Brd2 haploinsufficiency extends lifespan and healthspan in C57B6/J mice

Shilpa Pathak; William C. L. Stewart; Christin E. Burd; Mark E. Hester; David A. Greenberg

doi:10.1371/journal.pone.0234910

Peer Review History

Original SubmissionNovember 19, 2019
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Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this manuscript, the authors observed that reduced Brd2 expression benefits for extending lifespan and healthspan of C57B6/J mice. Despite Brd2 haploinsufficient mice have been generated by several groups in different ways, the pro-longevity phenotype in HET mice has never been identified. The authors also concerned the downstream effectors, e.g. Sirt1, HO-1, and P53, were upregulated in Brd2 HET tissues. These genes are apparently cytoprotective and longevity-related, partially explaining the pronounced role of Brd2 haploinsufficiency in longevity. However, there are a number of issues the authors need to address before publication. In the second paragraph, the authors introduced Cisd2, a well-known longevity gene, prolongs the lifespan of mice to the extent that Brd2 haploinsufficiency does. Does the authors aim to exclude the benefits of Brd2 HET from Cisd2’s role? Are there some functional overlaps between these two proteins? However, I could not find any test on Cisd2 function in Brd2 HET mice. Since the authors’ observation is so distinct to the other groups based on their mouse models, please provide the generating strategy and characterization of the Brd2 HET mice, e.g. scheme of gene trap and Brd2 protein expression profile in various tissues. Again please discuss a bit more the reason why your mouse models are phenotypically different to other groups. In supplementary Fig2, the author showed the bodyweight was unchanged in Brd2 HET mice when comparing to WT. However, it has been reported that Brd2 disruption in mice causes severe obesity. Please discus this difference. In addition to kidney, the authors also definitely examined the pathologies in other organs, like liver, spleen and testis (data not shown). I suggest the authors provide these data to strengthen the conclusion that Brd2 HET improves mouse healthspan. In this manuscript, the authors provided large descriptive information on mouse aging phenotype, yet no evidence. For example, in line 262-271, the whole paragraph demonstrated the observation of symptoms of aging in WT and HET mice without any data. The authors should provide some scientific evidence, like the pictures of aged WT and HET mice, quantifications of mouse activities, or the movies on mouse behavior … Please label the medium lifespan of mice in figure 1. In Result section, there is no data description of Figure 2C. Reviewer #2: This paper describes a novel discovery, that reduced expression of Brd2 leads to increased lifespan, which is associated with reduced pathology and improved health. Although this is an important observation, I have the following major concerns with the manuscript: 1. The increase in lifespan is impressive and is the key to this paper. However, the lifespan reported for the WT female and male mice (which are in the C57BL/6 background) of 23 and 26 months is very short compared to other reports (e.g., see data from Jackson Laboratory, de Cabo, and Richardson) for C57BL/6 mice. The lifespans the authors obtain with the Brd2 HET mice are actually about what is observed with normal WT mice. This is a major concern because the increased lifespan could arise from making the mice more robust to the conditions that gave rise to the shorter lifespan of the WT mice rather than retarding aging. This has been shown in previous studies, which reported increased lifespan when the median/mean lifespan of the WT mice was less thatn 26 months of age, and the increased lifespan was not replicated when conducted in colonies where the WT mice had a median/mean lifespan of over 29 months. 2. A great deal of emphasis was placed on the pathological data. The major cause of death in C57BL/6 mice is lymphoma in most aging colonies; however, the data on cancer was lumped together as tumors, i.e., it was not clear what the neoplastic lesions were in the mice. In addition, renal pathology appears to be a major problem in the mice in this study, which is in contrast to most other studies on aging in C57BL/6 mice, where renal pathology is relatively minor. 3. The data on the mice being ‘biologically’ younger was weak. For example, a great deal of emphasis was placed on the epigenetic clock. However, the epigenetic clock is a measure of chronological age, not physiological age. In addition, these data were generated with only 4 mice per group and no data were given of the animal to animal variation in data (e.g., SD or SEM). The data on observational health of the animals on page 13 was unconvincing. Data on physiological functions such as grip strength, activity, rotarod performance, and cognition would have been stronger evidence that the HET mice were physiologically younger. Minor Concerns: 1. The authors state on page 4 (line 78-77) that there are “only a handful of candidate longevity genes for mice.” This is not correct; there have been at least two dozen different genes identified. The most cited are those that show reduced growth hormone/IGF. The Cisd2 mouse has not been studied greatly from an aging context. 2. In the methods, it was stated that the mice were either allowed to live out their lifespan or euthanized. It would be important to know the number (%) of mice euthanized for the WT and HET mice, i.e., where more WT mice euthanized. 3. Supplement: there is no list of references for the figures in the supplement, and it is not clear what the third figure is all about. 4. The introduction reads more like a discussion. For example, I found very little information about the Brd2 gene in the introduction or anywhere in the manuscript. I would have liked to know what protein this gene codes for and its biochemical/molecular function, etc. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. 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. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0234910.r001
Revision 1
8 May 2020 Author Response Reviewer # 1 In this manuscript, the authors observed that reduced Brd2 expression benefits for extending lifespan and healthspan of C57B6/J mice. Despite Brd2 haploinsufficient mice have been generated by several groups in different ways, the pro-longevity phenotype in HET mice has never been identified. The authors also concerned the downstream effectors, e.g. Sirt1, HO-1, and P53, were upregulated in Brd2 HET tissues. These genes are apparently cytoprotective and longevity-related, partially explaining the pronounced role of Brd2 haploinsufficiency in longevity. However, there are a number of issues the authors need to address before publication. Comment 1: In the second paragraph, the authors introduced Cisd2, a well-known longevity gene, prolongs the lifespan of mice to the extent that Brd2 haploinsufficiency does. Does the authors aim to exclude the benefits of Brd2 HET from Cisd2’s role? Are there some functional overlaps between these two proteins? However, I could not find any test on Cisd2 function in Brd2 HET mice. Response 1: First, our HET mice are identical to C57B6/J at every gene except Brd2, having been back-crossed for at least 10 generations. Thus, there is little or no probability that any meaningful Cisd2 expression differences between WTs and HETs exist. Second, there are no known functional overlaps between Cisd2 (which is chiefly involved in Calcium homeostasis) and Brd2 (which is an epigenetic reader). These points have now been included in the second paragraph of Introduction and Discussion (lines 430-435). Comment 2: Since the authors’ observation is so distinct to the other groups based on their mouse models, please provide the generating strategy and characterization of the Brd2 HET mice, e.g. scheme of gene trap and Brd2 protein expression profile in various tissues. Response 2: Respectfully, we must disagree with the Reviewer’s comment that our results are “so distinct” from other groups. First, Gyruis et al. (2009) (1) used the same ES cell line as we did (RRE050) and confirmed our findings (Shang et al. 2009) (2), including that Brd2-/- mice have impaired CNS development, an observation about which the Wang et al. (2009) (3) paper is silent. Second, Wang et al. used a Brd2 insertion/disrupter (e.g. ES cell line RRT234) different from the one we used or that Gyruis et al used. Third, Wang et al’s principal phenotype was obesity in the mice but neither Gyruis et al nor we observed obesity in our mice. Fourth, and most quizzically, there is residual Brd2 expression in their double knockout mouse, whereas neither Gyruis nor we observed any residual expression. Thus, the phenotype of Gyruis’s mouse and our mouse phenotype are not “distinct” whereas the Wang et al. phenotype differs markedly from our findings and those of Gyruis. As to generating strategy, the genetic engineering and characteristics of Brd2 HET mice has been described in details in Shang et al (2009) (2), and we have added the mouse generation strategy in detail in the revised manuscript (see Materials and Methods, lines 143-151). Gene expression profiles showing (among other things) the reduction of Brd2 expression in HET animals in kidney, liver, heart and mouse embryonic fibroblasts is a part of supplemental information (Supplementary Figure 1). These findings were also replicated by Gyruis et al. (2009)(1). Comment 3: Again, please discuss a bit more the reason why your mouse models are phenotypically different to other groups. Response 3: This comment was addressed above in Response 2 and we have modified our discussion incorporating the genotypic and phenotypic differences related to the different mouse models (Discussion, lines 437-444). Comment 4: In supplementary Fig2, the author showed the bodyweight was unchanged in Brd2 HET mice when comparing to WT. However, it has been reported that Brd2 disruption in mice causes severe obesity. Please discus this difference. Response 4: Again, this comment was addressed in Response 2, and in Discussion. Comment 5: In addition to kidney, the authors also definitely examined the pathologies in other organs, like liver, spleen and testis (data not shown). I suggest the authors provide these data to strengthen the conclusion that Brd2 HET improves mouse health span. Response 5: The histopathology data for liver, spleen and testis has been included in the supplementary information in the revised manuscript. Comment 6: In this manuscript, the authors provided large descriptive information on mouse aging phenotype, yet no evidence.. For example, in line 262-271, the whole paragraph demonstrated the observation of symptoms of aging in WT and HET mice without any data. The authors should provide some scientific evidence, like the pictures of aged WT and HET mice, quantifications of mouse activities, or the movies on mouse behavior … Response 6: We are somewhat surprised by the comment about the acceptability our description of the differences over time between the HET and WT mice. Such descriptive content has long been a staple of scientific publications going back to the very beginning of science journals. Not surprisingly, standard photographs are often inadequate to capture the differences (e.g. subtle differences in grooming and grizzled appearance). Furthermore, while lifetime surveillance of each mouse would permit quantification of differences in maintenance of youthful activity versus continued decline in actively resulting in lethargy, this is impractical. Therefore, the time-honored practice of reporting such observations in the scientific literature are crucial to the understanding of phenotype. As to mouse behavior, those results were published in Chachua et al. 2014 (4). Those studies showed that while HETs and WTs do have similar cognitive functioning, the HETs have decreased anxiety, and are more aggressive than WTs (especially in the case of females). Comment 7: Please label the medium lifespan of mice in figure 1. In Result section, there is no data description of Figure 2C. Response 7: The median lifespan has been labeled in Figure 1, and the Figure 2C has been mentioned in the revised manuscript (Line 350). Reviewer # 2 This paper describes a novel discovery, that reduced expression of Brd2 leads to increased lifespan, which is associated with reduced pathology and improved health. Although this is an important observation, I have the following major concerns with the manuscript: Comment 1A: The increase in lifespan is impressive and is the key to this paper. However, the lifespan reported for the WT female and male mice (which are in the C57BL/6 background) of 23 and 26 months is very short compared to other reports (e.g., see data from Jackson Laboratory, de Cabo, and Richardson) for C57BL/6 mice. Response 1A: In fact, there is no appreciable difference in the median lifespans of our WT males and the Jackson Lab (JAX) C57BL/6J mouse colony. In our longevity tabulations we included all mice that died before adulthood, which Jackson labs does not. Furthermore, there is notable variations in average lifespan of this strain of mice across vivaria, especially differences in deciding when and with what pathologies aging mice should be euthanized (5, 6). Furthermore, although the salient comparison is the one between our HETs and our WTs, it is also true that our HETs live longer (on average) than Jackson Labs C57BL/6J WT mice (p<0.002) (7). This is included in Discussion section in the revised manuscript. (See also the answer to 1C, below.) Comment 1B: The lifespans the authors obtain with the Brd2 HET mice are actually about what is observed with normal WT mice. Response 1B: We think the reviewer is mistaken on this point. 31.5 months (HET median) is 23% longer than 26.5 months (WT median; p<0.0001). Comment 1C: This is a major concern because the increased lifespan could arise from making the mice more robust to the conditions that gave rise to the shorter lifespan of the WT mice rather than retarding aging. This has been shown in previous studies, which reported increased lifespan when the median/mean lifespan of the WT mice was less than 26 months of age, and the increased lifespan was not replicated when conducted in colonies where the WT mice had a median/mean lifespan of over 29 months. Response 1C: There are several reports in the literature of median lifespan for WT approx. 26-27 months, so by comparison, our HET animal with median lifespan of 31-32 months is still quite remarkable. The HET males showed a 19% extension in median lifespan (925 vs 781 days; p<0.0001) but the WT females appear to have shorter lives than the WT males: WT females show a median lifespan of 23 months, and we agree it would be interesting to know how Brd2 haploinsufficiency might compensate for it compared to 26 months for WT males, but we hypothesize that the reason for the difference is not genetic. The median lifespan of our WT males is the same as reported by other vivaria, but the mean figure for WT females is lower than the median lifespan of JAX C57. We hypothesize that the hyper-aggressiveness of the HET females (4), which are more aggressive than even the WT males, leads to increased stress on the WT females, which are housed with the HET females, and that this accounts for the somewhat shorter lifespan of the WT females, which also reduces the overall WT survival rate. We plan to test this hypothesis but currently, such investigations are beyond the scope of the current paper. Moreover, it is highly unlikely that the shortened lifespan of WT females is genetic in origin since our HETs (which were backcrossed for 10 generations with WTs) do not have hidden mutations or deletions (as determined by whole genome sequencing). Comment 2: A great deal of emphasis was placed on the pathological data. The major cause of death in C57BL/6 mice is lymphoma in most aging colonies; however, the data on cancer was lumped together as tumors, i.e., it was not clear what the neoplastic lesions were in the mice. In addition, renal pathology appears to be a major problem in the mice in this study, which is in contrast to most other studies on aging in C57BL/6 mice, where renal pathology is relatively minor. Response 2: Our lab has considerable expertise in the area of kidney and liver pathology, and there is strong evidence in the aging literature (8, 9) that dysmorphic kidney and liver structure (e.g. lesions) is a normal part of the aging process in mice. Therefore, differences in the organ pathology of HET and WT mice are a natural place to start: both from the perspective of documenting what was observed, and from the perspective of generating mechanistic hypotheses to test. For instance, given the abnormal structure of both liver and kidney in WT (Supplementary Figure 3), it is possible that HET animals may have fewer senescent cells than WT. This would be an interesting hypothesis to test in future studies. The overwhelming point, however, is that the HETs show considerably less age-related organ pathology than the WT. While lymphoma is most certainly an important cause of death in C57 (somewhere between 10% and 50% (10), our primary endpoint for this study is lifespan, not cancer. It was only incidentally (ie, at the time of death), that we noticed a difference in HETs and WTs with respect to the presence of solid tumor(s). While determination of the primary tumor type in these animals would be interesting, this is beyond the scope of this current work. Comment 3A: The data on the mice being ‘biologically’ younger was weak. For example, a great deal of emphasis was placed on the epigenetic clock. However, the epigenetic clock is a measure of chronological age, not physiological age. Response 3A: The methylation clock (DNAge) was first demonstrated to be highly correlated with chronological age, and could then be used as a good predictor of biological age (11-15). Furthermore, as we illustrate in Table 1, WT mice of the same chronological age as HET mice (i.e., 770 days) have dramatically higher biologically measured age (DNAges) than the longer-lived HET mice. Since we know that HETs live longer than WTs (in general), this confirms several reports in the aging literature that DNAge is a better measure of biological age than is simple chronological age. Comment 3B: In addition, these data were generated with only 4 mice per group and no data were given of the animal to animal variation in data (e.g., SD or SEM). Response 3B: Even though there are only 4 mice per group, the methylation clock analysis showed statistically significant younger liver tissue in HET’s than WT’s (p<0.012 and SEM(WT): 4.5 and SEM(HET): 7.1), and the DNAges of each animal are shown in Table 2. We have included SEMs in the revised manuscript. Comment 3C: The data on observational health of the animals on page 13 was unconvincing. Response 3C: Please see Response 6 to Reviewer 1. Comment 3D: Data on physiological functions such as grip strength, activity, rotarod performance, and cognition would have been stronger evidence that the HET mice were physiologically younger. Response 3D: We have previously tested HET and WT mice in a battery of behavioral tests (open field, tube dominance test, elevated plus maze, Morris water maze and Barnes maze) that showed increased aggressiveness in HETs without cognitive impairment (4). Relative to the biological indicators of age-related differences that we already have described (e.g. HETs have increased lifespan, HETs have reduced cancer incidence, and HET have reduced organ pathology), tracking the rate of cognitive decline will not give us a better indication of the HET-WT aging difference. Testing such physiological functions in HET mice is currently beyond the scope of this manuscript, and is contingent upon future funding. Minor Concerns: Comment 4: The authors state on page 4 (line 78-77) that there are “only a handful of candidate longevity genes for mice.” This is not correct; there have been at least two dozen different genes identified. The most cited are those that show reduced growth hormone/IGF. The Cisd2 mouse has not been studied greatly from an aging context. Response 4: By our count, we find that there are only 11 candidate longevity genes for C57 mice (16), and we now express this explicitly in the revised manuscript in the second paragraph of Introduction. Moreover, of the aforementioned 11 candidates, only one has greater median lifespan than our HETs, and two actually have reduced lifespan compared to JAX labs. Comment 5: In the methods, it was stated that the mice were either allowed to live out their lifespan or euthanized. It would be important to know the number (%) of mice euthanized for the WT and HET mice, i.e., where more WT mice euthanized. Response 5: In the WT cohort, 68% mice were euthanized as opposed to 45% mice in HET group. Comment 6: Supplement: there is no list of references for the figures in the supplement, and it is not clear what the third figure is all about. Response 6: Figures in the Supplementary information has been appropriately cited in the manuscript and the third figure in the supplementary information has been deleted. Comment 7: The introduction reads more like a discussion. For example, I found very little information about the Brd2 gene in the introduction or anywhere in the manuscript. I would have liked to know what protein this gene codes for and its biochemical/molecular function, etc. Response 7: The reviewer raises an important stylistic point. We did not include much information about Brd2’s biochemical/molecular function because, as interesting as that would be from a basic science point of view, we have yet to find a plausible explanation for the wide range of biological processes in which this molecule is involved. Aside from its two most cited roles: a transcription factor element in copying RNA from DNA, and an acetylated histone effector, it is associated with a number of disparate biologic observations, such as: over-expression in cancer; regulation of neuron migration during development; linkage and association to a specific form of epilepsy; over-expression in hormonally modulated epithelia (e.g., mammary gland, ovary, kidney, and uterus); and it is even reported to act as a scaffold for TATA binding. Thus, in our view, what the protein “does” is not at all well understood. Furthermore, because it takes a great deal of space to explain what individual processes the protein is involved in, and because this explanation is unlikely to provide an overarching understanding of how Brd2 brings about these observations, we did not feel it at all appropriate to go into detail about topics that do not, as far as we can see, increase understanding of Brd2’s effect on mouse lifespan and health. Instead, we opted for explaining only how we came to serendipitously observe its effect on longevity and to substantiate the depth of that effect with observations, for example, on changes in kidney pathology between HETs and WTs. In truth, we regret not being able to review Brd2’s biologic functions in greater detail than the few lines allotted in Discussion, but that task is best left to a review article, not in this report—which emphasizes Brd2’s dramatic and surprisingly beneficial effect on mouse longevity and health. References: 1. Gyuris A, Donovan DJ, Seymour KA, Lovasco LA, Smilowitz NR, Halperin AL, et al. The chromatin-targeting protein Brd2 is required for neural tube closure and embryogenesis. Biochim Biophys Acta. 2009;1789(5):413-21. 2. Shang E, Wang X, Wen D, Greenberg DA, Wolgemuth DJ. Double bromodomain-containing gene Brd2 is essential for embryonic development in mouse. Dev Dyn. 2009;238(4):908-17. 3. Wang F, Liu H, Blanton WP, Belkina A, Lebrasseur NK, Denis GV. Brd2 disruption in mice causes severe obesity without Type 2 diabetes. Biochem J. 2009;425(1):71-83. 4. Chachua T, Goletiani C, Maglakelidze G, Sidyelyeva G, Daniel M, Morris E, et al. Sex-specific behavioral traits in the Brd2 mouse model of juvenile myoclonic epilepsy. Genes Brain Behav. 2014;13(7):702-12. 5. Yuan R, Peters LL, Paigen B. Mice as a mammalian model for research on the genetics of aging. ILAR J. 2011;52(1):4-15. 6. Yuan R, Tsaih SW, Petkova SB, Marin de Evsikova C, Xing S, Marion MA, et al. Aging in inbred strains of mice: study design and interim report on median lifespans and circulating IGF1 levels. Aging Cell. 2009;8(3):277-87. 7. Flurkey K. The Mouse in Biomedical Research. 2nd Edition ed. Burlington, MA: Elsevier; 2007. 8. Lim JH, Kim EN, Kim MY, Chung S, Shin SJ, Kim HW, et al. Age-associated molecular changes in the kidney in aged mice. Oxid Med Cell Longev. 2012;2012:171383. 9. Pettan-Brewer C, Treuting PM. Practical pathology of aging mice. Pathobiol Aging Age Relat Dis. 2011;1. 10. Ward JM. Lymphomas and leukemias in mice. Exp Toxicol Pathol. 2006;57(5-6):377-81. 11. Bocklandt S, Lin W, Sehl ME, Sanchez FJ, Sinsheimer JS, Horvath S, et al. Epigenetic predictor of age. PLoS One. 2011;6(6):e14821. 12. Zhang Y, Wilson R, Heiss J, Breitling LP, Saum KU, Schottker B, et al. DNA methylation signatures in peripheral blood strongly predict all-cause mortality. Nat Commun. 2017;8:14617. 13. Petkovich DA, Podolskiy DI, Lobanov AV, Lee SG, Miller RA, Gladyshev VN. Using DNA Methylation Profiling to Evaluate Biological Age and Longevity Interventions. Cell Metab. 2017;25(4):954-60 e6. 14. Field AE, Robertson NA, Wang T, Havas A, Ideker T, Adams PD. DNA Methylation Clocks in Aging: Categories, Causes, and Consequences. Mol Cell. 2018;71(6):882-95. 15. Horvath S. DNA methylation age of human tissues and cell types. Genome Biol. 2013;14(10):R115. 16. Pedro de Magalhaes J, Thompson L, de Lima I, Gaskill D, Li X, Thornton D, et al. A Reassessment of Genes Modulating Aging in Mice Using Demographic Measurements of the Rate of Aging. Genetics. 2018;208(4):1617-30. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0234910.r002
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Formally Accepted
10 Jun 2020 Acceptance Letter - Christoph Englert, Editor PONE-D-19-32190R1 Brd2 haploinsufficiency extends lifespan and healthspan in C57B6/J mice Dear Dr. Pathak: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Christoph Englert Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0234910.r004

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