Latently infected C57BL/6 mice were induced to reactivate in vivo by hyperthermic stress. Infectious virus was detected in a high percentage (60–80%) of the trigeminal ganglia from these mice at 20 hours post-reactivation stimulus, but declined by 48 hours post-stimulus (0–13%). With increasing time post-reactivation stimulus, the percentage of reactivating neurons surrounded by a cellular cuff increased, which correlated with a decrease in detectable infectious virus and number of viral protein positive neurons. Importantly, in addition to intact viral protein positive neurons, fragmented viral protein positive neurons morphologically consistent with apoptotic bodies and containing cleaved caspase-3 were detected. The frequency of this phenotype increased through time post-reactivation. These fragmented neurons were surrounded by Iba1+ cells, consistent with phagocytic removal of dead neurons. Evidence of neuronal destruction post-reactivation prompted re-examination of the previously reported non-cytolytic role of T cells in controlling reactivation. Latently infected mice were treated with anti-CD4/CD8 antibodies prior to induced reactivation. Neither infectious virus titers nor neuronal fragmentation were altered. In contrast, when viral DNA replication was blocked during reactivation, fragmentation was not observed even though viral proteins were expressed. Our data demonstrate that at least a portion of reactivating neurons are destroyed. Although no evidence for direct T cell mediated antigen recognition in this process was apparent, inhibition of viral DNA replication blocked neuronal fragmentation. These unexpected findings raise new questions about the resolution of HSV reactivation in the host nervous system.A fundamental question in herpes simplex virus (HSV) pathogenesis is the consequence of viral reactivation to the neuron. Evidence supporting both post-reactivation survival and demise is published. The exceedingly rare nature of this event at the neuronal level in the sensory ganglion has limited direct examination of this important question. In this study, an in-depth  Herpes simplex virus (HSV) is an endemic human pathogen that establishes latency in neurons and can periodically reactivate over the lifetime of the host. Whether or not neurons survive post-reactivation is controversial and has significant implications for long-term infection. HSV reactivation events can be characterized in mice, which maintain the complexity of host-pathogen interactions, with the goal to provide insight into how the virus behaves in the nervous system. In this report, it is shown that the elimination of infectious virus following reactivation in vivo corresponded with a highly focused cellular response and destruction of the neuron containing HSV proteins. The role of T cells in this response was investigated. Previous work identified T cells as major regulators of HSV reactivation. However, neuronal destruction was still observed when T cell antigen recognition co-receptors were absent and infectious virus titers and viral spread was not different from isotype treated control ganglia. Conversely, destruction of viral protein expressing neurons was not observed when viral DNA replication was inhibited. These findings suggest that reactivation is resolved through destruction of the neuron, which appears to be independent of antigen-mediated T cell cytotoxicity, but does require viral replication. Herpes simplex virus (HSV) is endemic in the human population . Infecti+ T cells can interact with latently infected neurons in a non-cytolytic manner, which suggested that T cells function to preserve the neuron and represent a potential barrier to reactivation  and mice latently infected with HSV-1 strain 17syn+ [HSV(+)] were treated with control IgG or anti-CD4 and anti-CD8 depleting antibodies. Mice were given a second dose of antibodies 3 days later and the latently infected mice were re-infected with 1 x10(TIF)Click here for additional data file.S7 FigMice latently infected with 17syn+ were treated with ACV  or saline (mock) control (n = 5 mice/group) starting one day prior to hyperthermic stress and continued until the time of sacrifice. At 46 h phs, ganglia were removed and HSV proteins detected by WGIHC. (A) Bars represent the number of intact and fragmented viral protein expressing neurons (n = 10 TG per group). Fragmented neurons were not detected in the ACV treated group. *ACV treated and untreated groups are different, p = 0.0004, Fishers exact test. (B) Photomicrographs of fragmented, HSV protein positive neurons detected in mock treated mice. (C) Photomicrographs of intact, HSV protein positive neurons detected in ACV treated mice.(TIF)Click here for additional data file. 14 Aug 2019Dear Dr Sawtell,Thank you very much for submitting your manuscript "Resolution of herpes simplex virus reactivation in vivo results in neuronal destruction" (PPATHOGENS-D-19-01139) for review by PLOS Pathogens. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, and that the study has the potential to make a substantial contribution to the literature. There are however some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.All reviewers acknowledge the high quality of your experimental work and that your study addresses a very important long open standing topic. However, particularly reviewer 1 and reviewer 3 feel that there are significant gaps that need to be filled.All reviewers ask for more information on the reactivation state and the fate of HSV positive neurons, and the potential temporal relationship of neuronal apoptosis and nodules. Furthermore, reviewer 1 and reviewer 3 request a further characterization of the immune cells surrounding the HSV positive neurons. In particular, I would like to highlight the comments of reviewer 3 on the T cell depletion experiments. She/he questions whether it has been sufficiently investigated which cell types have been depleted, and whether there may be a remaining T cell activity that might contribute to the resolution of the reactivated HSV infection.The suggestion of reviewer 1 for an additional animal model seems to be beyond the scope of the present study. However, we agree with reviewer 3 that further references to similarities and difference to the mechanisms of HSV reactivation in patients and in other animal models with particular reference to the role of T cells in the discussion would make the study more accessible to a broader readership.All reviewers have further specific suggestions to improve the clarity of the manuscript.We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.In addition, when you are ready to resubmit, please be prepared to provide the following:(1) A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. 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Revised manuscripts received beyond 60 days may require evaluation and peer review similar to that applied to newly submitted manuscripts.[LINK]We are sorry that we cannot be more positive about your manuscript at this stage, but if you have any concerns or questions, please do not hesitate to contact us.Sincerely,Beate SodeikGuest EditorPLOS PathogensKlaus FrühSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XGrant McFaddenEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-2556-3526***********************Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: Report on the manuscript by Doll et al., entitled “Resolution of herpes simplex virus reactivation in vivo results in neuronal destruction”This report addresses a very important and relevant question: what is the fate of the neuron upon of herpes simplex virus type 1 (HSV-1) reactivation. To answer this question the authors infect C57BL/6 mice through the cornea with two HSV-1 strains, induce reactivation by hyperthermic stress 45 days post-infection and determine the number and fate of reactivated neurons using staining techniques, quantitative PCR and plaque assays. They define latency by the lack of detection of viral protein and infectious viral particles. Reactivation is defined here by the detection of viral protein and/or infectious viral particles. The authors infect female and male mice with the McKrae and 17syn+ strains, respectively and reach similar conclusions despite the differences in strain and gender.Initially, they authors perform a thorough characterization of the viral replication kinetics, the time of latency establishment and reactivation. They find out that the number of neurons positive for HSV-1 antigens reaches a peak around 24 hours post-reactivation and then decreases. This correlates with the viral titres in TG, which are highest between 20-24 hours post-reactivation. Interestingly, some neurons have an intact morphology whereas others seem fragmented or damaged and are positive for the apoptotic marker caspase 3. The percentage of such neurons, but not their total number, increases with time post-reactivation. The authors find that these neurons are surrounded by infiltrating cells, many of them expressing Iba1 and considered by the authors to be microglia. They also find nodules in spaces possibly occupied previously by apoptotic neurons. Since T cells are thought to be relevant on inhibiting HSV-1 reactivation the authors deplete them prior to hyperthermic stress. Surprisingly, the data suggests that T cells do not play a role in controlling HSV-1 reactivation in this model.The main findings of this study are: (i) the discovery of apoptotic neurons upon HSV-1 reactivation; (ii) the formation of nodules surrounded by Iba1+ cells, suggesting that they are involved in the clearance of reactivated, apoptotic neurons; (iii) CD4 and CD8 positive T cells do not play a key role in controlling HSV-1 reactivation in the mouse model despite previous results suggesting the contrary.The manuscript is very well written and clear, most figures are of high quality. The experiments are performed well, including the relevant controls. Most conclusions are supported by the data, although others are not.Reviewer #2: The authors are known for their painstaking and careful morphologic work, leadership in the hyperthermic stress model, and have a track record of influencing the field. A plus is that they study two different viral strains. The authors nicely check for latency at day 30 vs 45 and for differences in latent HSV DNA load between models. McKrae, even at a lower inoculum, replicated to higher titers in eye and TG at day 4 and resulted in more mouse death. Data are presented for one sex for each viral strain, with a note that McKrae was even more pathogenic in males.Reviewer #3: In the manuscript entitled “resolution of herpes simplex virus reactivation in vivo results in neuronal destruction”, Doll et al. present the results of a very extensive analysis of the fate of latently infected murine trigeminal ganglia neurons that have been induced to reactivate HSV-1 by hyperthermic stress in vivo. For this study, the authors use two HSV-1 strains that are efficient reactivators in the mouse model (17+ and McCrae) and examine reactivation by two classic criteria: the ability to detect infectious virus and the ability to detect viral antigen in individual neurons by immunohistochemistry. In order to address the question of neuronal fate after reactivation, ganglia are examined at several time points after thermal stress: 20, 34 and 48 hrs. Key findings of this study are: 1) reactivation can be detected by 20 hours post stress and some of the antigen positive neurons exhibit fragmentation (an indication of possible apoptosis), 2) the number of neurons exhibiting fragmentation increases at the later time points, 3) the fragmented neurons show evidence of caspase 3 cleavage, indicating they are undergoing apoptosis, 4) latently infected ganglia show increases in activated microglia, but reactivating ganglia show evidence of clustering of these microglia around antigen positive cells, but especially around fragmented cells, suggesting that these cells may play a role in clearance of these cells, and 5) blocking of HSV-1 antigen-specific T cells does not alter the number of reactivating neurons, nor their fate.This is a highly significant study as it attempts to address the age-old question regarding whether neurons that reactivate HSV latent infection die as a result of the reactivation, or whether they can survive. As the authors point out, there are publications that support both possibilities. In particular, explant-induced reactivation does not tend to show robust evidence for neuronal death as a direct result of reactivation. This is the first study to take this question on looking at reactivation in vivo. The study is very well-designed and carefully performed. Because reactivation is a relatively rare event, large cohorts of mice were examined to allow statistically defendable conclusions to be drawn. This is an outstanding study and one that will be highly cited and have a large impact on the field. There are only a few relatively minor points that should be addressed for precision and clarity.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: 1. The main objective of this manuscript is to determine the fate of the neurons in which HSV-1 reactivated. This is a very difficult task due to the low number of neurons in which reactivation is detected. This is not a criticism to the authors, it is more a description of the challenge faced by them. The authors find both neurons that have a normal morphology and others that look “fragmented” upon reactivation. Although in lines 452-454 the authors suggest that in some neurons the viral cycle will progress and apoptosis will be inhibited, in other sections of the paper such as lines 463-465 it seems that the authors promote the idea that fragmentation and apoptosis of the reactivated neuron is the main mechanism to control reactivation. Even the title of the manuscript suggests this. However, I do not think that this can be concluded from the current data.Similarly, based on data shown in Figure 2, the authors conclude that there is a transition from non-fragmented to fragmented neurons because the percentage of the latter increases with time. However, the total number of fragmented neurons does not increase as one would expect if most non-fragmented neurons become apoptotic. Since the authors also found the presence of nodules and suggest that these appear in places in which apoptotic neurons were located one would also expect that the number of nodules increases with time reaching a level similar to that of reactivated neurons. The authors show that the number of nodules increases with time (Table 3) but is difficult to compare these numbers with the numbers of reactivated neurons shown in Figure 2 since there is no information on the number of ganglia used for this figure. However, the low number of nodules shown in Table 3 suggests that many of the reactivated neurons did not become nodules. This would suggest that the fate of many reactivated neurons is not to undergo apoptosis and that reactivation is controlled by an alternative mechanism.The authors base their conclusions on a systematic analysis to identify neurons positive to HSV-1 antigen. However, it is possible that HSV-1 reactivated in more neurons without generating enough viral antigens to be detected by antibody staining. The use of hybridization techniques to identify HSV-1 genome and transcripts will detect such neurons allowing the quantification of the percentage of reactivating and apoptotic neurons. This will shed some light on how common the described phenotype is.2. As indicated in lines 254-257, several cell types are involved in removing cellular debris in peripheral ganglia. However, the authors focus on microglia, a cell type more commonly found in the CNS than in peripheral ganglia . It may help the reader who considers them as CNS-restricted cells if this is explained in the manuscript. A possible alternative to the authors’ conclusions is that the Iba1+ cells are not microglia but infiltrating macrophages since they also express this protein :844-56; Sasaki et al., Biochem Biophys Res Commun. 2001 Aug 17;286(2):292-7, among others). P2Y12 is expressed in microglia and not macrophages and is currently used as a marker to distinguish between these two cell types. Moreover, its expression is increased upon microglia activation. The authors should use this marker to clearly demonstrate that microglia and no macrophages surround the reactivating neurons. Are there other cells, not Iba1+, surrounding the neurons during reactivation? If so, which ones?One interesting question arising from this work is whether the Iba1+ cells are there to clear the apoptotic neuron or whether they are involved in control of reactivation or in the induction of apoptosis, since non-fragmented neurons are also surrounded by these cells, although to a lesser extent . Also, in Figure 5 the Iba1+ cells have a different morphology in the mock and latently infected ganglia. In certain areas of the latent ganglion there is more Iba1 expression and the Iba1+ cells have a rounded morphology indicative of activation, whereas in others they look as non-activated cells. A quantification of both cellular phenotypes would facilitate asserting the relevance of these observations. According to Figure 5B, the Iba1+ cells also surround the neurons before reactivation and cover nodules where presumably a neuron died during lytic infection. It would be helpful to detect the viral genome by FISH to determine whether there are activated Iba1+ cells already surrounding the latently infected neurons. Also, detection of HSV transcripts by RNA-scope or similar technique will inform whether reactivation already started in some of these neurons but did not progress to production of protein and infectious viral particles. This would indicate whether the Iba1+ cells participate in initial control of reactivation. Once the authors convincingly prove that the cells are microglia or macrophages it will be very interesting to determine their role in neuronal fate upon HSV-1 reactivation by depleting them prior to reactivation. There are protocols available in the literature to deplete either cell population in the mouse.3. Previous studies did not show efficient reactivation in C57BL/6 mice, contrary to the findings in this report. The authors discuss nicely the possible reasons for these discrepancies. Here, the analysis of protein expression in neurons and the presence of infectious virus in TG demonstrate reactivation. But there is no data showing the presence of HSV-1 in the eye or tear film upon reactivation. It is possible that C57BL/6 mice are particularly efficient in controlling HSV-1 reactivation and that a different phenotype may be observed in other mouse models in which reactivation is more efficient. Although I am aware of the difficulty of setting up a similar model with a new mouse strain, performing such experiments will allow determining whether the Observations reported here are mouse-strain specific or a general phenomenon. This will also provide information of the mechanism(s) leading to control of reactivation.Reviewer #2: 1. Overall there were relatively few HSV IHC (+) cell outlines in the entire study, emphasizing careful microscopy. Were the microscopists evaluating from intact vs fragmented HSV protein (+) cell outlines  blinded as to time point or viral strain? Similarly for Fig. 1C,D were the microscopists blinded for time point? The same blinding query extends to additional data sets and observations.2. I am concerned about the interpretation of the data about the temporal progression of inflammatory cuffs surrounding HSV antigen (+) zones over time discussed in lines 291-327. The authors note that at their early time point (20 hours after hyperthermia) there is a mix of cuffed and non-cuffed neuron-like cells, but that by later time points (48 hours) all of the HSV IHC (+) areas (interpreted as neurons) have an inflammatory cuff. Of concern, the absolute number of HSV IHC (+) areas at the late time points is very small, such that the ratio of cuffed to non-cuffed HSV IHC (+) areas is unlikely to be measured with much precision. Between McKrae and 17syn+, there were only 6 HSV IHC (+) areas observed at 48 hours (Tables 1 and 2). Lines 437-438 in the Discussion also concern the proportion of fragmented neurons and this again is based on a very limited number of observations. It is not clear if the nodules discussed in lines 328-335 are really areas of inflammation localized to (now cleared) reactivated neurons: were these nodules also HSV IHC (+),or just clumps of Ib1a1+ cells? ? Can we see an example in a micrograph? An average of one such nodule was seen per TG (Table 3) and the legend line 919 indicates that these were “likely” sites of previous neurons. Given the small numbers of these structures and the incomplete documentation that these nodules are sites of cleared neurons, I think these findings should be de emphasized.3. One of the key conclusions of the paper is that T cells are not involved in the morphologic disruption of HSV IHC (+) areas that probably represented dying reactivated neurons. This is a potentially paradigm-breaking finding in this field, as both prior human and mouse data indicate that HSV-specific T cells form halos around latently infected neurons, and depletion of T cells in various reactivation models leads to increased HSV replication. It would help to build the case for this statement by staining the cell halos around HSV IHC (+) cell areas  for T cell markers, in addition to the microglial marker. The simple presence of T cells could not imply a causal role in viral shutdown, but their convincing absence would build the case.4. The main technology used was administration of mAbs against CD4, CD8 alpha, and CD8 beta. In preliminary experiments , administration of anti-CD4 and anti-CD8 prior to acute infection leads to increased recovery of HSV at day 7 from eye and brain after eye inoculation. The authors then shift to administering the mAbs 3 days and 1 hour prior to hyperthermic stress in latently infected mice. Fig 7 shows that cells that are CD3(+) TCRbeta positive are still present in spleen and TG at 48 hours after the hyperthermic stress. Thus, the T cells are not depleted: at least some cells are still present, but the CD4 and CD8 epitopes are interpreted as masked by the overwhelming amounts of mAb administered previously to the mice. What we don’t know about in enough detail is what these residual cells are: are they CD4negCD8neg T-like cells (as in some gamma delta T cells or NKT cells) or are they traditional CD4+CD8- or CD4-CD8+ T cells with masked surface CD4/CD8 co-receptors as hypothesized by the authors. We also don’t; know if they are functionally blocked by the mAbs.The human literature contains well described CD8 co-receptor independent T cell clonotypes: for these T cells, the TCR itself can bind strongly to peptide-MHC, and CD8 co-receptor binding to MHC class I is not required for T cell signaling and function. Thus, the absence of stain-accessible CD8 does not mean that residual traditional CD4-CD8+ T cells, if present, are non-functional.The authors try to address this issue by incubating immune splenocytes with HSV-charged DC as APC in the presence or absence of mAbs . In these expts, the mAbs are added in vitro; it would have been better to use responder splenocytes from animals that had previously been given mAbs in vivo. They use inactivated HSV to charge the DC, and as expected, they see relatively little CD8 reactivity (little decrement on adding blocking only anti-CD8). DC cross-presentation of inactivated antigen can occur but is typically very challenging to detect. In the data shown, the addition of anti CD8 alone leads to lonely a partial decrement in T cell activation, while anti-CD4 leads to better blockade that is not increased by adding anti CD8 to the anti CD4.Thus, we are not really presented data from a system in which anti-CD8 could even potentially block HSV-specific CD8 T cell responses. It would be more convincing to isolate tetramer (+) CD8 T cells specific for the well-known HSV-1 gB immunodominant CD8 epitope and test if the anti-CD8 mabs would block their activation in response to peptide-pulsed or HSV-infected APC, which are more traditional APC for CD8 T cells. Regardless, the use of high doses of candidate blocking mAb in vitro is less convincing than testing responder cells recovered from mAb-treated mice. The finding of neuron fragmentation in the mAb-treated animals given hyperthermic stress could be due to residual CD8 T cell activity that was not depleted/blocked.5. In human medicine, there is no evidence of neuron drop-out in post-mortem exams of HSV (+) vs. HSV (-) ganglia. Many neurons in TG are DNA (+) or LAT (+) or both even in very elderly people, consistent with long term survival of at least some infected neurons. There is also no clinical evidence of anaesthesia in lip or genital areas supplied by HSV-infected ganglia that might occur if there was significant loss of neurons. The Discussion should acknowledge the differences in pathogenesis and the difficulty of drawing conclusions in humans.6. Neuron dropout/death in the mouse model was associated with a halo of glial-like cells. Since we don’t know if neuron dropout occurs in humans, the significance of this finding to humans is also unknown. Both host species share the finding of halos or rims of HSV-specific T cells (including HSV tetramer (+) CD8 T cells with markers of cytolytic effector function) that surround HSV-infected ganglionic neurons. Thus there is the potential for T cell recognition of HSV proteins ), and either cytolytic or non-cytolytic T cell effector functions influencing the reactivating neuron. In vitro, upon explant, neutralization of CD8 or IFN-g leads to increased lytic HSV reactivation of mouse neurons.Ghiasi et al. have also re-addressed this issue with findings that CD8+ DC may account for some anti-CD8 findings, an issue that should be discussed. This issue of whether or not acquired T cells contribute to control of reactivation in vivo is of great translational concern, as therapeutic vaccines for HSV are largely T cell oriented and current thinking is to try to get antigen-specific T cells into ganglia.Reviewer #3: None**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: 1. Mouse models are essential to understand HSV-1 pathogenesis. At the same time, some conclusions or observations obtained using mice do not apply to humans. In this report reactivation is rapidly controlled and whether infectious virus is present in the eye or tear film after reactivation is not indicated. Do the authors detect infectious virus in the eye upon reactivation? The authors indicate in lines 99-100 that “inflammatory foci have been detected in HSV infected human ganglia post-mortem, independent of viral protein expression ”. They also mention in lines 474-476 observations supporting death of neurons after HSV-1 reactivation in humans. However, it is also known that HSV-1 reactivates asymptomatically in humans quite frequently, and infectious virus is detected in the innervated mucosa. Moreover, HSV-2 reactivation in human sacral DRG is associated with increased neurite outgrowth and not with neuronal death . The data presented here provides support for the death of neurons upon reactivation but, to my knowledge, it is not clear at present what the percentage of reactivated neurons that undergo apoptosis upon reactivation in humans is. Is there a “reactivation threshold” that leads to neuronal apoptosis? Or does this also occur during asymptomatic reactivation? Although it is beyond the scope of this work to obtain answers to these questions, it makes sense to take all these previous observations into account and discuss the relevance in humans of the interesting findings reported here.2. Figure 4: The authors show that 3 out of 3 fragmented neurons are positive for caspase 3 whereas non-fragmented ones are negative. Although the data looks convincing it would be even more so if the n is increased. The caspase 3+ neuron is surrounded by Iba1+ cells. Was this observed in the other apoptotic neurons found?3. In some experiments, such as the ones in Tables 2 and 3, Figures 2 and 4, it is not clear the number of ganglia used. Also, what is the variation between different ganglia?4. Lines 432-435. Maybe I do not understand what the authors imply, but I do not agree with the current statement. The number of HSV-1 positive neurons associated with inflammatory cuff does not increase with time post-reactivation. Tables 1 and 2 show more neurons associated with cuff at 20 hours than later. The percentage is higher but this may be due to the fact that the number of events is highly reduced with time.5. Some references are not inserted with endnote or similar software. Instead, the surname of first author and year are included.Reviewer #2: 1. Was there a sex difference for 17syn+?2. There are some issues with the Fig. 4 legend. I don’t understand which of the 4 X marks in B correspond to which of the 3 X marked neuros in D. While I can intuit what E is, it is not called out in the legend. Similar for F. G and H appear to be lower power shots of a ganglia with an iba1(+) infiltrate around an HSV (+) cell but this is not called out in the legend. It is called out in the text around line 248 but better to also mention in legend.3. The morphologic findings in Fig. 5E seem to represent a single finding in a single ganglia, and as such I question whether they should be presented as a characteristic of post-hyperthermic stress samples. In addition, the interpretation in the results lines 289-290 is conjecture, with no visualization of egressing virus, so I suggest this either be relegated to Discussion, or better, dropped. Similarly the line 995-996 statement that the cell cluster might be the site of previously infected neuron in a pre-stress latent ganglia is not supported by data and should be dropped.4. They key Fig. 7B, C data showing no difference in reactivation parameters in mAB-treated mice is a little hard to interpret. What virus strain was used and what time point was studied? Of note the number of HSV IHC (+) cells was far lower in Fig 7C untreated animals (1-2 per ganglia) than in the Fig. 1 C, D lead-in experiments: why is this?5. The p value in line 447 appears to be based on differences in McKrae vs 17syn+ between Tables 1 and 2 at 20 hours. The mention of 24 hours in line 445 is confusing as 24 hours is not mentioned elsewhere. The numbers of fragmented outlines for McKrae at 20 hours  and for 17syn+  are very low absolute numbers, such that I question use of stats and drawing conclusions on such small numbers of observations.6. As a minor point in Sup 1D were brain dissections and titers done on day 10 in the 17syn+ mice?Reviewer #3: 1) The largest issue is that I believe that the manuscript gives the impression that HSV-1 reactivation itself induces the apoptosis that is being observed and arguing that immune factors (microglia and/or cytokines are not involved). The authors do elude to this possibility in the discussion, but I think it is important enough a point that it be more explicitly stated in the body of the manuscript whether this is discussed as well .2) line 184. It is stated that “less than 2 fold variation” in establishment is detected. This does not seem correct as the variation for each virus seems to be greater than that (lines 186 – 187)?3) Related to point 1 above, the way that the title, and conclusions in the abstract and throughout the text of the manuscript read, the authors seem to imply that all reactivating (or antigen-positive) neurons die by apoptosis. While I am convinced from the data presented that a majority of them seem to, the data do not exclude the (likely) possibility that some of them do not. This should be explicitly stated and discussed for completeness).4) Related to point 4, the sentence on line 528 should be modified to state that “at least some neurons supporting HSV-1 reactivation are destroyed by the event”.5) Lines 531 – 532. Probably should tine down the statement that suggests CD4+ and CD8+ T cells play no role in maintaining the latent state. As discussed in the paragraph above this statement, they may well play a role in cytokine signaling.**********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  Please note, once your paper is accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. 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The incorporated modifications, including new experiments and changes in the text, clearly improved the manuscript. The results provide substantial evidence for the authors' conclusions. I believe that this report will contribute to the understanding of the neuronal fate upon HSV-1 reactivation and it will spark scientific discussions and trigger more investigations in this exciting field.Reviewer #3: In the manuscript entitled “resolution of herpes simplex virus reactivation in vivo results in neuronal destruction”, Doll et al. present the results of a very extensive analysis of the fate of latently infected murine trigeminal ganglia neurons that have been induced to reactivate HSV-1 by hyperthermic stress in vivo. For this study, the authors use two HSV-1 strains that are efficient reactivators in the mouse model (17+ and McCrae) and examine reactivation by two classic criteria: the ability to detect infectious virus and the ability to detect viral antigen in individual neurons by immunohistochemistry. In order to address the question of neuronal fate after reactivation, ganglia are examined at several time points after thermal stress: 20, 34 and 48 hrs. Key findings of this study are: 1) reactivation can be detected by 20 hours post stress and some of the antigen positive neurons exhibit fragmentation (an indication of possible apoptosis), 2) the number of neurons exhibiting fragmentation increases at the later time points, 3) the fragmented neurons show evidence of caspase 3 cleavage, indicating they are undergoing apoptosis, 4) latently infected ganglia show increases in activated microglia, but reactivating ganglia show evidence of clustering of these microglia around antigen positive cells, but especially around fragmented cells, suggesting that these cells may play a role in clearance of these cells, and 5) blocking of HSV-1 antigen-specific T cells does not alter the number of reactivating neurons, nor their fate.This is a highly significant study as it attempts to address the age-old question regarding whether neurons that reactivate HSV latent infection die as a result of the reactivation, or whether they can survive. As the authors point out, there are publications that support both possibilities. In particular, explant-induced reactivation does not tend to show robust evidence for neuronal death as a direct result of reactivation. This is the first study to take this question on looking at reactivation in vivo. The study is very well-designed and carefully performed. Because reactivation is a relatively rare event, large cohorts of mice were examined to allow statistically defendable conclusions to be drawn. This is an outstanding study and one that will be highly cited and have a large impact on the field.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. 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The aim of our study was to compare the success rates of suture selection, recovery times and pain associated with local wound infection and seton placement in patients undergoing cutting seton placement for complex anal fistula.The study included a total of 90 patients who were admitted with the diagnosis of complex anal fistula between January 2015 and July 2018.The first session and other revision appointments demonstrated that the number of patients who required fistulotomy was significantly higher in group-1 as the seton failed to complete the transection (p = 0.001). When the patients were asked to rate pain for 3 different conditions according to numeric rating scale (NRS), the patients in group-2 had significantly higher pain in all 3 cases compared to the patients in group-1 (p 0.001). The impact of the suture material on local infection was examined and it was determined that the results of cultures for seton material were significantly more positive in group-1 (p = 0.001).We conclude that a multi-stage tight seton placement with silk material can lead to satisfactory results by aiming to shorten the cutting time of silk seton. Anal fistula is a surgical condition which can be defined as chronic abnormal communication between the epithelialised surface of the anal canal and the perianal skin. Although fistula is detected in 0.71% of patients in the colonoscopy series, the main diagnosis is usually made by anamnesis and physical examination.The study included a total of 90 patients who were admitted to the General Surgery Clinic of Afyon Medical Sciences University with the diagnosis of complex anal fistula between January 2015 and July 2018. An approval  was obtained from the Ethics Committee for the study. Informed consent was obtained from all patients included in the study group. The exclusion criteria included patients undergoing previous surgeries due to anal fistula, patients with abscesses, patients with inflammatory bowel disease and patients with multiple fistulas. The patients were randomized by the surgeon according to the Parks classification using a magnetic resonance imaging method. The patients undergoing 0 silk seton placement were classified as group-1 and the patients undergoing 0 polypropylene suture placement were classified as group-2. The patient demographics, surgery and recurrence data, follow-up time, seton fall-out time and surgical wound infection were recorded in the pre-designed information forms and electronic medical records.All patients were administered laxative enema to have an empty bowel before surgery. 1g of cefuroxime axetil was applied intravenously for surgical prophylaxis because of the contaminated wound according to ACS-NSQIP surgical wound classification. The patients were operated under general or spinal anesthesia in the lithotomy position. The anus was opened with the help of anoscope and hydrogen peroxide was administered through the external opening. After its exposure, the external opening of the fistula tract was then gently probed till the internal opening, at this time, the length of the fistula tract was measured over the probe wire. The fistula tract up to the sphincter was excised and curated. The suture was then attached to the probe and passed through the sphincter inserting a tight seton using 0 silk suture in group-1 patients and 0 polypropylene suture in group two patients. All surgical procedures were performed by the same surgical team with the same surgical technique. In the postoperative period, patients who had no complications due to surgery or anesthesia were discharged on the following day. They were told to have a sit bath with ethacridine lactate (rivanol sashe) after each defecation and were scheduled for follow-up at 1 month intervals.At the first follow-up appointment, a 1 cm piece from the end of the seton materials was dissected and sent for anaerobic culture and the e-coli colonies in culture results were compared by the number of colonies per millilitre (colony-forming unit/millilitre (cfu/mL)). Culture results of 100 000 cfu/mL and above were evaluated as positive and 5000 cfu/mL and below as negative.During the 1-month follow-up period, the absence of reduced discharge or spontaneous seton fall-out were defined as non-healing. Fistulotomy was performed in patients with up to a 1cm of tissue remaining after seton transection, whereas seton revision was performed for tightening of the seton in other patients. In addition, they were advised to apply to the service immediately if the seton fell out. During the 1-month follow-up period, spontaneous seton fall-out or disappeared discharge after fistulotomy were defined as healing. The re-diagnosis of anal fistula in non-symptomatic healed patients was defined as recurrence.The patients were asked to rate their pain sensations by using the numeric rating scale (NRS). The pain during daily activities, defecation and rest was noted separately. The scale was performed one month after surgery at the first follow-up appointment with the aim to distinguish the pain associated with seton placement from early postoperative pain.The data were analyzed using the Statistical Package for the Social Sciences 21  The variables were presented as mean, minimum-maximum and percentage. A value of p<0.05 was considered statistically significant.The study consisted of 90 patients who underwent a tight-cutting seton placement following the diagnosis of complex anal fistula. In Group-1, 11 (24.4%) of the patients were female and 34 (75.6%) were male. The mean age was 45.5 ± 10.2. 37 (82.2%) patients had transsphincteric fistula, 7 (15.5%) had suprasfinkteric fistula and 1 (2.2%) patient had extrasphincteric fistula.In Group-2, 12 patients were female (26.6%) and 33 (73.4%) were male. The mean age was 46.7 ± 10.9. 38 (84.4%) patients had transsphincteric fistula, 6 (13.3%) patients had suprasfincteric fistula and 1 (2.2%) patient had extrasphincteric fistula. There was no statistically significant difference in the mean age, gender, and distribution of patients according to fistula classification between the groups (p> 0.05). The duration of operation was determined as 22.1 ± 5.2 minutes in group-1 and 23.4 ± 4.3 minutes in group-2 after the end of anesthesia preparation (p> 0.05). The length of hospital stay was 1 day in 83 patients (92.2%). Of 7 patients with a hospital stay longer than one day, four patients were followed up for pain palliation and 3 patients were followed up for additional diseases  resulting in a prolonged hospital stay.The comparison of the groups according to healing and non-healing status revealed that the number of healed patients with spontaneous seton fall-out in a single seton session were similar in both groups (p> 0.05). In addition, the first session and other revision appointments demonstrated that the number of patients who required fistulotomy was significantly higher in group-1 as the seton failed to complete the transection (p = 0.001). The number of operations required for healing was also significantly higher in group-1 patients using silk seton than group-2 patients using polypropylene seton (p = 0.001). The mean duration of seton placement showed no significant difference between the patients (p> 0.05). There was no significant difference in the number of recurrence (p> 0.05). The mean follow-up time was 24.2 months. When the patients were asked to rate pain for 3 different conditions according to NRS, the patients in group-2 had significantly higher pain in all 3 cases compared to the patients in group-1 (p 0.001). The impact of the suture material on local infection was examined and it was determined that the results of cultures for seton material were significantly more positive in group-1 patients than in group-2 patients (p = 0.001). The number of negative cultures was significantly higher in group-2 patients (p = 0.001). In group-1, 12 (26.6%) patients had local wound infection accompanied by hyperemia and purulent discharge at the wound site, whereas this number was 10 (22.2%) in group-2. However, there was no statistically significant difference (p> 0.05). Setons are mostly used either as a loose seton or as a tighter cutting seton. Loose seton placement is usually performed for drainage in order to prevent the obstruction of the fistula tract due to chronic inflammatory diseases in the anal region and inflammatory bowel diseases. The perianal fistulas caused by other reasons constitute the majority of patients and they are treated with cutting seton. The correct selection of seton materials is important to ensure the quality of life of patients at a sustainable level and provide healing with a minimum number of operations possible. Silk and nylon sutures, metal wire, elastic band, penrose drain, pieces of surgical gloves, plastic clamps are among the seton materials reported to be used in the literature, but the majority prefer surgical sutures.21Although there was no significant difference in the outcomes, it was determined that the number of patients healed in a single session was similar in both groups using silk and PP sutures, however, the number of patients requiring multiple sessions was higher in the silk suture group. In addition, it was observed that the requirement for fistulotomy was quite high in most patients undergoing silk seton placement as it failed to transect after a certain level. It was also demonstrated that the pain and discomfort were significantly higher in group-2 patients than in group-1 even though the PP material provided a better cut. The results revealed that PP seton caused a significantly higher amount of pain compared to silk seton especially during daily activities, which was persistent even during defecation and rest.Banche et al.In the literature, there are no studies showing the effect of suture materials on bacterial adhesion in the perianal region. Consistent with the study of Banche et al., our study revealed significantly higher e-coli colonization in the silk seton group. We think that it is because the polypropylene and irregular surface of the silk yarn facilitates bacterial colonization. The wound site was similar in both groups unlike the culture results with regard to the local wound infection and discharge in patients undergoing silk seton and PP seton placement. We see that silk seton is predisposed to bacterial colonization but it is not reflected in the clinical outcomes.The limitations of our study are that the sample size is not large enough and the groups are not randomized. In addition, evaluation of only e-coli colonization in culture results shows fecal contamination adequately, but for optimal results, it is necessary to conduct studies considering other pathogens.It has been concluded that patients using silk sutures undergo a greater number of surgeries averagely, which results in a longer duration of seton placement. However, it has been observed that the level of comfort is much higher in the silk group than in the PP group. There was no difference in wound infection and recurrence between the two groups. In the light of these data, it may be appropriate to say that patients undergoing silk seton placement may need longer and repetitive procedures, whereas patients undergoing PP suture placement may experience persistent pain and discomfort in the following postoperative days. We think that a multi-stage tight seton placement with silk material can lead to satisfactory results by aiming to shorten the cutting time of silk seton.MA: Conceived, designed, did statistical analysis & editing of manuscript.MA, OE: Did data collection and manuscript writing.MA, OE: Did review and final approval of manuscript.MA: Is responsible for the accuracy and integrity of the work.


A transition at age 25 to the hypothetical dietary guideline diet reduces cumulative warming associated with diet by 7 to 9% at the 100th year compared with consuming the average New Zealand diet. The reduction in diet-related cumulative warming from the transition to a hypothetical meat-substituted diet varied between 12 and 15%. This is equivalent to reducing an average individual’s lifetime warming contribution by 2 to 4%. General improvements are achieved for nutrient intakes by adopting the dietary guidelines compared with the average New Zealand diet; however, the substitution of meat items results in characteristic nutrient differences, and these differences must be considered alongside changes in emission profiles.Dietary transitions, such as eliminating meat consumption, have been proposed as one way to reduce the climate impact of the global and regional food systems. However, it should be ensured that replacement diets are indeed nutritious and that climate benefits are accurately accounted for. This study uses New Zealand food consumption as a case study for exploring the cumulative climate impact of adopting the national dietary guidelines and the substitution of meat from hypothetical diets. The new GWP* metric is used as it was designed to better reflect the climate impacts of the release of methane than the  This et al.  has repoThere is ongoing research exploring potential solutions to the nutritional and sustainability issues that challenge the global food system. A substantial field of research has focused on consumption aspects, particularly in developed regions and, generally, replacing animal-source foods with plant-source alternatives as one way to reduce the food systems contribution to climate change –15, alonde facto standard in literature and explores the radiative contribution associated with the production life cycle of different food items [2), it does not clearly reveal the cumulative impacts of ongoing emissions of short-lived GHGs, such as methane (CH4) [4 emission component for some food items, particularly ruminant animal-sourced foods and rice [4, given its relatively short lifetime in the atmosphere, in addition to stock impacts related to slow climatic response [et al. [A key methodological limitation quantifying climate change impacts is in the accurate representation of atmospheric warming through the, so-called, GHG emission ‘metrics’; this contention has been the subject of ongoing debate –26. Currod items ,29 and mod items ,31. Whilne (CH4) –34. Thisand rice . A new mresponse –34. Therresponse  has appl [et al.  to dieta4 [Although global studies can provide general guidelines, it is important to explore region-specific nuances, as local food systems and societies have particular characteristics to appreciate. For example, New Zealand makes for an interesting case study to test global observations, as it is generally thought to have more efficient and less GHG emission intensive food production , with an4 . The New4 , and rec4 . This st4  highligh4 contributes to dietary climate impact, and contrasts this with the de facto standard GWP100. To the best knowledge of the authors, this represents the first study to use GWP* to explore the cumulative climate impact of dietary transitions. This work explores caveats for how different diets are compared  and what happens when hypothetical diets have significantly different GHG compositions . Context is provided by examining how the atmospheric warming impacts of dietary change compared with an individual’s wider consumption profile, how an individual’s dietary profile is related to a larger collective .In this study we explore the mitigation potential of dietary change in New Zealand, by analysing the cumulative atmospheric warming impact of one individual adopting the national dietary guidelines and the substitution of meat. The nutrient benefits and trade-offs of the dietary transition are also explored. This work uses the GWP* metric to explore how the warming impact of CH2Full methodological details for this study is provided in the appendix. In summary, the diet framework used in this work is based on the New Zealand Total Diet Study ,42, and 4 from the production and processing stages to so-called ‘methane-intensive’ foods, data from the original reference as listed in Drew, Cleghorn, Macmillan and Mizdrak [4 proportion which were not accounted for by the ‘methane-intensive’ foods from the Drew et al. 2020 database, or when the original literature was not accessible, as is the case for rice. Where applicable, this database also allowed use of the IPCC Assessment Report 5 GWP100, without carbon-climate feedback equivalence values [Life cycle assessment (LCA) data used in this study are based on the database accumulated by , with so Mizdrak , or as p Mizdrak . To conv Mizdrak  and US [ Mizdrak  data fro Mizdrak  global Le values  for conse values  were useth generation’s direct family line and for each successive generation after the 1st generation, the generational responsibility changes due to the requirement of partners to generate offspring  and long-lived GHG categories . Using these values, the cumulative climate impact was estimated using the GWP100 metric for long-lived GHGs and GWP* metric for the short-lived CH4. These metrics used the IPCC 2013 equivalence values without climate-carbon feedbacks, which correspond to 28 for CH4 and 265 for N2O. The GWP* metric is used with the recommended values [4 temperature response over Representative Concentration Pathways 2.6 to 6. The GWP* equation calculates CO2 warming equivalents (CO2we) and is used here is as follows: r equals the weighting for flow rate (0.75), 4ΔECH equals the difference in emissions of CH4 over the timescale of flow, t equals the timescale of flow in years (20), H is the time horizon corresponding to the GWP used (in this case100-years), s equals the weighting for stock (0.25), 4CHE is the emissions of CH4, and GWP100 is the 100-year Global Warming Potential of CH4.Annual emissions were calculated based on the consumption of a given diet for short- , consistent with literature reporting the positive relationship between meat and dietary emissions [4 in daily emission values .A summary of daily values for the diets explored here is provided in missions –15. Thes4 are explored . After this time, the contribution from the stock-term decreases, particularly after the 19 to 30-year life stage, when the annual energy consumption and the annual emission of CH4 begin to decrease. Throughout the modelled lifetime, the CO2 and N2O emission proportions are directly related to the annual energy consumption and the cumulative emission value for these gases undergoes a close to linear increase from birth to death.By applying the transition lifetime modelling scenario, the short- and long-term behaviours of CHexplored . Over th2we in the 50th and 100th years, respectively. For the transitions, divergence in the cumulative emission CO2we values is observed immediately after the 25th year. For the →DG diet transition this divergence involves a total of 123 and 130 cumulative tonnes of CO2we in the 50th and 100th years, respectively; a reduction of 6 and 7% compared with the Current diet, respectively. By comparison, for the →DG -Meat diet transition, this results in 108 and 119 cumulative tonnes of CO2we in the 50th and 100th years, respectively; a reduction of 17 and 15% compared with the Current diet, respectively, or 12 and 8% relative to the →DG diet. The variation in cumulative warming between scenarios slowly decreases over time and, on death of the modelled individual, there is a rapid reduction in the absolute difference and a small reduction in the relative difference between all diet scenarios , the cumulative impact reaches a plateau or a long-term limit that equals the cumulative total GWP100 values for CO2, N2O and 0.25x the cumulative total GWP100 value for CH4.In cenarios . This re4 on atmospheric warming are not clearly accounted for. Specifically, because it is a short-lived gas, the warming caused by CH4 is primarily determined by recent emissions, in contrast to the long-term cumulative effects from CO2 and N2O. Despite these relative changes, the order of climate impact remains the same: Current diet > →DG diet transition > →DG -Meat diet transition.When comparing the results from the daily GWP100 values to the lifetime modelling using both GWP100 and GWP*, two considerations arise . FirstlyWhile this analysis and similar analyses in the literature, such as Drew, Cleghorn, Macmillan and Mizdrak , have a 3.1To test and explore the assumptions and data associated with dietary modelling, several additional analyses were undertaken. These include compensating diets using protein, different proportions of short- and long-lived GHGs and considering how the results compare if the modelling is extended from an individual perspective to a collective or family perspective. To infer that, New Zealand can be an outlier in the global context due to relatively high production efficiencies (GHG/kg food produced), an additional analysis using median global LCA data is also carried out. However, due to limitations for comparisons between global and New Zealand LCA data, this has been included in the appendix for reference purposes only.3.1.1It is common practice in related literature to compensate diets based on their energy prior to comparison ,14,18, a2we in the 50th and 100th years, respectively. The →DG diet transition involves a total of 112 and 119 cumulative tonnes of CO2we in the 50th and 100th years, respectively: a reduction of 8 and 9% compared with the Current diet, respectively. By comparison, for the →DG -Meat diet transition this involves 105 and 116 cumulative tonnes of CO2we in the 50th and 100th years, respectively: a reduction of 14 and 12% compared with the Current diet, respectively, and 6 and 3% relative to the →DG diet transition, respectively. The changing of relative differences for the two transition scenarios arises due to an increased (→DG) or decreased (→DG -Meat) bioavailable protein density per kg CO2we for the diet when compared with the current diet. Despite these differences, the order of climate impact remains the same when using energy and protein compensation: Current diet > →DG diet transition > →DG -Meat diet transition; but, the effect of transitioning to the meat-free diet is more than 50% smaller than when considered on an energy basis.With protein compensation, the Current diet involves a total of 122 and 132 cumulative tonnes of COIt is notable that when compensating by protein, the Current and DG and DG -Meat diet amount to total energy values of 2211, 2152 and 2360 Kcal per day, respectively, to achieve an equivalent bioavailable protein value of 83 g per day. The above analysis highlights that there is a potential impact of using energy compensation on both the overall climate impact, but also for the oversupply of energy in the DG -meat diet relative the Current or DG diets, where it is important to maintain a constant protein consumption.3.1.24. In addition, it is likely that the allocation of meat items in an individual’s actual diet may vary considerably from those in the baseline average adult diet. This may be exacerbated by individual preferences, including those formed in accordance with recommendations made in literature, which encourage or discourage particular types of meat  [Given that the production of different meat items can emit different amounts and proportions of short- and long-lived GHGs, meat choice can serve as a good case study for contrasting the warming impacts of diets with significantly different proportions of CHd meats) . For thi4 and CO2, where the higher GWP100 value and CH4 proportion for beef is offset over time by the primarily CO2 and N2O emissions associated with pork.With meat allocation to either beef or pork, the cumulative impact differs from the baseline →DG diet transition. An interesting observation is that, in the short-term, the allocation to pork reduces the cumulative impact (6% at year 50), while in the long-term, the cumulative impact is increased for this diet (5% at year 100) compared with the baseline. In contrast, transition to beef has an increased short-term cumulative impact (15% at year 50), but no effect over the 100-year term (<1% at year 100) compared with the baseline. In other words, a transition to pork has a lower impact over an individual’s lifetime compared with a transition to beef; however, the long-term impact of the transition to beef is removed soon after consumption stops, while much of the long-term impact of a transition to pork remains indefinitely. Importantly, this behaviour is not reflected by the daily GWP100 values of DG Beef and DG Pork diets of 6.19 and 5.75 Kg CO2e per day, respectively, which would imply a reduced impact for the →DG Pork diet transition over the →DG Beef diet transition. These hypothetical diet transitions are good examples of the different climate impacts of CH4 and CO2, respectively. Failing to do this can risk causing unintended perverse outcomes, such as increased cumulative warming impact, particularly in the longer-term. To alleviate the uncertainty associated with the overall GWP100 values indicated above, an estimation of the long-term impact of CH4 from an individual’s food can be appreciated using the stock GWP* term (0.25 × GWP1004CH). By combining this stock term contribution and the total CO2 and N2O GWP100 values, a long-term index to compare foods on an estimated long-term impact can be allocated. This index correlated with the GWP100 values can differentiate the short- and long-term impacts of different foods  is that the dynamics and interpretations are not the same for a collective or family perspective. As an intermediate step towards a broader perspective, a generational dietary emission impact can also be considered. In this case, the responsibility of descendants is allocated to the direct family line  would experience substantial warming from CH44.11 averaged for the years 2011 to 2017 [4), an individual’s deliberate dietary shift to a the hypothetical DG or DG -Meat diets may decrease an individual’s consumption-based total warming footprint by 2 to 4% (depending on energy or protein compensation) at year 100. For context, the transition to the hypothetical DG or DG -Meat diet would equate to a reduction in ‘transport’ or ‘housing and household utility’ consumption-based emissions of around 5 to 11% or 14 to 29%, respectively [2we) in at age 25 would equate to avoiding around three return international flights (3.5 t CO2 per return) from Auckland, New Zealand to London Heathrow, United Kingdom [2we) would equate to avoiding around six. As the emissions context is highly dependent on the individual, it remains unclear how actual reductions in dietary emissions may compare with potential mitigation options in the other emission areas.When considering dietary emissions at an individual level it is useful to ground these in the broader individual emission context. In New Zealand the estimated proportion of household consumption-based emissions for ‘food and non-alcoholic beverages’ is around 26% to 2017 . Using tectively . Another Kingdom  over a lUnderstanding the climate change impacts of food production is an important part of wholistic sustainability approaches; however, the myriad of other environmental issues is also relevant. For example, LCA reviews have highlighted the various environmental impact categories that have been used to compare across different food items and food groups ,29. Thes4.24 emissions decreasing to near zero after the death of the individual. This results in a smaller absolute difference between the hypothetical diet transitions and Current diet at 100 years when compared with a daily GWP100 value or the cumulative GWP100 values. In simple terms, CO2 and N2O emissions accumulate over the long-term to become the dominant factor of the cumulative emission CO2we values. In the long run, an individual’s warming contributions are likely to be dominated by their CO2 emissions and dietary transitions evaluated in this study may allow a reduction of 2 to 4% to this total, based on New Zealand consumption-based emission patterns [4 emissions can constitute a major component of the cumulative emission CO2we values in the short-term, particularly from year 0 to around year 45. Both points relate to the ongoing discussion of how best to prioritise mitigation of short-, medium- and long-term climate impacts [The GWP* results highlight interesting dynamics, such as the cumulative warming impacts of the dietary CHpatterns . However impacts .4 emissions per unit of product will continue to reduce CH4 by this proportion. Due to the dominant impact of the flow term of GWP*, deviations from these assumed trends will have a strong impact on the estimate of dietary emissions. By contrast, this work used GWP* to explore the cumulative warming profile of an individual’s lifetime . This approach captures the strong short-term warming, when atmosphericAn important distinction should be made between this study and the recent application of GWP* to dietary emissions by Ridoutt, Baird and Hendrie . The Rid4 increase, neutral warming impacts as emission intensities are roughly stable, and the decrease in warming once emission intensity decreases or stops. These two unique applications highlight how GWP* is flexible in how it can be applied and that it can be used to provide different information .concentrations of CH4.3The key limitation of the modelling undertaken, is the application of one or two dietary composition throughout different life stages using consumption profiles, given that actual diet compositions change over time. While this approach provides a satisfactory approximation of dietary emission over various life stages , more deThe New Zealand Adult and Child Nutrition Surveys ,51, whicLCA data both globally and specific to New Zealand are limited. While works by Poore and Nemecek  and DrewDespite efforts to standardise ,67,71, cThe proportion of food wasted is poorly quantified. Due to the lack of New Zealand-specific waste data at a product-level the UK Waste and Resources Action Programme data  were useIt is acknowledged that various aspects included in this modelling are expected to show temporal trends. For example, agricultural efficiency increases larger than 1% per year were observed globally for the time period between 1990 to 2015 , therefo52e values). In this study, dietary guidelines are used to improve the average diet  and the climate impact of substituting meat  is explored for a consumer of New Zealand-produced meat. A transition at age 25 to the hypothetical DG diet reduces the cumulative warming associated with food consumption by 7 to 9% at the 100th year compared with consuming the average New Zealand diet. The reduction in cumulative warming associated with food consumption from the transition to a hypothetical meat-substituted diet varied between 12 and 15%. Using New Zealand consumption-based emission estimates as context, transitions to these hypothetical diets may reduce an average individual’s lifetime warming contribution from consumption-based activities by 2 to 4%. In addition to GWP*, the de facto GWP100 metric was also used and, regardless of methodology, the order of climate impact for the scenarios explored remained the same.There is significant interest for using dietary transitions to improve the nutrient and climate outcomes of the food system. Care is needed to ensure replacement diets are indeed nutritious and that climate benefits are adequately accounted for. Diet-related GHG emissions are an important part of an individual’s carbon footprint and, as many recent works have highlighted, they can be lowered by eliminating meat in the diet. However, the stock and flow behaviour of how dietary emissions contribute to warming over time is not always clear in these studies  different GHGs are prioritised for reduction or offsetting, as well as for improving the understanding of the long-term climate sustainability of the food system in the broader context of national and global emission accounting.While CHImportantly, there are general improvements in nutrient intake by adopting the dietary guidelines compared with the average New Zealand diet. However, the DG and DG-Meat diets are not nutrient equivalent as substituting meat results in characteristic nutrient differences. This work emphasises the need to ensure dietary transitions are sustainable from both nutritional and climate perspectives.Further work in this area should seek to understand nutrient considerations more-completely , account for developing areas related to the climate change impact category (particularly related to land-use) and consider a broader range of sustainability indicators (such as affordability). In addition, there is an opportunity to explore how individual actions and impacts estimated with GWP* compare and contrast with collective-level outcomes where factors such as population dynamics become important.Supplementary Material


Background: Since cone-beam computed tomography (CBCT) technology has been widely adopted in orthodontics, multiple attempts have been made to devise techniques for mandibular segmentation and 3D superimposition. Unfortunately, as the software utilized in these methods are not specifically designed for orthodontics, complex procedures are often necessary to analyze each case. Thus, this study aimed to establish an orthodontist-friendly protocol for segmenting the mandible from CBCT images that maintains access to the internal anatomic structures. Methods: The “sculpting tool” in the Dolphin 3D Imaging software was used for segmentation. The segmented mandible images were saved as STL files for volume matching in the 3D Slicer to validate the repeatability of the current protocol and were exported as DICOM files for internal structure analysis and voxel-based superimposition. Results: The mandibles of all tested CBCT datasets were successfully segmented. The volume matching analysis showed high consistency between two independent segmentations for each mandible. The intraclass correlation coefficient (ICC) analysis on 20 additional CBCT mandibular segmentations further demonstrated the high consistency of the current protocol. Moreover, all of the anatomical structures for superimposition identified by the American Board of Orthodontics were found in the voxel-based superimposition, demonstrating the ability to conduct precise internal structure analyses with the segmented images. Conclusion: An efficient and precise protocol to segment the mandible while retaining access to the internal structures was developed on the basis of CBCT images. Two-dimensional 2D) radiographs have been widely used in the field of orthodontics since 1922 D radiogr,4,5,6,7.As the only bone that connects to the cranium by unique temporomandibular joints and exhibits variable growth and remodeling responses to orthodontic treatment, the mandible has been one of the classic and central topics for evaluating patients’ growth and development and assessing the influence of orthodontic and orthopedic treatments. However, the most commonly cited method of investigating the mandible today is still 2D cephalometric radiograph-based superimposition . There iwww.itksnap.org) and 3D Slicer (www.slicer.org) or licensure-required software such as Mimics  [Indeed, multiple attempts at mandibular segmentation and superimposition based on 3D CBCT imaging have been reported ,15,16,17ise, NV) ,19,20. STo overcome the technical, time-consuming, and financial challenges thus far associated with evaluations of the mandible, it is necessary to establish an efficient and precise mandibular segmentation protocol based on 3D CBCT that can (1) be performed with commonly used orthodontic imaging analysis software, (2) be easy to follow (not technique-sensitive), (3) access the internal structures, and (4) allow novel 3D and traditional 2D superimpositions.CBCT scans for this study were derived from preexisting clinical databases and approved by the University of Pennsylvania Institutional Review Board (IRB protocol #843611). No additional CBCT images were taken for the current study. Five CBCT scans from 3 patients were selected on the basis of the following criteria:Patient #1: An adult female patient without craniofacial syndromes who underwent orthodontic treatment. The initial  and final  records were selected to(1)establish the initial mandibular segmentation protocol to exclude the potential influence of gross pathologies and significant skeletal asymmetry;(2)perform and validate the 3D voxel-based superimposition  with no Patient #2: A late adolescent female patient diagnosed with hemifacial microsomia. The initial record  was selected to validate the precision and efficiency of the current protocol to segment the mandible, which presented a significant amount of asymmetry.Patient #3: An adolescent male patient without craniofacial syndromes who underwent orthodontic treatment. The initial  and progress  records were selected to(1)further validate the current protocol to segment the mandible;(2)perform 3D voxel-based superimposition to determine the sensitivity of the current segmentation and superimposition protocol for detecting mandibular growth and development in the one-year interval;(3)observe the potential growth and remodeling trends of the mandible.(1)In the “Orientation” module, set the facial midline as the midline of the full-volume CBCT and ensure that the cranial base structures and key ridges on the left and right sides overlap. This step is the same as the orientation setting for routine craniofacial CBCT assessments.(2)In the “Sculpting tool” module, orient the CBCT to the right view, and sculpt the majority of the cranial and maxilla structures with the option “free form”, as shown in (3)Orient the CBCT to the bottom view in which the borders of the condyles are clearly visible. Sculpt the visible cranial and maxillary structures, as shown in (4)Orient the CBCT to the right oblique view E, enlarg(5)Repeat step 4 for the left condyle with the CBCT oriented to the left oblique view.(6)Return to the right oblique view, in which some remaining cranial structures can be found around the left condyle I.(7)Repeat step 6 for the right condyle with the CBCT oriented to the left oblique view.(8)The segmented mandible CBCT is ready for either “Export” as DICOM files or “Create surface,” which yields an STL file J.CBCT DICOM files were all imported into the Dolphin 3D software . The mandible was segmented from each CBCT with the following steps:www.slicer.org) to evaluate intra-examiner and inter-examiner reproducibility, as well as the reliability of the current protocol using the “Model-to-Model distance” module [Each CBCT was segmented by the same examiner twice, with a 1-week interval in between. Each CBCT was also independently segmented by another examiner. The 3 STL files of each mandible were imported into 3D Slicer  and green  interval surface colors in the mandibular body and ramus regions. In contrast, only the green color was observed in the lower anterior dentition, indicating no significant change of the mandible, while there was proclination of the mandibular anterior teeth caused by orthodontic treatment A. To furUnlike the observations from patient #1, the Dolphin 3D voxel-based superimposition of patient #3 showed that the majority of the mandibular surface was green, indicating the potential growth of the mandible from the initial (white) to progress (green) time points A. Again,For this short Communication article, our primary aim was to establish a simple protocol to segment the mandible from a full-volume craniofacial CBCT using the “Sculpting tool” provided with the Dolphin 3D Imaging software and to evaluate the precision and reliability of this protocol. In contrast to other newly established mandibular segmentation protocols, the current protocol only involves one commonly used orthodontic imaging software, which reduces the cost, time, and energy of purchasing software, multiple-software practicing, and file transfers among different software ,16,17,22The 3D Slicer model-to-model distance analysis demonstrates perfect volume matching of the mandibular body, ramus, and condylar regions in all five tested samples. This consistency confirms the precision and reliability of the current protocol, which is not affected by notable mandibular asymmetry, such as seen in patient #2.We also performed superimpositions using the chin and symphyseal regions  with DolAs demonstrated in the superimposition image of patient #3, we found that specifically for this patient (1) the anterior–inferior contour of the chin and the inner contour of the cortical plate at the lower border of the symphysis can be entirely superimposed for the initial and progress records, (2) there is deposition at the inferior border of the mandible at the first premolar region but not at the third molar region, and (3) the ramus demonstrates anterior border resorption and posterior border deposition. These observations align with the previously reported and accepted phenomena of mandibular growth and modeling . Interestingly, some previously unreported growth trends were also found in this patient’s records, such as the deposition along the buccal surface of the mandible A,D–G andIn fact, in the newly published article , by perfUsing the current protocol, we could not thoroughly remove the maxillary dentition without affecting the mandibular dentition. Thus, we decided to sculpt through half of the maxillary tooth crowns. Although the remaining maxillary dentition did not appear to influence the mandible analyses, further efforts are still needed to adequately separate the maxillary and mandibular dentitions.CBCT has also been proven to be an appropriate tool for evaluating the maxillary and mandibular bone marrow density ,25,26 anNo doubt, Dolphin 3D software is not inexpensive. However, we created this protocol based on Dolphin 3D because this software is not only routinely used in our clinic but is also widely utilized in graduate orthodontic programs. According to the PubMed database, an increasing number of researchers are employing Dolphin 3D software in their studies encompassing diverse areas of investigation, such as predicting the upper airway change in orthognathic patients , assistiLast but not least, the current study is a retrospective study using human data previously obtained for clinical treatment purposes. No additional radiographs were taken for the present study. Although technological developments have significantly reduced the radiation dosage associated with CBCT acquisition , we highIn summary, a user-friendly 3D-imaging-based mandibular segmentation protocol is introduced in the current study, with five CBCT images from three patients selected to preliminarily demonstrate the feasibility of the protocol. Without a doubt, many more samples are needed to draw definitive conclusions in growth and development investigations and assess treatment efficacy. However, by disseminating our protocol in a timely manner, we hope that this sharing of knowledge can significantly benefit global collaborative efforts to achieve a more detailed understanding of how to better evaluate growth and development and ultimately improve one’s ability to deliver better orthodontic clinical care.