Mediator of Paramutation1 (Mop1), a gene encoding a putative RNA dependent RNA polymerase. Here we show that although RdDM is essential for the maintenance of DNA methylation of a silenced MuDR transposon in maize, a loss of that methylation does not result in a restoration of activity. Instead, heritable maintenance of silencing is maintained by histone modifications. At one terminal inverted repeat (TIR) of this element, heritable silencing is mediated via histone H3 lysine 9 dimethylation (H3K9me2), and histone H3 lysine 27 dimethylation (H3K27me2), even in the absence of DNA methylation. At the second TIR, heritable silencing is mediated by histone H3 lysine 27 trimethylation (H3K27me3), a mark normally associated with somatically inherited gene silencing. We find that a brief exposure of high temperature in a mop1 mutant rapidly reverses both of these modifications in conjunction with a loss of transcriptional silencing. These reversals are heritable, even in mop1 wild-type progeny in which methylation is restored at both TIRs. These observations suggest that DNA methylation is neither necessary to maintain silencing, nor is it sufficient to initiate silencing once has been reversed. However, given that heritable reactivation only occurs in a mop1 mutant background, these observations suggest that DNA methylation is required to buffer the effects of environmental stress on transposable elements.In large complex plant genomes, RNA-directed DNA methylation (RdDM) ensures that epigenetic silencing is maintained at the boundary between genes and flanking transposable elements. In maize, RdDM is dependent on  MuDR element, it is essential for preventing heat-induced, stable and heritable changes in both H3K9me2 and H3K27me3 at this element, and for concomitant changes in transcriptional activity. These finding suggest that RdDM acts to buffer the effects of heat on silenced transposable elements, and that a loss of DNA methylation under conditions of stress can have profound and long-lasting effects on epigenetic silencing in maize.Most plant genomes are mostly transposable elements (TEs), most of which are held in check by modifications of both DNA and histones. The bulk of silenced TEs are associated with methylated DNA and histone H3 lysine 9 dimethylation (H3K9me2). In contrast, epigenetically silenced genes are often associated with histone lysine 27 trimethylation (H3K27me3). Although stress can affect each of these modifications, plants are generally competent to rapidly reset them following that stress. Here we demonstrate that although DNA methylation is not required to maintain silencing of the  Transposable elements (TEs) are a ubiquitous feature of all genomes. They survive in large measure because they can out-replicate the rest of the genome . As a coIn plants, heritable epigenetic silencing of TEs is almost invariably associated with DNA methylation –12. The de novo methylation of newly synthesized DNA from previously methylated DNA sequences is thought to be transcription by RNA POLYMERASE IV (POLIV) of short transcripts from previously methylated templates We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.Best regards, OrtrunOrtrun Mittelsten ScheidAssociate EditorPLOS GeneticsWendy BickmoreSection Editor: EpigeneticsPLOS GeneticsAs you will see from the detailed comments of the reviewers, they agree that your data about the cooperation of epigenetic factors at a specific transposon under different genetic and environmental conditions revealed interesting and novel insight into the complexity of transposon silencing. The MuDR element is documented as an excellent and well-defined model. It is to be hoped that the findings will prove valid for other elements, but evidence for this and a broader analysis is certainly future work.However, the reviewers have suggestions and concern that require revising the current manuscript thoroughly. They request full data availability, state-of-the-art quantification of gene expression and bisulfite conversion control, and access to internal controls for some of the assays. They recommend providing more and better references to previous work on the maize RdDM mutants and the role of the polycomb complex beyond FLC. An important point raised repeatedly is the striking difference of heat stress activation between younger and older developmental stages, which should be explained or at least discussed. The different kinetics of transgenerational methylation adaptation could be strengthened as suggested by data for the intermediate generation.Text and Figures need several modifications, additions, and careful editing to ensure congruency and correctness.Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: The manuscript by Guo et al., “RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea mays” shows how MuDR transposon silencing is maintained by overlapping epigenetic mechanisms: DNA methylation, H3K9 and K27 dimethylation (me2) as well as H3K27me3. Several findings presented the manuscript are novel and significant for the field of epigenetics and transposon regulation:- A single transposable element (TE) displays different epigenetic regulation on its 5’ and 3’ ends (TIRA and TIRB respectively) through histone modifications, H3K9me2 and H3K27me2 on TIRA, and H3K27me3 on TIRB. Although those marks are well associated with transcriptional gene silencing, they have been shown to play different roles . Here, the authors illustrate a very clear example of all those marks cooperating to maintain a functional TE under control.- The authors also show how loss of RdDM and DNA methylation, present at both TIRs together with histone modifications, does not result in the immediate transcriptional reactivation and transposition of MuDR. However, in the absence of DNA methylation MuDR reactivates upon heat stress. Many studies in Arabidopsis have shown that loss of RdDM is largely inconsequential to plant development or genome instability caused by TE reactivation under laboratory standard growth conditions. Here, it is shown how DNA methylation is not only required for the transgenerational stability of the TE silencing, but to prevent the mobilization of MuDR under environmental stresses that might represent a more natural situation.The manuscript is well written, methods are well described, and the claims are properly contextualized in light of current literature. There are only minor corrections that would improve the quality of the manuscript that are listed below:- As stated in the data availability policy: "numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information". I couldn't find any spreadsheet with data relative to qPCR and phenotyping graphs while the authors claim that "all data ara fully available without restrictions". Authors should check with editors whether the raw data should be provided for those figures to be on the safe side.- The introduction does a very good job at explaining the reader the biological model and working system. However, I have the impression it fails to introduce the biological question that the authors want to address. This is found within the first three sentences of the results section (lines 202-204).- In lines 208-210, the authors state that “in control plants … all cytosines in TIRA were unmethylated … indicated that bisulfite conversion was efficient”. I disagree, as bisulfite conversion is performed independently for each DNA sample, the only way to address conversion efficiency is to investigate DNA methylation levels from sequences with known methylation (no DNA methylation) within the same DNA sample or spiked unmethylathed DNA before conversion (such as plasmid or lambda DNA). Authors might want to rephrase or eliminate the statement.- In several instances  expression of MuDR is investigated by RT-PCR. qRT-PCR might have been more appropriated as means to properly investigate subtle effects on MuDR expression.- Given that the authors have used internal controls for the ChIP-qPCR it would be advisable to add those in the supplementary data as means for the reader to see the intrinsic variability of their ChIP experiments.Reviewer #2: RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea mays.Guo et al. provides a highly detailed study of the epigenetic regulation related to the activity of a single Mu element in wild type and mop1 mutant maize plants. The work describes the relations of DNA methylation and histone modifications of the element's terminal inverted repeats to transcriptional silencing and reactivation by the Muk element and heat, respectively. The latter relations are determined further in the context of transgenerational inheritance.A strength of the work is the clearly defined genetic test system. By combining it with RT-PCR, clonal bisulfite sequencing, and ChIP analyses, the authors convincingly demonstrate that loss of DNA methylation in mop1 mutants does not prevent heritable silencing. Instead, the stable silencing in mop1 mutants is associated with the increase of different histone modifications at the two terminal inverted repeats of the Mu element, which mediate promotor function for the respective genes. For TIRB H3K27me3 is newly attributed to transposon silencing in maize.As the main result the authors demonstrate that in contrast to wild type, missing DNA methylation in mop1 mutants allows for reactivation of the TE by heat treatment, which is associated with a reduction of the respective repressive histone modifications. This result indicates DNA methylation in functioning as a buffer against the effects of heat stress.The reactivation was seen in leaves of young, 14 day-old, but not in leaves of older, 28 day-old, plants. Given the relatively short time interval between the sampling and the similarity of the tissues, this result is somehow unexpected. Do the older plants respond to the heat treatment as tested by hsp90 expression in the younger ones?In the analyses of transgenerational stability of the reactivated state for TIRA both MuDR/-; +/+ active and H5 reactivated samples show extreme, fully demethylated DNA. In contrast the heat stressed (H2 reactivated) samples are strongly methylated in all contexts as the silenced MuDR*/-;mop1/+ are. For TIRB similar results were obtained but only related to asymmetric DNA methylation. These results are discussed as an adaption of the methylation state to the activity state of the locus, only after multiple generations. To strengthen this point it would be required to analyze DNA methylation in the generations in between i.e. H3 and H4.Although this work is a mainly descriptive work on a specific example of a single transposon type exemplar, the indications of this work that DNA methylation can rather respond to than determine the activity of a locus and that the balance of DNA methylation and histone modifications is dependent on transcriptional activity, adds important information on the variety of mechanisms involved in transgenerational silencing of transposable elements in maize. Especially, it points to new unexplored mechanistic relations and independence respectively between DNA methylation and histone modifications in this context. The main statement that RdDM is responsible for the prevention of the transposon silencing reversal is solely built on the involvement of MOP1 in RdDM. The analysis of non-coding RNA could have contributed to a mechanistic understanding of the findings.Minor comments:How do the author explain the loss of methylation at the 5' end of the TIRB in MuDR*/-; mop1/+ silenced plants in the generation following Muk exposure?Line 35: dimethylation instead of demethylationThat Aat is a housekeeping gene that was used as a positive expression control should be mentioned already in the legend of figure 1.In figure 3A, right scheme L7 instead of L3 was collected according to the main text.Sentence in line 322 needs rewordingThat MuDR~ indicates a reactivated MuDR element should be mentioned already in the legend of figure 4.In figure 7 B sequence orientation of TIRB is reversed compared to figure 1.Line 810: Ear ears (doubling)In the legend of figure 6 description of C is missingReviewer #3: In this work, Guo et al. use the maize Mutator system for studying the epigenetic silencing mediated by the RdDM pathway. For doing this, they produced maize plant families segregating for a single silenced MuDR element and homozygous or heterozygous for mop1 . In brief, by analyzing MuDR expression, DNA methylation and specific histone modifications (H3K9 and H3K27di-tri methylation) at TIRs of the Mu elements, they observed that loss of methylation does not result in a restoration of that transposon activity; instead, histone modifications are responsible for heritable maintenance of silencing. In addition, in mop1 mutants a short exposure to high temperature rapidly reverses both transcriptional silencing and histone modifications in a heritable way. Based on these observations, they concluded that DNA methylation is not necessary to maintain silencing although, they suggest, it is required to buffer the effects ofthe environmental stress on transposable elements.In general, I find the manuscript not accurately and not clearly written; it contains many typing errors that complicate the reading. There are also some incongruences in the Figures and in particular, some explanations regarding the gels are incomplete.I also have other concerns on the manuscript content.When describing the mop1 mutant (lines 91-99) the authors use the definition “largely phenotypically normal”.This definition is quite ambiguous, especially because the mutant plants are used in heat stress experiments and developmental/genetic defects might indirectly interfere with stress response.Moreover, much research work has been done and published on maize RdDM pathway and other maize epiregulator mutants, which can be either cited or at least discussed here. They also could be tested for validating the results of this work in different epigenetic background. In particular, the relationship between H3K9 methylation and DNA methylation is not sufficiently described, although this is fundamental to discuss the results of the work presented in the manuscript.Similarly, I find that citing the peculiar example of AtFLC locus, which is regulated by vernalization and many other epigenetic marks at its chromatin, for describing the role of H3K27me3 modification in gene transcriptional regulation does not give an appropriate and exhaustive information on what is known on this modification on different genomic context.Regarding the results of the work, my principal and general concern is that the authors investigated the variation in methylation and a few histone modification patterns specifically and only at the MuDr sequences; this approach is quite limiting in studying the regulation of transposons expression by pathways that are known to have a genome-wide role. I think that “control sequences” must be used in the experiments to validate the results obtained for MuDr. Although many genotypes and progenies were characterized for transcriptional activation, DNA methylation and histone modifications, no information on the effect of the MuDr integration on the mopi1 genetic background or of the stress treatments at are genomic loci are reported in the manuscript.The observation that the combination of heat stress and mop1 mutant reactivated the transposon transcription is quite interesting and very similar to the cited example of Onsen in Arabidopsis. However, the authors observed that the reactivation of MuDR occurred only in young maize plant leaves, while older plants behave differently. No explanations or discussion on these findings were provided by the authors.After reading the results of this work, we can be convinced that a couple of histone modifications have a principal role in controlling MuDr activation than DNA methylation in maize; however, we miss any evidence and sustainable hypothesis on the epigenomic context and on mechanisms which mediate this activation.Minor points:Grammatical errors in lines:41, 47, 48, 76, 95, 101, 109, 137, 173, 188, 233, 264, 321, 380, 395, 397, 409, 411, 427, 510, 517In all the gel images please explain the lane and use a ladder. In general, the quality of the figures must be improved.Fig 3.A MuDR*/- Fig 4 is a repetition of Fig3Reviewer #4: Review of Guo et al., 2020 - RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea maysREVIEWER SUMMARY:In this manuscript, the authors investigate the relationship between the loss of CHH methylation due to mutation in Mop1 and transcriptional activation of the stably silenced Mutator DNA transposon, MuDR, in maize. They found that loss of mop1 function and CHH methylation at MuDR TIRs does not alleviate silencing of MuDR but results in an increase in H3K9me2 at the 5’ TIR (TIRA) and an increase in H3K27me3 at the 3’ TIR (TIRB) which suggests that these marks are required to maintain silencing. In addition to differences in histone modifications between TIRs, the mop1-dependent DNA methylation pattern differs between TIRs where TIRA loses methylation in all sequence contexts while TIRB only loses CHH methylation suggesting that MuDR is regulated by different epigenetic pathways despite high sequence similarity between TIRs. Stably silenced MuDR (MuDR*) plants homozygous for a mop1 loss of function mutation display reactivation of MuDR upon exposure to a 4-hour heat treatment at the 14-day seedling stage that is associated with reduction of H3K9me2 at TIRA and H3K27me3 at TIRB, with a concomitant increase in H3K4me3. This reactivation is constrained to a specific developmental window and dependent on loss of mop1 function because reactivation is not observed in mop1/+ heterozygotes, +/+ wildtype, or at the 28-day seedling stage in the mop1/- background. The authors show that the heat-dependent activation of MuDR is somatically heritable in the absence of the inducing signal by analyzing leaf 10 and tissue from the tassel which are derived from primordial cells generated well after the stress was applied. This reactivation is also inherited transgenerationally for at least 5 generations in the presence of functional mop1 (mop1/+) and MuDR TIRs maintain lower levels of H3K9me2 and H3K27me3 and high levels of H3K4me3. The authors show that methylation patterns progressively resemble stably active MuDR elements over 4 generations in the presence of functional Mop1, with TIRA progressively losing all methylation and TIRB gaining methylation in all sequence contexts.SIGNIFICANCE OF FINDINGS:These results are significant because they dissect a well-characterized epigenetically regulated locus to show that mop1  functions to buffer MuDR silencing from alterations in chromatin modifications (H3K9me2 and H3K27me3) during heat stress. Loss of DNA methylation has been shown to alleviate silencing at many different TEs but the authors clearly show here that MuDR relies on two epigenetic silencing pathways that may have different requirements for DNA methylation. Although this work is focused on a single locus, it is novel because it describes the relationship between loss of methylation and transgenerational inheritance of heat-stress altered chromatin states and transcriptional activation of MuDR. The fact that RdDM is required for preventing heat-induced transgenerational epigenetic changes is fascinating and will require further investigation to determine the generality of these results. This work is important because it increases our understanding of the role of RdDM and builds on previous work investigating stress-responsive transposon activation and the function of Mop1 in maintaining silencing during stress.OVERALL QUALITY AND PRESENTATION OF THE WORK:The research methods described are certainly of sufficient quality to draw the authors’ conclusions. However, the presentation of the work, especially the figures, require correction and appear to be incomplete (see reviewer comments). Although the rationale and approach are clear and justified, the manuscript should be carefully edited for sentence structure and brevity before publication.SPECIFIC COMMENTS:Abstract/Introduction The authors refer to Mop1 as Modifier of paramutation but the commonly used name is Mediator of paramutation.Figure 2 Lines under the genotypes indicating silenced versus active are not positioned correctly. Figure 4 Add to legend that red text is heat stressed and black text is control.Figure 6 Figure caption is not correct. No caption for 6D, 6C caption incorrect.Figure 8 Lines under genotypes indicating silences versus active are not positioned correctly.Figure S3 It is important to show HSP90 expression in tissue-matched non-stressed control plants.74-78 Pol IV and V are DNA-dependent RNA-polymerases, not DNA polymerases.Introduction/Discussion The authors need to include discussion of mop1/RdDM in stress response. Although mop1 does not dramatically alter transcription under normal conditions, mop1 has strong effects on ABA-responsive transcriptional programs  and loss of rmr6 (Pol IV) also shows defective response to drought . The authors should also discuss Mikula, Genetics, 1995 which showed that heat-stress alters epigenetic inheritance of R paramutation in maize.General Reviewer Comments It would be interesting to test the heat-dependent MuDR* reactivation in MuDR*/-;MuKiller/-;mop1/- plants. This would be in the presence of the inducing factor and could provide information about how heat and loss of RdDM affects the establishment of silencing. This is not critical to the relevance of this manuscript.386-390 The author states that the increase of methylation at TIRA in the active MuDR/-; Mop1 WT lines is associated with the decrease in H3K9me2 but Figure 7 shows that TIRA is unmethylated in active wildtype MuDR lines and is not consistent with the previous paragraph.Discussion The authors do not discuss the heritable increase in H3K4me3 and how this mark, and the active transcription of the genes associated with it, are relevant to maintaining that activation even in the presence of RdDM and differences in methylation patterns of TIRA and TIRB.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the No: As stated in the data availability policy: "numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information". I couldn't find any spreadsheet with data relative to qPCR and phenotyping graphs while the authors claim that "all data ara fully available without restrictions". Authors should check with editors whether the raw data should be provided for those figures to be on the safe side.Reviewer #1: Reviewer #2: YesReviewer #3: NoneReviewer #4: Yes**********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 , there is only one point that needs another revision: reviewer 2 found some discrepancy between the new data added and the interpretation in the text. We therefore ask you to modify the manuscript accordingly. In addition we ask that you:1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. Note: we cannot publish copyrighted images.2) Upload a Striking Image with a corresponding caption to accompany your manuscript if one is available (either a new image or an existing one from within your manuscript). If this image is judged to be suitable, it may be featured on our website. Images should ideally be high resolution, eye-catching, single panel square images. For examples, please browse our plosgenetics@plos.org.We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to Submission Checklist.If present, accompanying reviewer attachments should be included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.While revising your submission, please upload your figure files to the data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.Please be aware that our 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 Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.PLOS has incorporated To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.[LINK]Please let us know if you have any questions while making these revisions.Yours sincerely,Ortrun Mittelsten ScheidAssociate EditorPLOS GeneticsWendy BickmoreSection Editor: EpigeneticsPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: The authors have answered to all my comments and I'm satisfied with the revised version of the manuscript. Therefore, I recommend it for publication.Reviewer #2: Guo et al. provides a detailed study of the epigenetic regulation related to the activity of a single Mu element in wild type and mop1 mutant maize plants. The indications of this work that DNA methylation can rather respond to than determine the activity of a locus and that the balance of DNA methylation and histone modifications is dependent on transcriptional activity, adds important information on the variety of mechanisms involved in transgenerational silencing of transposable elements in maize. Especially, it points to new unexplored mechanistic relations and independence respectively between DNA methylation and histone modifications in this context.In the revised version my comments are adequately addressed, except the concerns raised in the context of the transgenerational adaption of the methylation states to the activity states after stress induced reactivation:In the analyses of transgenerational stability of the reactivated state for TIRA both MuDR/-; +/+ active and H5 reactivated samples show extreme, fully demethylated DNA. In contrast the heat stressed (H2 reactivated) samples are strongly methylated in all contexts as the silenced MuDR*/-;mop1/+ are. For TIRB similar results were obtained but only related to asymmetric DNA methylation. These results are discussed as an adaption of the methylation state to the activity state of the locus, only after multiple generations. To strengthen this point it would be required to analyze DNA methylation in the generations in between i.e. H3 and H4.The authors now provide additional data for TIRA and TIRB on H4, which show the same full demethylation as in H5. The text is not changed accordingly (Line 377) to mention the analysis of the H4 generation neither the conclusion that multiple rounds of meiosis are likely required to adapt the methylation profile to the active state. Clearly this conclusion is weakened by the new results, but can be resolved only by analysis of H3 and the heat-stressed, reactivated H1, which the authors do not provide. The new results however indicate that a gradual loss of methylation in adaption to the activity state of the elements over multiple generations is less likely and it is questionable whether meiosis is required or related to the phenomenon at all. Alternatively, the adaption of the methylation state might be independent of meiosis and started already in H1. Indeed, although not quantitatively analyzed, the methylation profiles of reactivated H2 show demethylation compared to the silenced H1 generation. The new results should be described in the text and the interpretations adjusted.Examples of remaining grammatical or typing errors:Line 386 "latter" instead of "later"Line 332 "could" is doubledLine 485 "in maize" instead of "is maize"Reviewer #3: The authors have taken into consideration all concerns raised by my first report at theoretical level. I think that both introduction and discussion were greatly improved.I understand that the study of the their system/model in a different genomic background is not that simple as well as it is not the validation of the results for MuDr at histone modification level. However, I am convinced that these validations are essential to confirm their observations.Reviewer #4: All the major issues are addressed, minor issues are listed belowLine numbers Comment47 Change out-replicated to out-replicate145-147 Sentence is confusing, please clarify.543 Change understanding to understand566 Remove “for” or “in”595 Spelling error for collected**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: NoneReviewer #3: YesReviewer #4: Yes**********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 , and click on the Fetch/Validate link next to the ORCID field.  This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.In the meantime, please log into Editorial Manager at If you have a press-related query, or would like to know about making your underlying data available , please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!Yours sincerely,Ortrun Mittelsten ScheidAssociate EditorPLOS GeneticsWendy BickmoreSection Editor: EpigeneticsPLOS Geneticswww.plosgenetics.orgTwitter: @PLOSGenetics----------------------------------------------------Comments from the reviewers (if applicable):----------------------------------------------------Data DepositionDryad Digital Repository. As you may recall, we ask all authors to agree to make data available; this is one way to achieve that. A full list of recommended repositories can be found on our website.If you have submitted a Research Article or Front Matter that has associated data that are not suitable for deposition in a subject-specific public repository (such as GenBank or ArrayExpress), one way to make that data available is to deposit it in the The following link will take you to the Dryad record for your article, so you won't have to re‐enter its bibliographic information, and can upload your files directly: http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-20-01828R2http://www.datadryad.org/depositing. If you experience any difficulties in submitting your data, please contact help@datadryad.org for support.More information about depositing data in Dryad is available at data availability policy requires that all numerical data underlying display items are included with the submission, and you will need to provide this before we can formally accept your manuscript, if not already present.Additionally, please be aware that our ----------------------------------------------------Press Queriesplosgenetics@plos.org.If you or your institution will be preparing press materials for this manuscript, or if you need to know your paper's publication date for media purposes, please inform the journal staff as soon as possible so that your submission can be scheduled accordingly. Your manuscript will remain under a strict press embargo until the publication date and time. This means an early version of your manuscript will not be published ahead of your final version. PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via  10 Jun 2021PGENETICS-D-20-01828R2 RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea mays. Dear Dr Lisch, We are pleased to inform you that your manuscript entitled "RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea mays." has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.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 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. Soon after your final files are uploaded, unless you have opted out or your manuscript is a front-matter piece, the early version of your manuscript 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 PLOS Genetics and open-access publishing. We are looking forward to publishing your work! With kind regards,Zsofi ZomborPLOS GeneticsOn behalf of:The PLOS Genetics TeamCarlyle House, Carlyle Road, Cambridge CB4 3DN | United Kingdomplosgenetics@plos.org | +44 (0) 1223-442823plosgenetics.org | Twitter: @PLOSGenetics


Interestingly, viral RNA was detected throughout various extrapulmonary tissues and organs without visible tissue damage. The dissemination of SARS-CoV-2-RNA throughout the body supports the hypothesis that there is a maladaptive host response with viremia and multiorgan dysfunction.Clinical observations indicate that COVID-19 is a systemic disease. An investigation of the viral distribution within the human body and its correlation with tissue damage can aid in understanding the pathophysiology of SARS-CoV-2 infection. We present a detailed mapping of the viral RNA in 61 tissues and organs of 11 deceased patients with COVID-19. The autopsies were performed within the early postmortem interval  to minimize the bias due to viral RNA and tissue degradation. Very high viral loads (>10 Since the discovery of the new coronavirus that causes COVID-19, scientists have been scrambling to understand the different features of the virus. While a lot more is now known about SARS-CoV-2, several key questions have proved more difficult to answer. For example, it remained unclear where the virus travels to in the body and causes the most harm.To help answer this question, Deinhardt-Emmer, Wittschieber et al. performed postmortem examinations on 11 patients who had recently died of COVID-19. After sampling 61 different organs and tissues from each patient, several tests were used to detect traces of SARS-CoV-2. The experiments showed that the largest pool of SARS-CoV-2 was present in the lungs, where it had caused severe damage to the alveolae, the delicate air sacs at the end of the lungs’ main air tubes.Small amounts of the virus were also detected in other organs and tissues, but no severe tissue damage was seen. In addition, Deinhardt-Emmer, Wittschieber et al. found that each patient had increased levels of some of the proteins involved in inflammation and blood clotting circulating their bloodstream. This suggests that the inflammation caused by SARS-CoV-2 leads to an excessive immune reaction throughout the entire body.This research provides important new insights into which areas of the body are most impacted by SARS-CoV-2. These findings may help to design more effective drug treatments that target the places SARS-CoV-2 is most likely to accumulate and help patients fight off the infection at these regions. Betacoronavirus called SARS-CoV-2 were first described in the city of Wuhan in China  and transmembrane protease serine 2 (TMPRSS2), two membrane-bound proteins, have been shown to be crucial for the entry of the virus into cells . ACE2 isIn the present study, we (1) included full autopsies, (2) performed autopsies in the early postmortem interval , (3) dissected organs and tissues without prior fixation in formalin, (4) measured SARS-CoV-2 RNA in a high number of samples, (5) correlated the viral load with tissue damage using comprehensive histopathological investigations, (6) visualized virus particles in pulmonal tissue samples by means of transmission electron microscopy (TEM), and (7) determined the postmortem serum levels of inflammatory cytokines and prothrombotic factors. Sampling was performed in the very early postmortem interval to provide reliable viral RNA (vRNA) measurements and enabled us to obtain blood serum and well-preserved tissue samples for ultrastructural analysis.The investigation of COVID-19 patients included a full characterization of the clinical characteristics and parameters . In detaOur results show that patients 1–10 died of COVID-19, whereas patient 11 suffered from a metastasized squamous cell carcinoma of the cervix and died of an ileus following peritoneal carcinosis  viral loads in at least two samples obtained from the lymphatic system. Lymphatic structures with topological relationships to the respiratory tract were always positive for vRNA.Of the patients subjected to intensive care treatment (patients 1–7), patients 1, 3, 4, and 5 exhibited moderate to very low viral loads in the cardiac samples  PatientsViral RNA was detected in the blood only for patients 3–5. Patients 1, 2, 6, and 7, who were treated with lopinavir/ritonavir, were negative for vRNA in blood. Viral RNA was present in the bone marrow of all three patients who tested positive for vRNA in blood and was also found in an additional three patients who tested negative for vRNA in blood.Patients 3–5 had vRNA in variable amounts throughout the small and large intestine. Patients 6 and 7 tested negative for all 12 gastrointestinal samples. Of note, in patient 9, almost all the gastrointestinal samples exhibited moderate to very high viral loads.Viral RNA could also be detected in endocrine organs, in the urinary tract and in the reproductive organs. Only patients 3–5 tested positive for vRNA in the central nervous system. Skin , subcutaneous tissue  and skeletal muscle (psoas major) tested negative in all patients.Due to the very early postmortem interval in which the autopsies were performed, we were able to verify the vRNA findings via TEM of a lung sample from patient 3 by detecting intact SARS-CoV-2 viral particles within lung fibrocytes .To analyze the proinflammatory responses of the 11 patients, we measured interleukin (IL)−6  and IL-8Since abnormalities in the coagulation system were described in COVID-19 patients , we analOur results showed significantly higher serum levels of tissue plasminogen activator (tPa)  in COVIDThe level of a biomarker with potential for cardiovascular risk stratification, sCD40L , was sigMacroscopic signs of severe and extensive lung damage were found in all patients. In patients 1, 3–5, 9, and 10, the lung tissue was full of blood (hyperemia) and fluid (edema) and showed weakened consistency . In contSeveral features of coagulopathies were found, including infarction of the lung  and the Microscopically, the lung tissues from patients 1, 3–5, 9, and 10 revealed changes consistent with the early (exudative) phase of diffuse alveolar damage (DAD). The consistent acute changes included severe intraalveolar and interstitial hemorrhages , architeWhile the upper lobes of the lung of patient 2 showed only moderate emphysema , the hemA common histological feature in all patients was the loss of the follicular architecture of the lymph nodes due to architectural changes . In the In clinical practice, many critically ill COVID-19 patients show multiple organ involvement in addition to lung failure, in particular vascular dysfunction, including thrombosis and/or impaired microcirculation . A dysreSome aspects of the viral pathogenesis and the toxicity of the novel virus SARS-CoV-2 are known based on previous studies of SARS-CoV . The vir4to 9 x 109 copies/ml, while the highest values in our study were ~107 vRNA copies/ml. From Quite a few morphological studies have been published so far. Some are single case reports based on necropsies of the lung, liver and heart , partialWe detected the highest viral loads and the most severe tissue damage in the lungs. The lung samples of all patients showed large cells, sometimes multinucleated giant cells, that were similar to the giant cells described in cases of respiratory syncytial virus (RSV) infection. The preliminary immunostaining pattern of the enlarged cells indicated that they represented affected pneumocytes. Squamous (metaplastic) large cells and clusters of giant cells have been reported by most morphological studies, with one exception . The remAll patients who died due to COVID-19 (patients 1–10) had viral RNA in at least some samples of the lymphatic tissue. Lymphatic tissue with a topological relationship with the respiratory tract  was more likely to be positive overall than lymphatic tissue without such a topological relationship . One remarkable finding in the lymph node samples of all patients was the loss of the follicular structure . AtrophyViral loads in the cardiac tissue were moderate to very low and systematically  detected in the patients with early lung damage, while patients with later lung damage displayed high viral loads only sporadically or not at all. The cardiac histology of the left ventricle, anterior wall, and basal portion in patient one with a moderate viral load is presented in The viral loads in the samples from the vascular tissue (aorta and pulmonary artery) followed a similar distribution pattern depending on the stage of lung damage but were higher compared to those in the cardiac tissue. The unremarkable histology of the thoracic aorta of patient 5 with a high viral load is presented in Interestingly, the presence of detectable vRNA in the gastrointestinal tract was variable. The very high viral loads in patient nine throughout the upper gastrointestinal tract as well as in the small and large bowels were noticeable. The histology of the corresponding tissue samples was unremarkable. According to the clinical documentation, patient 9 did not exhibit any gastrointestinal symptoms.Viral RNA could also be detected in low to very high amounts in the samples from the endocrine organs, urinary tract, nervous system, and reproductive system. Interestingly, the samples of patients 1, 2, 6, and 7, who were treated with lopinavir/ritonavir, tested negative. Our results support the findings of The same distribution pattern among the patients was observed regarding viral RNA in blood. Apart from patients 8–11, who were not subjected to intensive care (patients 8–10) or did not die of COVID-19 (patient 11), it is noticeable that among the patients who received intensive care prior to death (patients 1–7), only patients 3, 4, and 5 tested positive for vRNA in blood, while patients 1, 2, 6, and 7 tested negative. The latter patients were treated with lopinavir/ritonavir, so the effect of antiviral medication on preventing viremia may be indicated. In our study, 4 of 11 patients were treated with an antiviral medication. None of these four patients showed the presence of vRNA in the blood, but one patient showed the presence of vRNA in the bone marrow (patient 1). The application of antiviral drugs is currently being investigated in many studies. However, it has not yet been shown that a significant effect can be achieved by their application . HoweverThe patients with vRNA in the blood also showed vRNA in the bone marrow. Patients 1, 8, and 9 were negative for vRNA in the blood but positive in the bone marrow. Patient 9 showed the highest viral loads by far in the bone marrow. The histology of the bone marrow, apart from hypercellularity, a left shift and an increased number of megakaryocytes, showed a significant amount of hemophagocytosis . HemophaA positive test result based on qRT-PCR can be determined with the Ct-value. Due to the further spread of the pandemic, clinicians have discussed the release of this value as part of the test result, as the viral load has been identified as an important prognostic indicator . The autHowever, the College of American Pathologists reminds us to be cautious in interpreting the results . As a muAnother important reason for the detection of Ct-values is the statement about infectiousness. Jaafar et al. showed that the culture of the virus is successful up to a Ct-value of 25 (70%). At a Ct-value of 30, this value drops to 20%, and values above 35 as associated with only a low likelihood (3%) of the possibility of culture . These vTo further elucidate the immunological host response, we measured IL-6 and IL-8 in postmortem serum samples. The serum levels of interleukin six were significantly elevated in all patients, including patient 11, who died of multiple organ failure following ileus. The serum levels of IL-8 were also significantly elevated. In accordance with other autopsy studies , we obseIn summary, we presented an autopsy series of 11 patients with COVID-19. The autopsies were performed in the early postmortem interval to avoid bias due to the degradation of vRNA, virus particles, and tissue structures. SARS-CoV-2 RNA could be detected in very high to high amounts in the lungs and in very high to very low amounts in the lymphatic tissue. TEM visualized SARS-CoV-2 particles in the lung tissue. Viral loads and histological tissue damage were strongly correlated in the lungs even at the organ level (patient 2). Histological structure changes were also present in the lymph nodes (atrophy and loss of follicles). High viral loads were detected in many other tissue samples from different extrapulmonary organs and tissues without evident tissue damage based on light microscopy.The study was approved by the local ethical board (registration no.: 2020–1773). Complete autopsies  of 11 patients with SARS-CoV-2 infection  and a clinical diagnosis of COVID-19 were included in this study. As soon as possible after death, the closest relatives were contacted, who gave their informed consent. The autopsies in this study were performed 1.5–15 hr (mean 5.6 hr) postmortem, and the organs were dissected directly without prior fixation. The same team including two experienced forensic pathologists conducted all autopsies. The lungs, with expected high viral loads, were removed first and dissected last to avoid the transfer of vRNA to other organs/tissues. At each autopsy, a total of up to 61 native and nonfixed samples  were collected after rinsing them in clean tap water. Tissue samples were transferred for virological processing immediately after autopsy; blood samples were centrifuged to obtain serum. Samples from the same anatomical locations were fixed in 5% buffered formalin solution for comparative histopathological analysis, and selected samples were placed in a special fixative (see below) for TEM. Since the correct electron microscopic identification of virus particles in tissue obtained by autopsy can be challenging , all posAll tissues were homogenized in RPMI medium by using the FastPrep-24 5G Instrument , and disposable homogenizer beads  were used to avoid contamination. We placed 200 mg of each tissue/organ in 1000 µl RPMI-1640 . After a centrifugation step , the supernatants were collected for the determination of the viral load. RNA extraction was performed by using the QIAcube RNeasy Viral Mini Kit  according to the manufacturer's guide. qRT-PCR was performed using RIDAgene  with the Rotor-Gene Q  to detect the E-gene of SARS-CoV-2 by determining the cycle threshold (Ct) value. The RNA standard curve, prepared by amplification of the positive control with the RIDAgene  kit, was applied for quantification. SARS-CoV-2 RNA is represented as the decadic logarithm of the number of copies/ml. The following scale was applied: very high (>104 copies/ml), high (103–104 copies/ml), moderate (102–103 copies/ml), low (101–102 copies/ml), and below the detection limit (bdl).For the measurement of proinflammatory cytokines and coagulation parameters, a Legendplex Human Thrombosis Panel (13-plex)  was used. Twenty-five microlitres of each serum sample was transferred in duplicate into a 96-well filter plate, and the Legendplex panel was performed by following the manufacturer’s instructions. The samples were measured on the same day on a flow cytometer , and the protein amount was calculated by comparison to a standard curve. Serum samples from five healthy volunteers without any signs of infection were age-correlated and analysed as a control.After fixation for at least 24 hr in 10% neutral buffered formalin, the tissue samples were dehydrated in a graded series of ethanol and xylene, mounted in paraffin and cut into 3-μm-thick sections. In addition to hematoxylin and eosin (HE), Elastica-van-Gieson (EvG), Berlin Blue (Fe), periodic acid Schiff stain (PAS), Alcian Blue-periodic acid Schiff stain (abPAS), Giemsa, Gomori Trichrome, and Kongo Red stain were used by following routine protocols. For immunohistochemistry, the following antibodies were used: AE1/3 (Dako/IR053), TTF-1 (Dako/IR056), CK7 (Dako/IR619), CK5/6 (Dako/IR780), p40 (Zytomed/MSK097), Ki67 (Dako/IR626), CD68 (Dako/M0876), CD61 (Dako/M0753), CD31 (Dako/IR610), CD34 (Dako/IR623), ASMA (Dako/IR611), CD3 (Dako/IR503), CD20 (Dako/IR604), MUM1 (Dako/IR644), collagen IV (Dako/M0785), and tenascin (Chemicon/MAB19101). All immunostaining were performed with the Dako Omnis immunostainer (Agilent) by following routine procedures. The sections were examined microscopically , and representative photographs were obtained , Carl Zeiss Microscopy GmbH).During each autopsy, several small pieces of lung tissue (2 mm cubes) were immediately fixed with freshly prepared modified Karnovsky fixative  for 24 hr at room temperature. After washing three times for 15 min each with 0.1 M sodium cacodylate buffer (pH 7.4), the tissue was further cut into 1 mm cubes and postfixed with 2% w/v osmium tetroxide for 1 hr at room temperature. During the subsequent dehydration in an ascending ethanol series, poststaining with 1% w/v uranyl acetate was performed. Afterwards, the samples were embedded in epoxy resin  and sectioned using a Leica Ultracut S . Based on the examination of semi-thin sections, regions of interest of approximately 500 µm x 500 µm in size were selected and trimmed. Finally, the ultrathin sections were mounted on filmed Cu grids, post-stained with lead citrate, and studied in a transmission electron microscope  at 80 kV and 3000–50,000x magnification. For image recording, a 2K slow scan CCD camera  was used. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This study of 11 COVID-19 full autopsy cases, describes histopathology in relation to TEM detection of intact virus particles in lung, PCR virus RNA detection in other organs, and cytokines and coagulation markers in serum. The study represents a thorough body of work that may enrich our insight into COVID-19 pathogenesis. The main message is that COVID-19 is a systemic disease as determined by presence of virus RNA, yet not related to tissue damage outside the lungs. This adds to the field in its extensiveness of testing within each patient. A side message is that COVID-19 in selected cases may show a loss of follicular structure.Decision letter after peer review:eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Katrien Grunberg (Reviewer #2).Thank you for submitting your article "Early postmortem mapping of SARS-CoV-2 RNA in patients with COVID-19 and correlation to tissue damage" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has summarized the essential points to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:This study of 11 COVID-19 full autopsy cases, describes histopathology in relation to TEM detection of intact virus particles in lung, PCR virus RNA detection in other organs, and cytokines and coagulation markers in serum. The sample size is small but relevant and sufficient for a descriptive study. The study represents a thorough body of work that may enrich our insight into COVID-19 pathogenesis. The main message is that COVID-19 is a systemic disease as determined by presence of virus RNA, yet not related to tissue damage outside the lungs. This adds to the field in its extensiveness of testing within each patient. A side message is that COVID-19 in selected cases may show a loss of follicular structure.Revisions:1) All the data reported in the first paragraph of the Introduction must be updated.2) Please remove "extraordinary" from the phrase "measured SARS-CoV-2 RNA in an extraordinary high number of samples".3) Given the statements made by the authors "Contrary to recommendations of the pathological professional societies not to perform autopsies earlier than 24 hours post-mortem and to store the organs in formalin before dissecting them, the autopsies in this study were performed 1.5 – 15 h (mean 5.6 hours) post-mortem and the organs were dissected directly without prior fixation. The same voluntary team including two experienced forensic pathologists conducted all autopsies considering all necessary precautions and using the comprehensive personal protective equipment as recommended by professional societies"; it is necessary to mention the numerous national and European sources that dictate these precautionary rules.4) The stains EvG, Fe, PAS, abPAS, must be preceded by the whole word  before the acronym.5) "Macroscopic signs of severe and extensive lung damage were found in all patients. In patients 1, 3-5, 9 and 10, the lungs with prominent hyperemia and edema displayed a fragility of the tissue". What does it mean?6) I do not think it is appropriate to keep these descriptive self-satisfaction phrases in the text as the number of cases is still small. "The sample size of our case series can be compared to the larger autopsy case series from literature".7) "multifocal acute injury of cardiomyocytes which is frequently observed in critically ill patients under catecholamine therapy". The authors must specify the type of cardiac damage by representing whether it is a coagulation myocytolysis or other.8) Discussion paragraphs thirteen and fourteen can be easily removed.9) Figure 4 is very trivial.10) Figure 7C-D I do not understand the usefulness of showing a normal myocardium in c and I do not see any alteration of the myocardium in d.11) The references must also be expanded for the autopsy case reports that have been published and that are not mentioned in this paper.The main message is that COVID-19 is a systemic disease as demonstrated by presence of virus in organs outside the lungs. The description of tissue damage is mostly about lung. Apparently, a correlation viral load – tissue damage could not be established outside lung, while some lesions where present in other organs. The claim that these data allows the study of a correlation between viral load and tissue damage is not fully justified. A few limitations:12) Quantification of viral load insufficiently explained. Materials and methods/Sars-cov2 detection quantification expressed as copies/ml: how did the investigators standardize for amount of tissue in medium13) No TEM in other organs beside lungs. PCR detects all RNA, not necessarily intact viral particles, so unclear if this is just degraded virus, being transported from lung though the body.14) The digestive tract and pancreas not exactly great for post mortem studies, even when the interval between exitus and autopsy is short. Subtle changes may be missed.15) It is unclear from what area of organs exactly and whether lesional areas were specifically sampled or not. For example, was kidney cortex was properly sampled? So insight in viral tropism and where damage is to be expected is limited.16) Role of antiviral drugs  is not discussed.None of these limitations is discussed. Both parts of the conclusion that COVID-19 is 1 a systemic disease and 2 that there is no correlation with tissue damage outside the lung are in my view overstretched.17) The authors present no (plan to study) data on interconnectedness of inflammation, thrombosis and infection/viral load in their Materials and methods. Correlation of IL-6, coagulation markers, presence of virus is unclear from results and figures. E.g. hemophagocytosis in bone marrow observed in one case and is discussed as possibly related to MAS and cytokine storm. Yet, no mention of IL-6. Hence, the presentations of any correlation comes across as a haphazard fishing expedition.18) The conclusion ("Virus may be able to infect different cell types in different organs and tissues but may not be able to replicate in non-respiratory organs/tissues as efficiently as in the respiratory ones") is not based on the findings themselves hence not a conclusion but it may be discussed as a speculative explanation of the findings. Revisions:1) All the data reported in the first paragraph of the Introduction must be updated.Modification has been made. The data in the first paragraph have been updated.2) Please remove "extraordinary" from the phrase "measured SARS-CoV-2 RNA in an extraordinary high number of samples".Modification has been made. “Extraordinary” was removed from the sentence.3) Given the statements made by the authors "Contrary to recommendations of the pathological professional societies not to perform autopsies earlier than 24 hours post-mortem and to store the organs in formalin before dissecting them, the autopsies in this study were performed 1.5 – 15 h (mean 5.6 hours) post-mortem and the organs were dissected directly without prior fixation. The same voluntary team including two experienced forensic pathologists conducted all autopsies considering all necessary precautions and using the comprehensive personal protective equipment as recommended by professional societies"; it is necessary to mention the numerous national and European sources that dictate these precautionary rules.We want to thank the reviewer for this suggestion.The recommendation in question is a standard operation procedure (SOP) distributed among the members of the professional societies, which has not been published. Unfortunately, it can thus not be cited properly. The reference to the recommendations of the professional societies was removed from the two sentences.4) The stains EvG, Fe, PAS, abPAS, must be preceded by the whole word  before the acronym.We would like to apologize for the inaccurate description and have made all necessary additions. The full term was added to the acronyms of the stains.5) "Macroscopic signs of severe and extensive lung damage were found in all patients. In patients 1, 3-5, 9 and 10, the lungs with prominent hyperemia and edema displayed a fragility of the tissue". What does it mean?We want to thank the reviewer for the comment.The sentence was intended to express, that the lung tissue – alveoli as well as interstitium – was filled with blood (hyperemia) and fluid (edema) and was at the same time fragile or brittle to pressure. We rewrote the sentence hoping, that the meaning became somewhat clearer.6) I do not think it is appropriate to keep these descriptive self-satisfaction phrases in the text as the number of cases is still small. "The sample size of our case series can be compared to the larger autopsy case series from literature".We want to apologize for the phrases in the text and thank the reviewer for bringing this topic to our attention. We rewrote the sentence focusing on the limitation due to the small numbers. We have now deleted the sentence and added:“The small number of patients is a limitation of the study. However, we have analyzed a large number of organs and tissues and the present study focused on keeping the post-mortem interval as small as possible to avoid bias due to degradation of SARS-CoV-2 virus particles, SARS-CoV-2 RNA and tissue ultrastructure.”7) "multifocal acute injury of cardiomyocytes which is frequently observed in critically ill patients under catecholamine therapy". The authors must specify the type of cardiac damage by representing whether it is a coagulation myocytolysis or other.We want to apologize for the inaccurate description. We specified the type of cardiac damage in the text (contraction band necrosis).8) Discussion paragraphs thirteen and fourteen can be easily removed.Modification has been made; we omitted the last summarizing paragraph as suggested.9) Figure 4 is very trivial.We understand the concerns of the reviewers, but with our article we also want to address scientists who are not pathologists and for them the presentation of a lung damaged by SARS-CoV-2 is very important and impressive. Figure 4 demonstrates macromorphological findings in unfixed organs, which is a complement to the histopathology. We hope that the reviewer agrees with this.10) Figure 7C-D I do not understand the usefulness of showing a normal myocardium in c and I do not see any alteration of the myocardium in d.Figure 7D represents a higher magnification of Figure 7C. The patient showed high viral loads in the cardiac samples. The figures emphasize our main result of normal micromorphology or unspecific findings despite high viral loads.11) The references must also be expanded for the autopsy case reports that have been published and that are not mentioned in this paper.We have conducted a recent pubmed research using "autopsy and COVID-19". The most important papers have been included in the discussion and quoted accordingly. For this, we have added four case reports and six autopsy studies.The main message is that COVID-19 is a systemic disease as demonstrated by presence of virus in organs outside the lungs. The description of tissue damage is mostly about lung. Apparently, a correlation viral load – tissue damage could not be established outside lung, while some lesions where present in other organs. The claim that these data allows the study of a correlation between viral load and tissue damage is not fully justified. A few limitations:12) Quantification of viral load insufficiently explained. Materials and methods/Sars-cov2 detection quantification expressed as copies/ml: how did the investigators standardize for amount of tissue in medium”We want to thank the reviewer for the comment and want to apologize for the insufficient description. We have added subsection “SARS-CoV-2 RNA detection13) No TEM in other organs beside lungs. PCR detects all RNA, not necessarily intact viral particles, so unclear if this is just degraded virus, being transported from lung though the body.We want to thank the reviewer for the comments. We added a detailed discussion of this issue.“TEM investigations of the lung samples revealed the presence of morphologically intact virus particles in the tissue which corresponds to the vRNA mapping, which shows the highest viral loads in the lungs. The morphologically intact virus particles were localized in lung fibrocytes.49 viral inclusion bodies were detectable using TEM. Whereas in lung tissues the morphology of viral particles was clearly given , in other tissues like liver, heart, and intestine viral inclusions were not visible with TEM. The loss of structural hallmarks could be due to the post-mortem cell and tissue turnover with reduced integrity of virus particles for TEM-related preparations. It has to be stressed that the molecular detection of the virus depends not on the morphological intact virus particle.”In agreement with data published by Varga et al. 14) The digestive tract and pancreas not exactly great for post mortem studies, even when the interval between exitus and autopsy is short. Subtle changes may be missed.Both patients showed high viral loads in the gut and pancreas. The figures emphasize our main result of normal micromorphology or unspecific findings despite high viral loads and demonstrate the very well preserved postmortem histomorphology due to the early postmortem sampling.15) It is unclear from what area of organs exactly and whether lesional areas were specifically sampled or not. For example, was kidney cortex was properly sampled? So insight in viral tropism and where damage is to be expected is limited.The samples were taken following routine protocols. In case of the kidney cortex and medulla were sampled. The institute of forensic medicine and the section of surgical pathology are accredited and follow process-oriented quality management.16) Role of antiviral drugs  is not discussed.None of these limitations is discussed. Both parts of the conclusion that COVID-19 is 1 a systemic disease and 2 that there is no correlation with tissue damage outside the lung are in my view overstretched.Thank you very much for the comments of the reviewers. We have added the following essential points in the discussion:“In our study, 4 of 11 patients were treated with a virustatic medication. None of these four patients showed vRNA in the blood, but one patient showed vRNA in the bone marrow (patient 1). The application of virustatic drugs is currently being investigated in many studies. However, it could not yet be shown that a significant effect can be achieved by application . However, drugs are often used in severe cases in the ICU with controversial recommendations . Our data could indicate that lopinavir/ritonavir leads to a reduction of viremia. However, our group size is too small for such a statement, as we could not detect vRNA in the blood, even in untreated patients in some cases.”We would also like to apologize for the correlation statement.17) The authors present no (plan to study) data on interconnectedness of inflammation, thrombosis and infection/viral load in their Materials and methods. Correlation of IL-6, coagulation markers, presence of virus is unclear from results and figures. E.g. hemophagocytosis in bone marrow observed in one case and is discussed as possibly related to MAS and cytokine storm. Yet, no mention of IL-6. Hence, the presentations of any correlation comes across as a haphazard fishing expedition.Hemophagocytosis was observed in more than one case but was not the focus of this study. It will be addressed in another study with reference to histiolymphocytosis-related clinical symptoms. The need of further investigation is also mentioned in the text.18) The conclusion ("Virus may be able to infect different cell types in different organs and tissues but may not be able to replicate in non-respiratory organs/tissues as efficiently as in the respiratory ones") is not based on the findings themselves hence not a conclusion but it may be discussed as a speculative explanation of the findings.We want to thank the reviewer for the comment and want to apologize for the insufficient description which has been removed.


Alpelisib is an orally selective PI3K alpha inhibitor recently available for the treatment of advanced breast cancer. PI3K pathway is an intracellular signaling pathway that plays an important role in regulating glucose metabolism. Hyperglycemia is the most common adverse event associated.We describe the case of a severe hyperglycemia associated with alpelisib treatment in a patient with metastatic breast cancer and previously near-normal glycemia. We analyze the clinical presentation, PI3K inhibitor pharmacodynamic aspects, its influence in glycemic control and the required treatment approach.An important impairment of glycemic control was observed after initiation of alpelisib. In addition to insulin sensitizers drugs, intensive insulin regimen was necessary. Flash glucose monitoring (FGM) information has been helpful in understanding the pharmacodynamic aspects of alpelisib and insulin titration. Development of hyperglycemia is fast, already observed 24 hours after initiation of therapy. FGM shows severe and persistent hyperglycemia during most of the day, with a significant downward effect in the 4 hours after each daily intake, which evidences the strong but transitory effect of the drug enzyme blockade. C-peptide level is remarkable in accordance with drug pharmacodynamics, consistent with a significant insulin resistance.Glucose monitoring should always be performed in patients treated with alpelisib, especially in patients with diabetes and prediabetes. It is crucial to anticipate in these patients. Any delay can lead to a worsening in metabolic control resulting in the discontinuation or reduction of alpelisib, which would lead to a decrease in its effectiveness, and consequently would deny patients an effective treatment with an impact on survival. Breast cancer is the most common cancer worldwide with more than 2.2 million cases in 2020. There is 1 in 12 chance women will develop breast cancer sometime in their lives -positive, human epidermal growth factor receptor 2 (HER2)-positive breast cancer and ER/HER2 and progesterone (PR)-negative breast cancer (triple negative). More than 70% of breast cancers are HR-positive and HER2-negative, and 40% of them have activating mutations in the phosphatidylinosiyol 3-kinase (PI3K) gene  have synergistic antitumor activity and are used in patients with PIK3CA-mutated, HR positive, HER2-negative advanced breast cancer who had received endocrine therapy previously as cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors  and severe (grade 4 toxicity - fasting plasma glucose > 500 mg/dl) forms . Even ifWe describe the case of a severe hyperglycemia associated with alpelisib treatment in a patient with metastatic breast cancer. We analyse the clinical presentation, PI3K inhibitor pharmacodynamic aspects, its influence in glycemic control and the required treatment approach that we believe is crucial to anticipate in these patients with an effective oncologic treatment.2. Given the presence of mild dysglycemia with prediabetes criteria, we prospectively used flash glucose monitoring system (FGM) FreeStyle Libre 2® Abbott and a sequential therapy with antidiabetic drugs was established in case hyperglycemia rose to 160 mg/dl. Hyperglycemia was already observed on the first day of treatment with alpelisib . The onset of hyperglycemia was similar to baseline. The treatment course and the insulin dosage that were required to achieve an optimal metabolic control did not differ from the previously treatment regimen described. At 7-month follow-up, HbA1c was 8.1% and alpelisib tolerance was adequate, with no other adverse events attributable to the drug.Hyperglycemia is the main adverse effect of alpelisib and the most frequent factor leading to discontinuation or dose decrease, being the cause of permanent withdrawal in 6.3% of the patients . It is nThe safety of alpelisib in patients with type 1 and uncontrolled type 2 diabetes has not been established as these patients have been excluded from clinical trials . Severe Despite the lack of detailed information available on the pharmacokinetics of alpelisib, FGM has been helpful in understanding the pharmacodynamic aspects of alpelisib and provides valuable information that correlates with its pharmacokinetic properties. There are barely any studies describing in sufficient detail the day-to-day pattern related to alpelisib administration, the rapid onset alpelisib-induced hyperglycemia and its prompt reversibility . In thisWe report the case of a severe hyperglycemia associated with alpelisib treatment in a patient with metastatic breast cancer, which highlights PI3K inhibitor pharmacodynamic aspects, its influence in glycemic control and the required treatment approach. Glucose monitoring should always be performed in patients treated with alpelisib, especially in patients with diabetes and prediabetes. It is crucial to anticipate in these patients. Any delay can lead to a worsening in metabolic control resulting in the discontinuation or reduction of alpelisib, which would lead to a decrease in its effectiveness, and consequently would deny patients an effective treatment with an impact on survival.The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.All authors listed in the manuscript have contributed to the work: BP and AA contributed to the preparation of the manuscript. FS, AA, and AB contributed to the patient’s clinical care. AA and MM contributed to revision and final approval of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The participants filled out electronic background and feedback questionnaires. The agreement between the methods was evaluated using intraclass correlation coefficients (ICCs) and Bland–Altman plot analyses. The agreement between the methods was moderate to excellent for the energy intake : 0.66 to 0.97, p < 0.001) and good to excellent for total carbohydrate intake . The adolescents considered photographing easier and faster than keeping a food diary. In conclusion, the image-based method appeared comparable to the food diary for dietary intake estimation among adolescents with T1D. The photographing of meals may become a useful dietary assessment tool for adolescents with T1D, but must be further developed and validated.Valid and useful dietary assessment methods for adolescents with type 1 diabetes (T1D) are needed. In this study, we compared an image-based method with a written food diary for dietary intake estimation among adolescents with T1D and evaluated the adolescents’ experiences of the methods. Adolescents with T1D aged 13 to 18 years ( The reliable measurement of dietary intake is necessary in nutrition research. Accurate dietary intake data are especially necessary in the treatment of type 1 diabetes (T1D), where nutrition counselling supports healthy eating habits and is an important part of diabetes self-treatment and good metabolic control . These mPrevious studies have underlined challenges in the measurement of dietary intake in adolescents, such as the tendency to misreport ,5,6,7,8.Adolescents with T1D have to count meal carbohydrate amounts several times a day to match insulin doses and to manage varying blood glucose levels. Thus, feasible dietary assessment methods are particularly important among young people with diabetes. Using technology, such as smartphones and image-based methods, in dietary assessment could reduce the burden on adolescents and improve compliance with reporting their dietary habits . Some imIn image-based dietary assessment studies, in general, the participants are asked to photograph their meals before eating and to send the images with further information to a researcher. Thereafter, the image data are analyzed to specify the foods, to estimate portion sizes, and to count the dietary intake. Recent studies suggested that image-based methods could provide more accurate dietary data compared with traditional methods ; howeverAmong children and adolescents, image-based dietary assessment methods also appear to have similar validity compared to traditional methods ,24,25,26The study population comprised sixteen adolescents with T1D who were aged 13 to 18 years. The participants were recruited from the Pediatric Diabetes Outpatient Clinic at Oulu University Hospital . The study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects were approved by the Northern Ostrobothnia District Hospital Ethics Committee, Oulu, Finland . Written informed consent was obtained from all the participants and also from their parents or guardians if the child was under 15 years old.In this comparative study, the image-based method was evaluated and compared with the written food diary as a standard method. The participants received written and oral instructions to take photographs of meals at a 45-degree angle before and after eating and to concurrently maintain a food diary over four consecutive days. The subjects were asked to make sure that all ingredients were clearly seen in the photograph and to photograph second servings and snacks too.The participants were asked to judge the clarity of photographs they had taken and to retake photographs that were unclear. Paying attention to the lighting and using the camera flash, if necessary, were advised. If a meal was not eaten completely, the leftovers also had to be photographed. The ingredients of mixed foods were asked to be provided in detail . In the written food diaries, the participants were instructed to estimate portion sizes as pieces , household measures , size , or weight (grams).The participants sent the photographs to a researcher using the WhatsApp application  the day after photographing. Further details were sent as a text or an audio message. The participants provided additional details regarding the food quality  and cooking methods. The researcher verified the pictures after the four-day study period, sent a feedback message, and asked for additional information from the participants if necessary. The participants returned the written food diaries by mail at the end of the study period. Additional questions about the quality and amount of food in the written food diaries were not asked.The adolescents replied to electronic background and feedback questionnaires . The background questionnaire included information on the date of birth, gender, date of diabetes onset, latest hemoglobin A1c , and use of a mobile phone’s camera. The actual height and weight measurements were drawn from the medical records. The participant experiences of the usability of the image-based method and sending photographs and further dietary information were evaluated with the feedback questionnaire.The ease and quickness of the methods were estimated using four questions with a 5-point Likert scale (1 = very easy to 5 = very difficult or 1 = very quick to 5 = very slow). The factors restricting the use of the image-based method were asked using a multiple-choice question: “Did you have any problems with using the image-based method?” The answer options were as follows: (1) I forgot to take photos; (2) I did not carry my mobile phone with me; (3) my mobile phone’s battery was out of charge; (4) my mobile phone’s camera was poor; (5) lightning was poor; (6) mobile phone use was not allowed; (7) I was too embarrassed to take photos; (8) something else, what?; and (9) I had no problems. The enabling factors were queried using an open-ended question: “Which factors made the image-based method easy?” In addition, the adolescents were asked which methods they would prefer in the future.The same nutritionist (L.H.) analyzed both the photographs and food diaries using the AivoDiet software program, version 2.0.2.3 . The photographs were analyzed first, and the food diaries at least four weeks later. The foods in the photographs were identified visually and using further detailed information provided by the participants.® software as grams. The AivoDiet® software uses the Finnish food composition database Fineli , which includes the average energy and nutrient contents of foods and food products used in Finland.Two picture booklets of the portion sizes were employed in estimating the food amounts in the photographs ,29,30,31The following assumptions were made: (1) if exactly the same picture was received in duplicate, it was considered to depict the same meal; (2) the absence of a leftover photograph indicated the meal was finished; (3) if the meal was not photographed, but its components were given in further detail as a text or audio message, it was included in the analysis; (4) if the ingredients of a mixed dish were not provided in further detail, it was assumed to have been cooked with a conventional Finnish recipe; and (5) if a portion size was not provided in the food diary, standard portion sizes were used.t-test was used to estimate the differences between the methods in the energy and nutrient intake estimation when the difference between the two variables was normally distributed. The Wilcoxon signed rank test was employed when the difference between the variables was non-normally distributed.The mean daily energy and nutrient intakes were calculated from the photographs and from the food diaries. First, the analyses were made using the original data. Second, to evaluate the image-based method’s validity in energy and nutrient intake estimation, the meals that the adolescents forgot to photograph were deducted from the food diary data . The normality of the continuous variables was tested. The paired samples Agreement between the methods was estimated using Bland–Altman plots and intraclass correlation coefficients (ICCs). Differences between the methods on the Y-axis were plotted against the mean of the two methods on the X-axis. The limits of agreement  were marked on the Bland–Altman plots. The presence of a proportional bias  was tested using linear regression analysis.p-value <0.05 was considered statistically significant. The statistical analyses were performed using IBM SPSS Statistics, version 23 .The ICC estimates and their 95% confidence intervals (CIs) were analyzed based on a single-rating, absolute-agreement, two-way mixed-effects model. For non-normally distributed variables, the ICCs were calculated using logarithmic values. ICCs less than 0.50 were interpreted as poor, 0.50 to 0.75 as moderate, 0.75 to 0.90 as good, and greater than 0.90 as excellent agreement . A p-valSixteen adolescents with T1D aged 13 to 18 years participated, and thirteen of them were included in this study . Three fThe participants photographed on average 18 ± 2 and recorded 20 ± 2 meals in their food diaries during the four-day study period. Two participants forgot to photograph one meal and three participants forgot more than one meal (range 7 to 10). Altogether, 237 meals from the photographs and 264 meals from the food diaries were analyzed in this study.p = 0.010). The difference in the median energy intake between the image-based method and the food diary was 34.4%  in boys and 7.2%  in girls. In the original data, the image-based method underestimated the median energy intake by 8.5% compared with the food diary  251, p < 0.001) (p = 0.033). Underestimation of the mean energy intake was 9.8%  in boys and 3.4%  in girls. The ICCs between the image-based method and the food diary were at least moderate for most nutrients in the deducted data . Regarding the carbohydrate intake estimation, agreement between the methods was good to excellent . The mean carbohydrate intake did not differ between the methods .In the deducted data, the agreement between the methods was moderate to excellent in the energy intake estimation  . The imap < 0.001) and protein intake estimation . The daily fat intake estimates did not differ between the methods . The image−based method underestimated the mean protein intake .Agreement between the methods was poor to excellent in the fat  and carbohydrate  intakes showed slight positive trends, indicating that the image-based method tended to underestimate energy and carbohydrate intakes when the mean intakes were low a,b. SimiAccording to the feedback questionnaire, 11 of the 13 subjects (85%) considered recording food intake by a mobile phone very or quite easy, and 12 (92%) assessed the method as very or quite quick. All the participants (100%) experienced sending photographs as being very or quite easy, and 11 subjects (85%) considered sending further information to be very or quite easy.The reported reasons for the ease of the use of the image-based method were reported as (1) I think the method was quick to use (46%); (2) I always carry my mobile phone with me (38%); (3) I am used to taking photos with a mobile phone camera (23%); and (4) I received clear instructions to take photographs (15%). The two most common reported problems with the image-based method were forgetting to take photos (62%) and poor lightning (31%).When the participants were asked which of the methods they would prefer if they were to record their food intake in the future, seven (54%) participants chose the image-based method, five (39%) participants accepted both methods, and one (8%) participant preferred the food diary. The reported reasons for preferring the image-based method were the easiness and quickness.In the present study, the image-based method appeared to be comparable to the written food diary regarding the energy and nutrient intake estimation of adolescents with T1D. The image-based method slightly underestimated the mean energy intake, while agreement between the methods was good in the carbohydrate intake estimation. We found that the image-based method was a feasible method among adolescents with T1D; however, young people’s tendency to forget to take photos weakened its accuracy. Furthermore, the adolescent participants preferred the image-based method in dietary assessment according to the feedback questionnaire. Although there was a relatively low number of participants in the present survey, the adolescent participants recorded a total of 264 meals, snacks, and drinks during the study period.The present study showed that the image-based method had moderate to excellent agreement with the food diary in energy intake estimation and good to excellent agreement in carbohydrate intake estimation among adolescents with T1D. Our results are in accordance with previous studies where the intakes of energy and carbohydrate were compared using image-based methods and a written food diary among children and adolescents ,34.In the present study, the mean difference in the carbohydrate intake between the methods was 8 g per day (4.4%); thus, the methods can be considered similar in practice. The mean energy intake was 88 kcal (5.5%) per day lower when assessed using the image-based method compared with keeping a food diary. This systematic bias was equal to earlier studies that reported 53–222 kcal per day underestimation of energy intake among children and adolescents ,25,34.The ICCs between the two dietary assessment methods were at least moderate for most nutrients, reflecting that the methods were comparable in estimating the adolescents’ dietary intake. The image-based method was equal to the food diary in fat intake estimation but underestimated the protein intake. However, it can be debated whether the mean underestimation of protein intake in this study (5 g/day) would have clinical or practical relevance.The protein intake estimates had weaker correlations than those of other macronutrients in this study. Davison et al.  and WangIn this study, we removed the foods that adolescents forgot to photograph from the food diary data to evaluate more accurately the image-based method’s ability to estimate dietary intake. According to the original data, the image-based method underestimated the median daily energy intake by 153 kcal (8.5%). The underestimation in our results was comparable with those of some previous studies among children and adolescents ,34. AdulUnderestimation of the mean carbohydrate intake in the original data  was greater when compared with one recent study [Based on the present and previous studies, commitment to studies might be challenging for adolescents. However, people with T1D are generally used to following instructions to maintain good glucose control, which can also improve their ability to follow protocols. In the present study, forgetting to take photographs was one source of misreporting, and we found that this weakened the feasibility of the method. Forgetting to photograph has also been found to be a source of underreporting in previous surveys ,36.The typical forgotten foods in the present study were drinks and extra snacks, for example, chips or meatballs. Systematic misreporting also existed: for example, a bread spread was not always seen in the photographs. A reduction of missing photographs could possibly be reached with prompts and systematic contact with the subjects during the survey. Backup methods, more detailed instructions, and final inspections of food diaries might also be helpful to diminish missing data in future studies.According to the present study, the image-based method appeared to be more accurate among the girls than in the boys. One explanation might be that adolescent girls were more interested in recording their dietary intake than boys, and therefore had a higher compliance with using the image-based method. In previous studies, underreporting of dietary intake has been more common among girls than in boys . If the One strength of the image-based method is that it exerts a lower respondent burden, as adolescents do not have to specify all consumed food and drinks and estimate portion sizes. On the other hand, the responsibility for portion size estimation is transferred to the researchers, which increases their burden and requires well-trained professionals for consistent and accurate imaged-based dietary assessment. Regular photograph inspection and contact with the participants also increases the researchers’ work. In this study, the researcher estimated portion sizes visually and using picture booklets of the portion sizes. Additionally, package labels and dishes seen in the photographs were of use. A fiducial marker was not used in this study but would be recommended for future studies to ensure the accuracy of portion size estimation.The image-based method appears to be a feasible tool in dietary intake estimation. The development and improvement of feasible dietary assessment methods could be very important for people with T1D as they have to count the carbohydrate contents of their meals several times a day to adjust insulin doses and to estimate blood glucose changes. Advanced methods for intensive insulin delivery and continuous glucose monitoring by sensor devices are already available.Novel automatic carbohydrate counting methods have already been developed; however, further research is needed to bring them into clinical practice ,14. OwinTo our knowledge, the present study is the first image-based method validation study in adolescents with T1D. The fact that participants with T1D in this study were used to paying attention to their dietary habits and, thus, were experienced in estimating their food content and portion sizes might limit the generalizability of the results for healthy adolescents. In this study, we compared the image-based method with a food diary, which has limitations, such as being prone to reporting errors .The written food diaries were returned by mail, and additional questions regarding the quality and amount of food were not asked, which may have weakened the validity of the diaries. In addition, if participants had sent photographs of the filled out written food diary to the researcher, the loss of some data may have been avoided. Only one rater analyzed the photographs, and a fiducial marker was not used, which may have limited the accuracy of the portion size estimation in this study. The feasibility and validity of the image-based method should be confirmed in larger studies.The image-based method seemed to have good agreement with the written food diary for estimating the dietary intake of adolescents with T1D. According to the findings in the present study, the image-based method appeared to be more accurate among the girls than in the boys; this finding may be due to a small sample size and would need more clarification in future studies. The young participants considered photographing easier and faster compared with the food diary and were more willing to use it in their dietary assessment. However, more detailed instructions and frequent prompts are required to maintain a good compliance with using this method. The image-based method may be a useful dietary assessment tool for adolescents with T1D, but additional research in larger samples is needed.