contains exciting data on these two conserved herpesvirus proteins that will be of interest to herpesvirologists in general and beyond. This natural virus-host model provides intriguing data that could not have been obtained for other herpesviruses. Only the following points should be addressed prior to publication.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: No-tag control for Co-IP shown in Figure 3F.No-tag control for US10 arrival at follicles, transmission and disease development.Phosphatase treatment of pUS10 to show multiple bands are due to phosphorylationReviewer #2: It is not well resolved why intracellular levels of the US10 protein decline with virus point mutated in the CHPK, and why they are nearly non-existent in the CHPK deleted virus. It is apparently not proteosome mediated degradation (not affected by MG132) or at the transcriptional level, but seems to be by another mechanism. This is speculated about in the discussion, but is rather an unfinished. Does stability measured by a protein pulse chase approach show increased degradation of US10 with the lack of CHPK? It may not be just the proteosome, but rather another means of turnover (autophagy). Is the protein being made? The CHPK could be targetting the translation machinery to affect translational levels. How does expression of other late genes become affected? A wider protein study is needed.Regarding the importance of US10, which is stated as being important for disease induction in chickens, the lack of US10 also affects the cellular localization of the Wt CHPK in cells. As such there are many potentials for lacking the US10 to be the cause of the modest attenuation of the virus in vivo. Thus the role of the US10 with the CHPK is complex and not resolved. It is not clear if the CHPK directly phosphorylates the Us10 or acts through another (cellular?) kinase… also please provide a bit more info on what US10 is in related viruses. Any suggestion of function? Is there a HSV equivalent? The statement made in line 392 is far too strong given the lack of the resolution of the relationship of CHPK and US10.In figure 1, virions are analyzed. However only antyibody blots are shown. It is critical to show a total protein stain of the viruses so one can judge the purity of the virus preparations and the relative abundances of the capsid proteins to other virus proteins, and the consequences of CHPK mutation on the virion protein profile. A blot of a non-structural protein should be shown, which would not be in the virions. And Why is there no major capsid protein seen in the WT infected cell extracts? This is not explained and does not make sense.Reviewer #3: 1) The authors generated an exciting set of recombinant viruses for their study. Unfortunately, they did not exclude the presence of secondary mutations in these mutants. This would be particularly important for the US10 mutant that provided very important data on the role of this protein in a natural infection. The authors (e.g. Dr. Spatz) should sequence at least this crucial mutant to ensure that this exciting phenotype is not due to a secondary mutation that can occur during mutagenesis.**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: Legend for Figure 6A should specify abbreviations. After a minute of confusion, I figured out that the -Con suffix denoted the results for chicken infected by contact with experimentally infected chickens, but this information should be in the legend.Reviewer #2: There are many sections that contain statements with data not shown. This does not conform with PLOS policy. Either the data is shown or the statement must be removedA lot of the first part of the work seems to be confirmatory of that published in reference 23, where it was shown that the CHPK influenced the in vivo spread and replication of MDV. This reduces importance and novelty of the findings presented here. By the looks of it, figure 1A largely is repetitive of that seen in publication 23. Please specify clearly which work is not precedented and what repeats previously published work.166-170 details the BAC analyses. However it would be helpful to add the restriction sites for the regions concerned to the figureLine 182 please show the single fluor channels as the nuclear location is not obvious in the figureHave the authors considered making a virus that has the US10 protein that simply lacks the sites of phosphorylation  could help explain the transcription of these genes without the functional CHPK.Figure 5A it is clear to this reader that the magnification of CHPK mutation is different from that of the WT and deletion mutants. Either that, or the mutation has a consequence on nuclei size!!!! And only a single infected cell is shown. Yet the cellular distribution patters seem quite variable in infected cell culture….Did the authors look autophpagy as a basis for the loss of ORF10There was no other mention of other targets for the CHPK in the phosphoproteome analyses studies……Reviewer #3: 1) Line 177ff & Figure 3: The authors identify that US10 expression severely impaired . But instead of continuing with this aspect, they jump to the localization of CHPV and its mutant . Only then they come back to the US10 expression validating it with a western blot . To keep the story line straight, the authors could optimally show the western blot earlier as 3C before addressing the CHPV localization.2) The authors use the Swiss-Model engine to predict the structure of the CHPK as no crystal structure is available. They should reevaluate their prediction with AlphaFold 2 as this has a much greater accuracy with its AI-based predictions.3) In their in vivo experiments, they only show the disease incidence, but not the tumor incidence. It would be great if they could provide this data in their manuscript, as they certainly have it. At least they should state if all animals that developed disease also had tumors.**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: Reproducibility:https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocolsTo enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at  6 Jan 2023AttachmentUL13onUS10Rebuttal(6-I-23).pdfSubmitted filename: Click here for additional data file. 30 Jan 2023Dear Dr. Jarosinski,We are pleased to inform you that your manuscript 'The alphaherpesvirus conserved pUS10 is important for natural infection and its expression is regulated by the conserved Herpesviridae protein kinase (CHPK)' has been provisionally accepted for publication in PLOS Pathogens.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.Best regards,Robert F. KalejtaAcademic EditorPLOS PathogensBlossom DamaniaSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens <span style="font-family: "Noto Sans", sans-serif; font-size: 12px;"> orcid.org/0000-0002-7699-2064 </span> ***********************************************************Reviewer Comments :Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: The revised manuscript of Ponnuraj et al., includes important new data and controls including phosphatase treatment to prove phosphorylation of viral proteins. They ahem also provided important standards of comparison from the literature and expanded the discussion as requested. All of my previous reservations have been satisfied.Reviewer #2: good response to most of the three reviewers comments overallReviewer #3: In this manuscript, the authors investigated the targets of the conserved herpesvirius protein kinases (CHPK) of Marek’s disease virus (MDV) using LC-MS/MS. They identified the US10 capsid/tegument-associated phosphoprotein as a target of CHPK in MDV infection in vivo. Expression of US10 was shown to be dependent on the CHPK activity. In addition, they examined the role of US10 in this model of virus induced tumor formation and could demonstrate that the protein is dispensable for transmission but plays an important role in the disease induction in naturally infected chickens. The manuscript is very well written and contains exciting data on these two conserved herpesvirus proteins that will be of interest to herpesvirologists in general and beyond. This natural virus-host model provides intriguing data that could not have been obtained for other herpesviruses. In this resubmission of the manuscript PPATHOGENS-D-22-01876, the authors addressed many comments of the reviewers and improved the manuscript, which could now be published.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: NoneReviewer #2: am pleased with the response to the previous reviews. im am still a little concerned about the purified virions preparations and the lack of a response to a request for a protein stain of the virions to be convincing that they are indeed virions and not some other intracellular substructure or secreted elements, but i can sympathize that this is probably difficult to do. It still leaves a bit of a concern, but i will abide by the editor on this issue if they think a protein stain is not nescessarymy only requirement is line 225, it should be figure 5A not 4AReviewer #3: none**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: NoneReviewer #2: (No Response)Reviewer #3: none**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article ," has been formally accepted for publication in PLOS Pathogens.We are delighted to inform you that your manuscript, "The alphaherpesvirus conserved pUS10 is important for natural infection and its expression is regulated by the conserved We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles  are generated on a different schedule and may not be made available as quickly.Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards,Kasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064


In this work, we identify two issues that can significantly affect the accuracy of AFM measurements of the diameter of single-wall carbon nanotubes (SWCNTs) and propose a protocol that reduces errors associated with these issues. Measurements of the nanotube height under different applied forces demonstrate that even moderate forces significantly compress several different types of SWCNTs, leading to errors in measured diameters that must be minimized and/or corrected. Substrate and nanotube roughness also make major contributions to the uncertainty associated with the extraction of diameters from measured images. An analysis method has been developed that reduces the uncertainties associated with this extraction to <0.1 nm. This method is then applied to measure the diameter distribution of individual highly semiconducting enriched nanotubes in networks prepared from polyfluorene/SWCNT dispersions. Good agreement is obtained between diameter distributions for the same sample measured with two different commercial AFM instruments, indicating the reproducibility of the method. The reduced uncertainty in diameter measurements based on this method facilitates: (1) determination of the thickness of the polymer layer wrapping the nanotubes and (2) measurement of nanotube compression at tube–tube junctions within the network. Advances in methods for the scalable manufacture, purification, and dispersion of single-wall carbon nanotubes (SWCNTs) have dramatically accelerated the development of applications based on this material. In particular, various approaches for separating semiconducting and metallic tubes have enabled demonstration of a variety of SWCNT-based electronic devices ,4,5,6,7.Atomic force microscopy (AFM) is a widely used technique capable of measuring the vertical dimensions of nano-objects with subnanometer accuracy . While ATo demonstrate the utility of our protocol for AFM-based diameter measurements, we applied it to measure the diameter distribution of individual nanotubes in networks of highly semiconducting enriched polyfluorene/SWCNT dispersions on silicon oxide substrates. Good agreement was obtained for diameter distributions measured on the same sample with two different commercial AFM instruments and analyzed by two different analysts. The reduced uncertainty associated with diameter measurements using this method facilitates determination of the thickness of the polymer layer wrapping the nanotubes, which has not been reported previously. In addition, analysis of height measurements at tube–tube crossings within the network demonstrate compression at the junctions. Quantifying this compression is expected to contribute to further understanding of electron transport at these junctions that limit the performance of devices based on SWCNT networks . The metFour different SWCNT dispersions were utilized to prepare nanotube networks in this study. Ultrahigh purity SWCNTs (>99.9% semiconducting) poly (PFDD)-wrapped nanotubes (IsoSol S-100) were purchased from NanoIntegris  in two forms, a PFDD/SWCNT dispersion (10 mg/L) in toluene and solvent-free bucky paper which was redispersed in toluene to the same nanotube concentration. The diameter range of the SWCNTs in both these samples are specified as 1.2–1.4 nm. The polymer-to-nanotube ratio was measured by UV–Vis–IR absorption to be 4:1 and 1:1, respectively. Semiconducting SWNCTs in an aqueous surfactant solution  were also obtained from NanoIntegris. The final tube type measured was SWCNTs dispersed in dimethyl sulfoxide (DMSO) at 100 mg/L , for which the diameter range was given as 1.4–2 nm.2 silicon substrates (Si(100) with 100 nm thermal oxide, Silicon Quest International, San Jose, CA, USA). PFDD/SWCNT dispersions were deposited directly onto clean SiO2 for 5 to 15 min, followed by rinsing with toluene for 20 to 60 s and drying with nitrogen. Aqueous surfactant SWCNT dispersions were deposited on Poly-L-Lysine (PLL)-coated SiO2 for 15 min, rinsed with water, and dried with nitrogen. The Linde SWCNT dispersion in DMSO was handled in a nitrogen-purged glovebox, where 100 µL of 2 mg/L DMSO-nanotube dispersion was deposited on a freshly cleaved 1 cm2 square of highly oriented pyrolytic graphite (HOPG). The sample was then heated to 100 °C for approximately 15 min, causing the DMSO to evaporate. For all samples, AFM measurements were performed within a day of preparation, but several samples were measured multiple times at different dates during several months following their preparation with no significant changes in overall morphology or measured diameters.The PFDD/SWCNTs in toluene and aqueous SWCNT dispersions were deposited onto piranha-cleaned ~1 cmMost of the AFM data were obtained with a MultiMode AFM with a NanoScope V controller  in Peak Force QNM mode. In this mode, the AFM tip oscillates at a given (2 kHz) frequency, with the highest (peak) repulsive interaction force used as the feedback signal. Silicon nitride ScanAsyst-Air AFM probes  were used in all peak force feedback measurements. The AFM tip diameter and cantilever spring constants (as specified by the supplier) were 2 nm and 0.4 N/m, respectively.To demonstrate the wide applicability of the measurement methods presented here, a Nanowizard II BioAFM  in intermittent contact mode was also used to measure SWCNT diameters. Silicon cantilevers  were used for imaging in air at RT (20–23 °C). Force was carefully minimized by first gradually reducing the free amplitude to the highest level where stable imaging could be performed  and adjusting the feedback set point to a value as close as possible to the free amplitude. The typical set point obtained in this way was 85–90% of the free amplitude, which was set to 600 mV.2 in size, acquired with 512 × 512-pixel resolution and at a scan rate of 1 Hz. In this way, the lateral pixel size was approximately 2 × 2 nm2. The measured diameters of single nanotubes were in the range from 1.2 to 2 nm, and based on our tests, the radii of AFM tips for peak-force AFM were in the 2 to 15 nm range . Based on the combination of the tip and nanotube radii, the pixel size of 2 × 2 nm2 was deemed sufficient to have reliable nanotube height measurements. The pixel size should not be larger than the smallest tip size since that could lead to two adjacent pixels across the nanotube being both off the highest point of the nanotube, leading to underestimation of the tube height.Images used to measure diameters were typically 1 × 1 µmTraceable calibration grids  were used on a regular basis (approximately every 6 months) for calibration verification and adjustments of our AFM instruments. On top of that, we regularly imaged atomic steps on HOPG. These steps have a known height of 0.34 nm. In our tests, we always measured the height of the smallest steps to be 0.34 nm +/− 0.01 nm, consistent with the expected value, further confirming the precision of our calibration see . The steAll image processing and analyses were performed using Gwyddion , a free, widely available open-source software . For the2 substrate. The lowest force that consistently resulted in stable imaging, regardless of the AFM tip or of the particular nanotube sample, was 0.2 nN. Using this as the initial force, sequential images were obtained at increasing peak-force feedback values followed by a final image back at 0.2 nN, as shown in To investigate the effect of applied force on measurements of nanotube diameter, the force was gradually increased while imaging the same network area. For each image in The radial deformability of CNTs in AFM imaging has been demonstrated previously within a similar force range ,29,30, bWhile the method used above is useful for rapidly demonstrating the average diameter of an ensemble of nanotubes, it does not permit measurement of the diameter of an individual SWCNT (except in the case where there is only a single tube in the image). The conventional method to determine the heights of individual features from an AFM topography image involves extracting a height profile perpendicular to the object being measured. Here we show how this approach can lead to significant variations in the measured diameter due to the roughness of both the substrate and the nanotube.2 AFM image of a PFDD/SWCNT network on SiO2 along with parallel pairs of height profiles extracted from the measured data. For each pair, one profile is along the top of the nanotube and the other is on the substrate adjacent to the tube. The profiles on the bare SiO2 substrate (the red points in the figure) are observed to be more uneven, with height variations sometimes exceeding 1 nm. The standard deviation of the substrate height profile data ranges from 0.31 to 0.18 nm for the four profiles shown in the figure. The nanotube profiles (blue points) also appear uneven, but with considerably smaller height variations that seldom exceed 0.5 nm and with standard deviations ranging from 0.13 to 0.10 nm. While this is less roughness than that of the substrate, it is still significant and appears to be uncorrelated to that of the nearby substrate. This is consistent with studies quantifying the persistence length of carbon nanotubes, showing they behave like rigid rods within a length range that exceeds one hundred nanometers [nometers . The heiThe profiles in One way to decrease the diameter measurement errors that result from roughness is to use an averaged diameter value based on multiple cross-sections. This can be accomplished easily using the Gwyddion analysis software, which offers an automated way to average up to 128 contiguous, single-pixel-wide cross-sections in one click. We find that using even 10-pixel-wide cross-sections reduces the uncertainty significantly, with larger widths offering even greater reduction. To further minimize this uncertainty, the averaging of the substrate baseline height can be improved by utilizing all the substrate pixels over the entire image. This can be done by choosing a height threshold to select the nanotube features (define a mask), as described above. Then, the average height value of all the pixels that are not masked is used as the average height of the substrate, which is then subtracted from the maximum height of the averaged nanotube profile to obtain the diameter.2, and then repeating the analysis on it several times, each time extracting the diameters of the same tubes. In each of the images, diameters of a number of nanotubes  were measured 5–7 times using the above-described methods. Finally, the diameter standard deviation was calculated for each of the nanotubes using all of the extracted diameter values for that nanotube. The same diameter values for each of the nanotubes could be reliably obtained, with the standard deviation reduced to below 0.1 nm , using the conventional intermittent contact mode with amplitude-based feedback. For these measurements, the AFM applied force was minimized by minimizing the free amplitude, and then using the smallest amplitude set point that facilitated stable imaging. The same analysis method (averaged cross-sections and average substrate baseline extraction) was used to measure diameters of an additional 126 nanotubes. Upon designing an effective method to increase the accuracy of the AFM measurements of SWCNT diameters by optimizing both our experimental (minimized forces) and analysis (averaging) approach, we set out to apply it to measure the diameter of PFDD/SWCNTs, providing insight into the interaction of the polyfluorene polymer with the nanotubes. PFDD/SWCNT networks were formed on SiOThe nanotubes in the PFDD/SWCNT dispersion have been previously measured to have a narrow diameter distribution ranging from 1.2 to 1.4 nm . Raman oThe protocol for accurate measurements of carbon nanotube heights can also be used to investigate junctions between tubes in these random networks. These junctions are of interest since electron transport across these junctions is the factor limiting the conductivity of random nanotube networks. Compression at SWCNT junctions has been modelled theoretically  and obseTo measure compression at a nanotube junction, the maximum height at the crossing point is compared with the sum of the two diameters of the tubes that cross. 2 that are functional as a channel material in transistors. While lower uncertainties may be achieved for isolated, pristine (no polymer or surfactant used for purification and dispersion) nanotubes deposited on ideally flat substrates such as mica or HOPG, the goal here was to develop a method for diameter measurements on “real-world” samples such as those used for device fabrication.Two issues that can significantly affect the accuracy of AFM height measurements of single-wall carbon nanotubes have been identified. Measurements on several different SWCNT samples indicate that under forces commonly used for AFM imaging, nanotubes can be readily compressed, leading to a significant underestimation of the diameter. A simple procedure, consisting of imaging the same area several times at different forces and plotting height vs. force graphs, can verify the extent of the compression effect and recover the actual nanotube height via extrapolation to zero-force height. In addition, we showed that the common AFM height analysis method based on drawing a cross-section across each of the nanotubes to extract the diameter of nanotubes can lead to significant errors arising from the roughness of both the nanotubes and the substrate. An analysis protocol based on averaging a larger number of pixels was proposed and shown to be easily implemented using a free and open-source SPM analysis software. This enables decreasing the variation of measured heights to less than 0.1 nm. It is important to note that these results were obtained for moderately dense semiconducting SWCNT networks on SiOTo illustrate the utility of this diameter measurement method, it was applied to measure the diameter of commercially available, highly enriched semiconducting nanotubes. The measured diameter for the nanotubes in a PFDD/SWCNT sample was shown to be larger than expected based on the diameters of the tubes present in the dispersion, suggesting the polymer remained on the SWCNTs upon deposition on the substrate and rinsing with a solvent. The average thickness of the polymer on the nanotubes was estimated to be ~0.4 nm. In addition, measured heights of tube–tube junctions were found to be less than the diameter of the two tubes that are crossing, indicating compression of ~20% at the junctions. Finally, we note that the measurement protocol developed here should also be generally applicable to measurement of vertical dimensions of other 1D or 2D nanomaterials.


Loneliness and social isolation are associated with anxiety and psychological discomfort, especially amongst the oldest and fragile persons.SILVER evaluates the acceptance of video calls by old hospitalized patients and their relatives during the ban on visits due to the COVID-19. Moreover, SILVER evaluates if the use of different communication technology is associated with different outcomes in terms of anxiety, fear of self and of others’ death and mood.SILVER is an observational multicentre study. Patients hospitalized in two geriatric units in Switzerland and in one orthogeriatric unit in Italy and their relatives were enrolled. Participants can freely choose to use phone or video calls and were evaluated over a week. We measured anxiety, fear of death and mood at baseline and at the end of the study with standard scales. The use of video or phone calls was associated to a change in these parameters by two-way ANOVA for repeated measures.Sixty-four patients and relatives were enrolled, 26.5% used phone calls and 73.5% video calls. The use of video calls was associated with a reduction in anxiety and fear of death in patients and relatives as compared to participants using phone calls.Old patients and their relatives accepted and appreciated the use of video calls during hospitalization; moreover, participant using video calls appears to be less anxious and less afraid of death.Video calls may be a useful communication tool for hospitalized older patients to keep social relationships with relatives and reduce their anxiety and fear of death.Retrospectively registered on 1st September 2021 in ClinicalTrials.gov (NCT05000099).The online version contains supplementary material available at 10.1007/s40520-022-02188-8. Quarantine or physical isolation are used for centuries to control and limit the spread of contagious diseases . NowadayDuring the COVID-19 pandemic, the use of social technologies and, in particular, of video calls have been proposed to nursing home residents , 11 and The SILVER study aimed to evaluate the acceptance of video calls in hospitalized older patients and in their relatives during the ban on visits due to the COVID-19 pandemic. In addition, we compared patients' baseline levels and changes in anxiety, in fear of death of self, in fear of others' death, and in mood across groups choosing a different type of communication technology. Finally, we evaluated the levels of anxiety and fear of death of their loved one in relatives according to the different types of communication technology chosen.SILVER is an observational multicentre study. Patients were hospitalized in two acute and post-acute geriatric units in French-speaking Switzerland , and in one orthogeriatric unit in Italy  and their relatives were enrolled in the study from 2 December 2020 to 19 May 2021.Study population All patients aged 65 years and older hospitalized in the participating units over the period of visit ban due to the COVID-19 pandemic were invited to participate. Patients who agreed to participate were asked to identify a specific relative who had then agreed to participate.Exclusion criteria Patients or relatives who refused to participate, and patients diagnosed with major neurocognitive disorders and a Clinical Dementia Rating [a Rating  score ofTo evaluate the acceptance of video calls as communication technology by patients and relatives.To compare the levels of patients’ anxiety; fear of self-death; fear of others’ death; mood according to the type of communication technique used.Primary endpoints of SILVER were:Standardization of communication: Patients and relatives were asked to select their preferred communication support . Patients who selected video calls were provided with a tablet available in each unit whereas their relatives were asked to use their own device for the calls. Video calls were performed using Skype©, WhatsApp© video, or Face-time©, according to subjects’ preferences. The process of video calls was standardized across study centers according to the position paper COMUNICOViD [Secondary endpoints of SILVER were to compare relatives’ anxiety; and fear of others’ death according to the type of communication technique used. All end points were evaluated twice: at baseline and after a 1-week period of study. UNICOViD . At leasEach patient benefited from a comprehensive geriatric assessment at baseline; the following data were recorded: performance in basic activities of daily living (ADL) , in instTo evaluate their acceptance of video calls, patients were asked to rate on a 4-point Likert scale their agreement (from 1 strongly disagree to 5 strongly agree) on the following four dimensions: perceived utility; improvement in the feeling of loneliness; improvement in anxiety level; improvement in their fear of death. Total scores ranged from 4 to 20 with higher scores indicating stronger acceptance of video calls as a communication technique.Patients’ general anxiety was assessed using the Geriatric Anxiety Scale, a 10-item form (GAS-10). The GAS-10 is a self-reported measure used to assess and quantify symptoms of anxiety in older persons; it includes 10 questions about feelings of anxiety and symptoms related to anxiety with yes/no answers. Scores range from 0 to 10, with higher scores indicating higher anxiety. A cut-off score of 6 or more defines the presence of clinically significant anxiety .Anxiety related to the fear of death was evaluated by the Collett–Lester Fear of Death Scale (CL-FODS) revised form . The CL-The 5-item Geriatric Depression Scale (GDS-5) was used to assess patients’ mood. The GDS-5 comprehends 5 questions that evaluate depressive symptoms with yes/no answers . Total sRelatives’ acceptance of video calls was assessed using the 4-point Likert scale, previously described for patients.The Clinical Anxiety Scale (CAS), a 25-item questionnaire about anxiety symptoms, was used to assess relatives’ anxiety. Possible scores range from 0 to 100, with higher scores indicating greater anxiety. A cut-off score of 30 or more defines the presence of abnormal anxiety  related All patient’s and relative’s outcome measures were assessed at baseline and at the end of the study period (7 days).No previous study compared the efficacy of video vs phone calls on patients’ anxiety, mood, and fear of death, precluding to performing a power calculation based on existing data. Hence, a post-hoc power analysis was conducted at the end of data collection on the primary and secondary endpoints by the G*power analyses software  to provi2 test for country of enrolment.Baseline characteristics of patients who selected video vs phone calls were compared by one-way ANOVA for continuous Gaussian variables, the U Mann–Whitney test for continuous non-Gaussian variables and Fisher exact test for gender and χp < 0.05 was considered statistically significant. Graphs were drawn using GraphPad 8.0 for Windows.The effects of video calls as compared to phone calls were evaluated by two-way ANOVA for repeated measures. SPSS 25.0 was used for the statistical analyses and p = 0.371), gender , as well as across countries . General characteristics of the enrolled population are reported in Table Amongst the 91 patients and relatives asked to participate in the study, 20 patients (21.9%) and 7 relatives (7.7%) refused, leaving a final sample of 64 enrolled patients, 27 recruited in Italy and 37 in Switzerland (8 in Geneva and 29 in Lausanne). No significant difference was observed between patients who refused and those who agreed to participate in mean age .Among recruited patients, 17 (26.5%) chose to use phone calls and 47 (73.5%) video calls. Patients using phone and video calls did not differ significantly in their clinical characteristics except for the level of general anxiety that was higher in patients choosing video calls. Indeed, all four patients with GAS-10 scores above the cut-off suggests abnormal anxiety selected video calls  and of their role in reducing the feeling of loneliness (p = 0.009), whereas there was no significant difference in patients using phone calls.  within as well as across groups Fig. E; howevep < 0.001) that is able to reduce their anxiety (p = 0.013) . Post-hoc power calculation for the 4-point Likert scale with a significant alpha at 0.05 is 89%.Relatives using video calls during the study increased their appreciation toward this communication technology; in particular, they found that there is a useful communication tool . In families, CAS was significantly correlated with fear of the loved one’s death both at baseline  and at follow-up .General anxiety and fear of death were weakly correlated in both patients and families. SpecificallyGAS-10 was significantly correlated with fear of the loved one’s death in patients  vs phone (stable or increased fear) call users. We hypothesize that seeing one's own loved one is more effective than just listening to him/her voice in reassuring about his/her state of health. An additional important contribution of SILVER is to highlight the link between the reported benefit of video calls on fear of the loved one’s death and the observed benefit on patients’ and relatives’ anxiety resulting from the visits ban. Although results from the GAS-10 showed decreased anxiety over time in both groups of patients, this improvement was more pronounced among those using video calls. Similarly, relatives who used video calls were also more likely to positively appreciate the role of video calls in reducing their anxiety than those using phone calls.Overall, these results lend support to using video calls in showing added value to patients’ and their relatives’ fear of death and anxiety. Furthermore, these results strongly challenge the assumption that older patients are refractory to video calls as a communication tool with their relatives .The main limitation of this study is its sample size; nevertheless, we achieved adequate statistical power for 4 out of six outcomes. Moreover, the exclusion of patients with moderate to severe form of dementia exclude a non-negligible part of patients hospitalized in the acute care geriatric unit. Considering the peculiarity in the communication and evaluation of patients living with dementia, a study planned ad hoc may be more adequate. A randomized and controlled approach would have been more appropriate to avoid selection bias, however, we found it ethically inacceptable to impose a type of communication to patients and their relatives. Furthermore, the comprehensive geriatric methodology is the gold standard approach to drive healthcare-related decisions in older adults and it accounts for patients’ goals and preferences to maximise participation . The incResults of the SILVER study show that old adults and their relatives accept and appreciate the use of video calls as a communication tool during hospitalization; moreover, SILVER suggests that video calls may be useful in relieving general and fear of death anxiety in old hospitalized patients. Similarly, SILVER suggests that the use of video calls may reduce the anxiety due to the fear of their loved one’s death in relatives.Thus implementing video calls as a communication tool in hospitals may be useful for old adults even without visit restrictions to keep social relationships with relatives unable to come to the hospital to visit their relatives.Supplementary file1 (DOCX 18 KB)Below is the link to the electronic supplementary material.


This cohort study examines the postapproval processes and timing for making palbociclib, ribociclib, and abemaciclib available to patients with metastatic breast cancer in a high-income country. How have cyclin-dependent kinase 4/6 (CDK4/6) inhibitors reached patients in need of these treatments in the Netherlands?In this retrospective cohort study, approximately 1847 patients have adopted the CDK4/6 inhibitors  by the end of the study in 2021. Although these medicines, particularly palbociclib, reached many Dutch patients who were eligible for this treatment, adoption rates were lower than expected in the first 2.5 years after approval.Findings of the study suggest that, although CDK4/6 inhibitors rapidly reached many patients who were eligible for these treatments, the speed of adoption of innovative medicines may be further optimized and better transparency of the availability of these medicines is needed. The number of new cancer medicines that are being approved by regulatory agents is increasing exponentially. Yet little is known about the pace at which these medicines reach eligible patients in daily clinical practice during different phases of the postapproval access pathway.To describe the entire postapproval access pathway of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors in the Netherlands, from regulatory approval to reimbursement and to investigate the adoption of these medicines in clinical practice among patients with metastatic breast cancer.ERBB2 (formerly HER2)–negative metastatic breast cancer who were treated with CDK4/6 inhibitors from November 1, 2016, to December 31, 2021, were included.This cohort study reviewed approval and reimbursement decisions of the CDK4/6 inhibitors palbociclib, ribociclib, and abemaciclib and estimated the number of patients with metastatic breast cancer who were eligible for these medicines compared with the actual use in clinical practice. The study used nationwide claims data that were obtained from the Dutch Hospital Data. Claims and early access data for patients with hormone receptor–positive and Description of the postapproval access pathway, monthly number of patients who were treated with CDK4/6 inhibitors in clinical practice, and estimated number of patients who were eligible for treatment. Aggregated claims data were used, and patient characteristics and outcomes data were not collected.ERBB2-negative metastatic breast cancer since November 2016. In the Netherlands, the number of patients who have been treated with these medicines increased to approximately 1847 (based on 1 624 665 claims over the entire study period) from approval to the end of 2021. Reimbursement for these medicines was granted between 9 and 11 months after approval. While awaiting reimbursement decisions, 492 patients received palbociclib, the first approved medicine of this class, via an expanded access program. By the end of the study period, 1616 patients (87%) were treated with palbociclib, whereas 157 patients (7%) received ribociclib, and 74 patients (4%) received abemaciclib. The CKD4/6 inhibitor was combined with an aromatase inhibitor in 708 patients (38%) and with fulvestrant in 1139 patients (62%). The pattern of use over time appeared to be somewhat lower compared with the estimated number of eligible patients (1847 vs 1915 in December 2021), especially in the first 2.5 years after approval.Three CDK4/6 inhibitors have received European Union–wide regulatory approval for the treatment of HR-positive and This study found that CDK4/6 inhibitors rapidly reached many eligible patients with metastatic breast cancer and were adopted gradually over time in the Netherlands. Adoption of innovative medicines may be further optimized, and better transparency of the availability of new medicines during different phases of the postapproval access pathway is needed. These postapproval procedures take time and are often blamed for delayed or restricted patient access to innovative medicines.7After innovative medicines are approved by regulatory authorities, there are several procedures that need to be completed before the drugs become available to patients. For instance, in most high-income countries, new expensive cancer medicines are subject to health technology assessment (HTA) procedures to guarantee that scarce resources are spent on treatments that offer value for money.9 Monitoring access is thus desirable to identify possible delays in use and disparities in uptake and to assist with improvement. Previous studies on the access of patients with cancer to new medicines have covered different phases of the access pathway. For instance, some studies evaluated the degree to which new medicines were available, reimbursed, or subsidized in countries of varying economic status.12 Other studies evaluated the time from approval to first sales and described sales over time.7 These studies revealed delays in use and variations in access and uptake, but to our knowledge, no study has yet assessed the entire access pathway, from regulatory approval to reimbursement to adoption among eligible patients.Even among the wealthiest countries in the world, achieving timely and sustained access to cancer drugs has been proven difficult.ERBB2 (formerly HER2)–negative metastatic breast cancer. With improvements of approximately 10 months in progression-free survival and up to 14 months in overall survival reported in randomized clinical trials (RCTs),14 these 3 CDK4/6 inhibitors are seen as important breakthroughs in the treatment of metastatic breast cancer.In this context, the cyclin-dependent kinase 4/6 (CDK4/6) inhibitors palbociclib, ribociclib, and abemaciclib, which are indicated for the treatment of patients with metastatic breast cancer, are interesting to study. These medicines were approved starting in 2016, and thus sufficient follow-up data are available for studying adoption rates over time. Moreover, these medicines offer renewed options for patients with hormone receptor (HR)–positive and In this cohort study, we evaluated the entire postapproval access pathway of CDK4/6 inhibitors from regulatory approval to reimbursement in the Netherlands, a high-income country in Europe, and investigated the adoption of these medicines in clinical practice. To identify the extent to which these medicines have reached eligible patients, we compared use with estimated use among eligible patients with metastatic breast cancer.15 Second, expensive medicines require an HTA. Depending on the results of these analyses, the Ministry of Health, Welfare and Sport in the Netherlands decides whether the medicines can be reimbursed.17 Third, if reimbursement is granted, the pharmacy should guarantee a sufficient supply and the government needs to ensure that declaration codes are available so that hospitals can bill insurance companies (logistic and administrative procedures). Ultimately, adoption in clinical practice should take place. New medicines may be available via alternative routes during the postapproval processes. This study was deemed exempt from review by Dutch legislation, which does not require informed consent from patients or approval by a medical ethics committee for this type of study, which did not contain directly identifiable patient data. We followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.In the Netherlands, different postapproval procedures need to be completed before newly approved, solid oncological medicines reach patients in need  a review of postapproval decisions for the CDK4/6 inhibitors, (2) data analyses of the number of patients who were treated in clinical practice, and (3) an estimation of the number of eligible patients. Aggregated claims data were used, but patient characteristics and outcomes data were not collected.ERBB2-negative metastatic breast cancer who were treated in clinical practice. First, the manufacturer of palbociclib provided data on the number of new patients (incident cases) who were treated each month during the wait for reimbursement. During this period of approximately 9 months, the manufacturer initiated an Expanded Access Program (EAP) for patients with HR-positive and ERBB2-negative metastatic breast cancer. The number of patients who received treatment  per month were estimated .Two data sources, claims data and early access data, were consulted to identify the number of patients with HR-positive and ERBB2-negative metastatic breast cancer per treatment line by applying proportions that were obtained from the literature23 ; who died while receiving first-line ET; who had an aggressive course of the disease, and thus cannot undergo ET; and who did not want any anticancer treatment. We assumed that patients who did not meet the criteria for use of CDK4/6 inhibitors in the first or second line of treatment at the time of reimbursement would not receive them in a later line.The monthly number of patients who were eligible based on the indications from the EMA label  was esti26 We assumed that the efficacy of the other CDK4/6 inhibitors (ribociclib and abemaciclib) was similar. Because the reported follow-up in the RCTs was too short, we extrapolated the survival curves beyond the observed survival time using the approach of Hoyle and Henley28 ; Microsoft Excel, version 2016 (Microsoft Corp); and R, version 4.2.2 .ERBB2-negative metastatic breast cancer since November 2016. In the Netherlands, the number of patients who have been treated with these medicines increased to approximately 1847 (based on 1 624 665 claims over the entire study period) from approval to the end of 2021. Palbociclib received EMA approval in November 2016; ribociclib received initial EMA approval in August 2017, and its extended indication was approved in December 2018; and abemaciclib received EMA approval in September 2018. After regulatory approval, all 3 medicines were recommended for clinical use, but reimbursement decisions were postponed. After price negotiations, reimbursements were granted for palbociclib in August 2017, ribociclib in May 2018, and abemaciclib in September 2019. The clinical assessment phase and HTA phase lasted 3 and 9 months, respectively, for palbociclib; 1 month (first use) and 8 months for ribociclib; and 3 months and 11 months for abemaciclib . The number of new patients (incident cases) who participated in this EAP increased the first 3 months (from 24 to 60 patients), remained stable in the 5 months thereafter (from 52 to 65 patients), and decreased in the last month (from 65 to 40 patients) of the early access phase.While awaiting clinical practice recommendations and reimbursement, 492 patients with HR-positive and In the access phase, the adoption of the first approved and reimbursed CDK4/6 inhibitor, palbociclib, in clinical practice increased over time from 0 patients being treated to approximately 1847 patients over a period of more than 50 months. Use of palbociclib increased steeply after reimbursement was acquired. By the end of the study period, 1616 patients (87%) were treated with palbociclib. Only a small number of patients were treated with the other medicines of this class, ribociclib (n = 157 [7%]) and abemaciclib (n = 74 [4%]), by the end of the study period. A majority of patients (1139 [62%]) received CDK4/6 inhibitors in combination with fulvestrant, and 708 patients (38%) received it with an aromatase inhibitor .The estimated number of patients eligible  who were treated with CDK4/6 inhibitors increased during the study period, in the base case analysis, from approximately 110 patients in the month that regulatory approval was granted (November 2016) to approximately 1915 patients in the final month of the study (December 2021) . The patIn the base case, of the 198 incident cases, we estimated that 38 (19%) were eligible for first-line and 75 (38%) for second-line CDK4/6 inhibitors each month. Additionally, 85 incident cases (43%) were estimated to be ineligible . We creaThis cohort study showed that CDK4/6 inhibitors were gradually adopted in clinical practice in the Netherlands despite reimbursement decisions lagging 9 to 11 months behind the EMA approvals. The medicines reached many Dutch patients with breast cancer within a short period, but the number of patients who were treated in daily practice appeared to be somewhat lower than the estimated number of patients who were eligible in the first 2.5 years after regulatory approval.29 Almost 500 patients participated in an EAP, which allowed them to benefit from this medicine even though it was not being reimbursed yet. This observation emphasizes 2 important points. First, it suggests that HTA procedures do not necessarily delay patient access to new medicines. This finding builds on earlier work that reported that different early access routes, either privately or publicly funded, offered options to patients in high-income countries.31 Second, it suggests that frequently used quality indicators of medicine accessibility  should be interpreted with caution. These indicators are used not only to rank European countries according to their medicine accessibility but also to urge policy makers to improve accessibility.32 Based on the study findings, in-depth studies that evaluate the entire postapproval access pathway are needed to identify and elucidate the disparities in access to innovative medicines.While previous studies have reported delays of 9 months, on average, between the approval of cancer drugs and their first use in the Netherlands (2017-2020), the present study found that many patients received palbociclib immediately after regulatory approval was granted.33 Because manufacturers are not allowed to advertise EAPs, not all clinicians may be aware of these programs. Lack of awareness can be a factor in inequalities in access between patients being treated by different physicians. Second, EAPs are not funded by the government in the Netherlands. Their implementation thus relies on the efforts and goodwill of pharmaceutical companies. While some companies may see benefits in EAPs , others may be unwilling or unable to allocate resources for implementing such programs.37 Because HTA procedures take time to complete, this delay likely plays a role in inequalities in access between different groups of patients. Countries surrounding the Netherlands have used different approaches to funding early access. For instance, in Germany, all newly EMA-approved medicines are reimbursed by default at the price set by the company, which ensures immediate access for patients. Pricing negotiations take place after the medicines have been launched.37 In France, the government funds different EAPs to allow patients to use medicines before reimbursement and marketing authorization.30 It remains unclear which approaches best deliver timely, sustainable, and affordable access to innovative medicines for all patients in need.Despite the rapid use of palbociclib, there are some caveats worth mentioning with regard to its early availability via an EAP. First, there is little transparency about the availability and use of EAP programs in most countries, including the Netherlands.42 Possibly the pattern of use can be explained by physician preferences, the marketing strategies of the manufacturers, hospital policies, or differences in price rebates. We also observed that the CDK4/6 inhibitors were mainly combined with fulvestrant, which likely reflects the recommendation in the Netherlands to primarily prescribe CDK4/6 inhibitors in the second line of treatment.24 The Dutch recommendation is contrary to that of other high-income countries , where they advise to prescribe CDK4/6 inhibitors in the first line of treatment.43 First-line treatment with these drugs has been associated with a longer progression-free survival but also with more toxic effects and higher costs due to longer treatment durations.44 Undoubtedly, the recommendation to use the CDK4/6 inhibitors mainly in the second line of treatment is interesting from a budget-impact point of view , which may help to ensure sustainable health care financing and prevent access problems in the long run. It remains to be seen what strategy is the most optimal from the effectiveness and cost-effectiveness perspectives. This topic is currently being investigated in the Dutch SONIA (Endocrine Therapy Plus CDK4/6 in First or Second Line for Hormone Receptor Positive Advanced Breast Cancer) trial.44We evaluated the use of 3 medicines of the same class that were approved for combination with either an aromatase inhibitor or fulvestrant. In this study, we observed that palbociclib, the first approved medicine of the 3 CDK4/6 inhibitors, remained the predominant medicine that was prescribed. This preference for palbociclib was also seen in other high-income countries, although there is no clear evidence that palbociclib is superior to ribociclib and abemaciclib.46 Therefore, it may be unsurprising that we observed a gap between the number of patients who were treated and the estimated number of patients who were eligible in the first 2.5 years after reimbursement was permitted. The disparities found in this study may partly reflect the uncertainty surrounding the estimated patient numbers, which relied on modeling rather than clinical practice data. However, we consider it plausible that the adoption of the 3 CDK4/6 inhibitors started more slowly than expected. Specifically, we observed a minor decline in the increasing use pattern from the moment that the EAP ceased and reimbursement was approved (August 2017). This decrease suggests that not all physicians responded directly to the changed reimbursement status of palbociclib. Raising awareness of this issue among treating physicians may further optimize the implementation of new medicines in the future.It is well known that the adoption of innovations in clinical practice takes time.47This study has some limitations. First, the number of patients who were eligible for the 3 medicines relied on decision analytic modeling, and the scenario analyses we performed showed that the results had substantial amount of uncertainty. Thus, the estimated number of eligible patients and adoption rates should be interpreted with caution. To fully understand access, detailed information is needed regarding patients with metastatic breast cancer, their treatments, and the decision-making process in daily practice. Second, we used aggregated claims data to evaluate the use of the CDK4/6 inhibitors, which means that we did not have data on patient characteristics or outcomes. As such, we could not assess whether patients who received the CDK/6 inhibitors immediately after approval and reimbursement differed from those who accessed the medicines later. Third, we studied the adoption of only 1 medicine class for the treatment of breast cancers in a single country. Future studies need to compare the adoption of medicines across different countries with different HTA policies. Fourth, the COVID-19 crisis was ongoing during the study period (2020-2021). Nevertheless, we did not expect a large association between the crisis and study results because treatment recommendations for patients with metastatic disease during the pandemic were similar to those from the years before.ERBB2-negative metastatic breast cancer and were adopted gradually over time in the Netherlands, a high-income country. Although many patients received the medicines during the period when reimbursement was pending, there appeared to be barriers to adoption other than reimbursement. To improve accessibility of innovative medicines, we call for better transparency of their availability during the different phases of the postapproval access pathway and for more in-depth cross-country studies on the adoption of medicines, which may further optimize the process.This cohort study found that the CDK4/6 inhibitors palbociclib, ribociclib, and abemaciclib were rapidly accessible to a group of patients with HR-positive and


Dunaliella salina at two distinct conformations. The conformers are also present in stacked PSII, exhibiting flexibility that may be relevant to the grana formation in chloroplasts of the green lineage. CP29, one of PSII associated light-harvesting antennae, plays a major role in distinguishing the two conformations of the supercomplex. We also show that the stacked PSII dimer, a form suggested to support the organisation of thylakoid membranes , can appear in many different orientations providing a flexible stacking mechanism for the arrangement of grana stacks in thylakoids. Our findings provide a structural basis for the heterogenous nature of the eukaryotic PSII on multiple levels.Photosystem II (PSII) generates an oxidant whose redox potential is high enough to enable water oxidation , a substrate so abundant that it assures a practically unlimited electron source for life on earth . Our knowledge on the mechanism of water photooxidation was greatly advanced by high-resolution structures of prokaryotic PSII . Here, we show high-resolution cryogenic electron microscopy (cryo-EM) structures of eukaryotic PSII from the green alga  In eukaryotes, the light reaction of oxygenic photosynthesis occurs in chloroplasts. Four protein complexes essential for the light reactions reside in an elaborate membrane system of flattened sacs called thylakoids . From thThylakoids form a physically continuous three-dimensional network, differentiated into two distinct physical domains: cylindrical stacked structures  and connecting single membrane regions (stroma lamellae). Photosystem I (PSI) is mainly located in the stroma lamellae, while PSII is found almost exclusively in the grana . Grana s2O oxidation and O2 release , quinones, carotenoids, and lipids which are coordinated by at least 20 protein subunits . In each release . The euk release . Two mon release  addition release .Although more than 3 billion years of evolution separate cyanobacteria, red algae, green algae and plants, and high-resolution PSII structures show that each PSII monomer along with its dimeric arrangement is highly conserved, especially in the membrane-bound regions of the PSII . StructuDunaliella salina. A high-resolution (2.43 Å) structure of PSII shows structural properties of the Dunaliella PSII supercomplex. The eukaryotic PSII appears to exist in two distinct core conformations that differ substantially in their inner dimer separation and the location of CP29, an important monomeric LHC. Structural analysis of stacked PSII dimers showed highly flexible interactions which can play a role in the dynamic organisation of chloroplast membranes. These findings introduce an additional, underlying, level of organisation, which can impact its excitation energy transfer (EET) properties and the overall organisation of the thylakoid membranes.In this work, PSII was isolated from the halotolerant green alga D. salina cells was applied on glow-discharged holey carbon grids that were vitrified for cryo-EM structural determination (see Methods). Initial classification of the dataset showed that approximately 20% of the particle population were in a stacked PSII configuration, containing two PSII dimers facing each other on their stromal side , and the high-specific activity of 816 μmol O2/(mg Chl * hr) measured for the preparation prior to vitrification suggest that both are highly active. The final reconstruction of the compact orientation refined to an overall resolution of 2.43 Å, the highest of any eukaryotic PSII structures  structures and maps suggests that the Chlamydomonas structure also contains these different conformers. This may explain the decreased local map resolution presented in the aforementioned subunits, compared to the rest of the cryo-EM map , or some side chains and ligands rearrangement associated with the movement of CP29. The structure and b-factor of CP29COMP and CP29STR are similar  showed that the first six principal components (PCs) explain more than 85% of the variance in the data and consist of continuous heterogeneity  – Ser84 appears to be phosphorylated and Cys218 seems to be sulfinylated (2.−) accumulation, which is subsequently converted by superoxide dismutase to hydrogen peroxide (H2O2) molecules (2O2. The map density around Cys218 suggests two cysteine oxidation events which result in the formation of sulfinic acid (RS-O2H).Several unique map densities were identified during model building, close to the OEC of both configurations a Naentified  binding entified . This ag of PSII . Two addinylated . Thus fainylated . Althouginylated . CP29 pholecules . CP47-Cy2. In addition to the two distinct conformations, large levels of continuous structural heterogeneity were discovered within each individual state. Multibody analysis  make EET between them inefficient. The closest protein contacts are found at the interface between core subunits from both PSII dimers and CP29, supported by peripheral interactions between LHCII trimer and CP26. Most of the PSII stromal surface is electronegative, and accordingly, most of the amino acids that seem to be involved in stacking interactions are either negatively charged or uncharged . Interacabilized . These istacking . Indeed,stacking . This hastacking . We suggstacking . Howeverstacking ; it is pDunaliella revealed an unexpected level of conformational flexibility in this highly conserved system. The two stable conformations appear to differ in their antennae connectivity and should be considered in PSII modelling attempts. Within each state, the large degree of structural heterogeneity also contributes to EET and may facilitate transitioning between the different states. In the stacked PSII dimer, we do not find any evidence for direct protein interaction connecting the two stacked systems, instead, long range electrostatic interaction between the core PSII subunits are flexible enough to allow for a wide range of motion, and their dependance on cation concentration provides a basis for light dependent regulation  cells were cultured in a 10 l BG11 medium, supplemented with 1.5 M NaCl, 6 μg/ml ferric ammonium citrate, and 50 mM NaHCO3 at pH 8 , 1 µM pepstatin, 60 µM bestatin, and 1 mM benzamidine  was added to a final concentration of 1%, and following stirring for 30 min at 4°C, the insoluble material was removed by centrifugation at 10,000 g for 5 min  under 560 µmol photons * m−2 * s−1 illumination  was applied on glow-discharged holey carbon grids (Cu Quantifoil R1.2/1.3) that were vitrified for cryo-EM structural determination using a Vitrobot FEI (3 s blot at 4°C and 100% humidity). The images were collected using a 300 kV FEI Titan Krios electron microscope, with a slit width of 20 eV on a GIF-Quantum energy filter, at the EMBL cryo facility, Heidelberg, Germany. A Gatan Quantum K3-Summit detector was used in counting mode at a magnification of 130,000 (yielding a pixel size of 0.64 Å), with a total dose of 51.81 e ÅtionCor2 . The contionCor2 . A totaltionCor2 . The pictionCor2 , for theCOMP) and stretched (C2S2STR). C2S2COMP was composed of 39,357 particles that undergone symmetry expansion, 3D homogeneous refinement, and multibody refinement  which gave the best overall fit to the map. All the reported resolutions were based on a gold-standard refinement, applying the 0.143 criterion on the Fourier shell correlation between the reconstructed half-maps. , showing both positions are occupied roughly by the same number of complexes. Focused classification was carried out on the upper dimer of the stacked PSII particles to determine if the compact and stretched conformations were also present in the stacked PSII arrangement. This analysis showed that the stacked PSII also contained a mixed population of the compact and stretched conformations. The compact set was composed of 9,567 particles, and these were pooled together and processed for 3D homogeneous refinement followed by multibody refinement  to give lf-maps. .PCA was performed using relion_flex_analyse as detailed in Focused maps obtained after multibody refinement were combined using the phenix_combined_focused_maps tool . CompletC. reinhardtii PSII model PDB 6KAC  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, and the evaluation has been overseen by a Reviewing Editor and Benoît Kornmann as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Helmut Kirchhoff (Reviewer #1).Thank you for submitting your article "Structure of Dunaliella Photosystem II reveals conformational flexibility of stacked and unstacked supercomplexes" 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 reviewers were enthusiastic about the quality of the data and its potential implications. Nevertheless, there were some concerns that need to be addressed, though these will probably not require additional experimentation.The authors should take extra care to explain what limits of their interpretations, and in particular be completely open about any possible artifacts introduced by the isolation and imaging in cryoEM. Keep in mind that many readers will have doubts about these kinds of artifacts, and it is better to both explain these potential issues, erring on the side of caution.Reviewer #1 (Recommendations for the authors):Chlamydomonas and higher plants, i.e. the distance between membranes in appressed grana regions. It would be very interesting to back-project the structure of the stacked PSII sandwich into a pair of stacked membranes found in native cells. The latter can be taken from the literature. Does the PSII sandwich predict the membrane-membrane separation for native membranes? That would add a very interesting aspect to this already compelling story.I have no concerns about the quality of the work, how the authors interpreted the data, and what they concluded about the structural organization of PSII with one exception. The authors, however, should consider that the two PSII structures and the 'stacked PSII' sandwich could be an artificial result of the isolation procedure. A critical discussion of this aspect would increase the credibility of the work. For example, there is quantitative information available on the stacking of thylakoid membranes in Reviewer #3 (Recommendations for the authors):Overall, this is a well-written manuscript with high-quality cryo-EM data processing, maps, and models. Below are several suggestions that I think would strengthen the overall arguments.1. Supplemental figure 1C should be expanded to show all classes from all rounds of 3D classification, and clearly delineate when symmetry was or was not applied at each individual step of classification/refinement.2. The ability to resolve water molecules is impressive, and the authors should capitalize on this opportunity to demonstrate the quality of their high-resolution reconstruction. The addition of a panel in Supplemental figure 3 clearly showing density for multiple waters would go a long way in demonstrating the map quality.3. Please ensure that the maps deposited to the EMDB are in an easily usable format for non-expert users of molecular visualization tools (ie: UCSF Chimera). In order to analyze the maps provided during the review I had to convert them out of float16 format in order to open them in Chimera, and at least for one of the maps I had to z-flip in order to invert the map handedness to match the atomic model. For people who are not intimately familiar with such processes, it would be very difficult to analyze the models/maps.4. Please consider depositing the individual maps for bodies from multibody refinement, in addition to the map of combined bodies. This allows future users to combine maps in alternate fashions if they choose.5. In Supplemental table 1, please consider providing a b-factor for water separate from ligands.6. Supplemental table 1, there seems to be an error in "Model composition". There are only ~50-100 protein residues listed. Reviewer #1 (Recommendations for the authors):I have no concerns about the quality of the work, how the authors interpreted the data, and what they concluded about the structural organization of PSII with one exception. The authors, however, should consider that the two PSII structures and the 'stacked PSII' sandwich could be an artificial result of the isolation procedure. A critical discussion of this aspect would increase the credibility of the work. For example, there is quantitative information available on the stacking of thylakoid membranes in Chlamydomonas and higher plants, i.e. the distance between membranes in appressed grana regions. It would be very interesting to back-project the structure of the stacked PSII sandwich into a pair of stacked membranes found in native cells. The latter can be taken from the literature. Does the PSII sandwich predict the membrane-membrane separation for native membranes? That would add a very interesting aspect to this already compelling story.Wietrzynski et al. as a reference. Although Wietrzynski et al. present evidence to the fact that stacked PSII dimers are rarely observed in vivo, this is one of the best examples of the in-vivo dimensions of the stacked PSII. Back projecting the stacked PSII into Chlamydomonas native cell tomograms shows an excellent alignment in all available features, including the stromal extrinsic subunits and the intermembrane space. This supports the similarity between the cryo-EM observed stacked PSII and an in-vivo stacked PSII configuration.We do not dispute that preparation conditions affect the formation of the stacked PSII dimer and this is noted in our manuscript, however, this does not imply that it is not biologically relevant. Several PSII stacked complexes were previously identified in vitrified thylakoid grana stacks using cryo-electron tomography. We selected a recent tomogram by Our suggestion is simply that stacked PSII dimers can play a role in supporting grana structure and that the large flexibility we observed in PSII orientations can lead to under-detection of these dimers in vivo.We added the definition for state 1 and state 10 in the legend of Figure 2.Reviewer #3 (Recommendations for the authors):Overall, this is a well-written manuscript with high-quality cryo-EM data processing, maps, and models. Below are several suggestions that I think would strengthen the overall arguments.1. Supplemental figure 1C should be expanded to show all classes from all rounds of 3D classification, and clearly delineate when symmetry was or was not applied at each individual step of classification/refinement.We have added these details to Figure 1—figure supplement 1 and they are now clearly indicated.2. The ability to resolve water molecules is impressive, and the authors should capitalize on this opportunity to demonstrate the quality of their high-resolution reconstruction. The addition of a panel in Supplemental figure 3 clearly showing density for multiple waters would go a long way in demonstrating the map quality.We have added panel d to Figure 1—figure supplement 3 to show all the water molecules coordinated by CP43 with their map densities.3. Please ensure that the maps deposited to the EMDB are in an easily usable format for non-expert users of molecular visualization tools (ie: UCSF Chimera). In order to analyze the maps provided during the review I had to convert them out of float16 format in order to open them in Chimera, and at least for one of the maps I had to z-flip in order to invert the map handedness to match the atomic model. For people who are not intimately familiar with such processes, it would be very difficult to analyze the models/maps.Conversion to float16 was done solely to reduce the file size in order to upload the maps. The maps deposited on the EMDB are at the correct orientation and in regular MRC format.4. Please consider depositing the individual maps for bodies from multibody refinement, in addition to the map of combined bodies. This allows future users to combine maps in alternate fashions if they choose.We prefer to deposit only a single map to avoid confusion although we acknowledge that this results in some data loss.5. In Supplemental table 1, please consider providing a b-factor for water separate from ligands.Done.6. Supplemental table 1, there seems to be an error in "Model composition". There are only ~50-100 protein residues listed.This entry mistakenly listed chains numbers, we corrected it to the show protein residues.