C. elegans to laboratory food sources using competition experiments on a panel of 89 recombinant inbred lines (RIL). Unexpectedly, we identified a single RIL with higher relative fitness than either of the parental strains. This strain also displayed a novel behavioral phenotype, resulting in higher propensity to explore bacterial lawns. Using bulk-segregant analysis and short-read resequencing of this RIL, we mapped the change in exploration behavior to a spontaneous, complex rearrangement of the rcan-1 gene that occurred during construction of the RIL panel. We resolved this rearrangement into five unique tandem inversion/duplications using Oxford Nanopore long-read sequencing. rcan-1 encodes an ortholog to human RCAN1/DSCR1 calcipressin gene, which has been implicated as a causal gene for Down syndrome. The genomic rearrangement in rcan-1 creates two complete and two truncated versions of the rcan-1 coding region, with a variety of modified 5’ and 3’ non-coding regions. While most copy-number variations (CNVs) are thought to act by increasing expression of duplicated genes, these changes to rcan-1 ultimately result in the reduction of its whole-body expression due to changes in the upstream regions. By backcrossing this rearrangement into a common genetic background to create a near isogenic line (NIL), we demonstrate that both the competitive advantage and exploration behavioral changes are linked to this complex genetic variant. This NIL strain does not phenocopy a strain containing an rcan-1 loss-of-function allele, which suggests that the residual expression of rcan-1 is necessary for its fitness effects. Our results demonstrate how colonization of new environments, such as those encountered in the laboratory, can create evolutionary pressure to modify gene function. This evolutionary mismatch can be resolved by an unexpectedly complex genetic change that simultaneously duplicates and diversifies a gene into two uniquely regulated genes. Our work shows how complex rearrangements can act to modify gene expression in ways besides increased gene dosage.Over long evolutionary timescales, major changes to the copy number, function, and genomic organization of genes occur, however, our understanding of the individual mutational events responsible for these changes is lacking. In this report, we study the genetic basis of adaptation of two strains of  rcan-1 gene and also modifies its expression in a number of tissues. Our work demonstrates how a complex structural change can duplicate a gene, modify the DNA control regions that determine its cellular sites of action, and confer a fitness advantage that could lead to its spread in a population.Evolution acts on genetic variants that modify phenotypes that increase the likelihood of staying alive and passing on these genetic changes to subsequent generations (i.e. fitness). There is general interest in understanding the types of genetic variants that can increase fitness in specific environments. One route that fitness can be increased is through changes in behavior, such as finding new food sources. Here, we identify a spontaneous genetic change that increases exploration behavior and fitness of animals in laboratory environments. Interestingly, this genetic change is not a simple genetic change that deletes or changes the sequence of a protein product, but rather a complex structural variant that simultaneously duplicates the  Structural variation, resulting in the removal, duplication, insertion, or rearrangement of large (> 50bp) genomic regions, makes up a significant component of natural genetic variation in many different species –6. The lE. coli or S. cerevisiae, elucidating the molecular basis of adaptation and profiling genome dynamics in evolving population under diverse laboratory settings. By identifying and studying causal genetic variants, important insights into beneficial mutations have been gained, such as their occurrence frequency, the complexity of their molecular basis, the role of contingency and genetic background into their effect, and their fitness effects in specific environments. A number of studies have demonstrated that genomic rearrangements can spread in these populations due to the actions of positive selection  Isolate 1.PTM553 kyIR1, qgIR1, kahEx170 Isolate 2.PTM554 kyIR1, qgIR1, kahEx171 Isolate 3.PTM555 kyIR1, qgIR1, kahEx172 Isolate 1.PTM556 kyIR1, qgIR1, kahEx173 Isolate 2.PTM557 kyIR1, qgIR1, kahEx174 Isolate 3.PTM558 kyIR1, qgIR1, kahEx175 Isolate 1.PTM559 kyIR1, qgIR1, kahEx176 Isolate 2.PTM560 kyIR1, qgIR1, kahEx177 Isolate 3.PTM561 kahEx185PTM566 CX12348 kahEx185PTM567 CX12348 To create the CX12311-CX12348 and LSJ2-CX12348 RILs, CX12311 males or LSJ2 males were crossed to CX12348 hermaphrodites. 96 F2 progeny  were cloned to individual plates and allowed to self-fertilize for 10 generations to create the inbred lines. One RIL line was lost from the LSJ2xCX12348 cross, creating 47 RILs.rcan-1 NILs (PTM413 and PTM414), CX12348 animals were backcrossed to CX12311 for 10 generations. Two completely independent sets of crosses were used to create two independent lines. Primers used to identify male animals containing the rearrangement were: 5’—gagacaatactctgatattagacgcacca -3’ and 5’–gctgacaccagcaatcattgttca -3’.To create the rcan-1 deletion strain (PTM505), two sgRNAs targeting the 5’ region of rcan-1 and two sgRNAs targeting the 3’ end of rcan-1 were created: sgRNA1: 5’-atttggaagatcatctttac-TGG-3’; sgRNA2: 5’-agtgctgatcaatgatccat-TGG-3’; sgRNA3: 5’-cgtggcatttcaattgctga-TGG-3’; sgRNA4: 5’-tcacatggagatgaagggcg-TGG-3’. CoCRISPR Please let us know if you have any questions while making these revisions.Yours sincerely,Harmit S. MalikAssociate EditorPLOS GeneticsKirsten BombliesSection Editor: EvolutionPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: Review uploaded as attachmentReviewer #2: In this manuscript, Zhao et al. characterize a structural rearrangement in the rcan-1 gene in C. elegans, and link this genetic change to alterations in foraging behavior and fitness. First, they use a panel of N2/LSJ2 RILs to investigate how genetic changes impact fitness, using a competitive fitness assay. They identify a single strain that appears to be an outlier and then show that this strain also displays in increase in exploratory foraging behavior. They then use genetic mapping and sequencing to show that this strain has a de novo genetic change in the rcan-1 gene that is linked to the fitness and foraging phenotypes. Zhao et al. characterize the genetic change in rcan-1 and show that it is a surprisingly complex genomic rearrangement that arose during generation of the RILs. Genetic studies show that the change in rcan-1 is causal of the fitness and foraging phenotypes. Moreover, they find that the rcan-1 genomic rearrangement alters expression levels of rcan-1.Overall, this is an interesting and convincing paper that should be of broad interest to the readers of Plos Genetics. This detailed study of a new genomic rearrangement and its strong ties to fitness and behavioral changes provides new insights into how structural variants cause phenotypic variability. I suggest just a few ways in which the manuscript can be improved and indicate the degree to which this would be preferred (vs. necessary), in my view.1. The authors characterize the structural rearrangement in rcan-1 through long read sequencing and confirm that the Illumina sequencing aligns well to the proposed rearrangement. They indicate that they were unable to PCR the entire region. If possible, I’d still suggest a series of smaller PCRs aimed at confirming their proposed rearrangement. I realize this is non-trivial given the repetitive nature of the rearrangement, but perhaps primers could be designed to span the new sequences at breakpoints (i.e. right around the chimeric reads). Overall, they already have strong evidence for their proposed rearrangement, but this structural variant is the central point of the paper and it would be preferable to have overwhelming confidence in their interpretation.2. The authors provide a solid first pass analysis of how the rearrangement of rcan-1 causes the foraging and fitness phenotypes, by comparing the NILs to a null mutant and examining how the non-coding changes impact expression (using fluorescent reporters). However, this is obviously still not fully resolved. For example, do the two truncated rcan-1 copies have any functional roles? Is this simply a gene dosage effect (seems unlikely…)? There are further analyses that could provide insights: A) what is the phenotype of the heterozygous NIL? B) Do CRISPR induced frameshift mutations in each of the four ORFs that comprise the genomic rearrangement impact the NIL phenotype? If possible, I’d suggest further exploration of this topic, but I do not view it is absolutely necessary for publication .Minor points:1. Is the distribution of datapoints in Fig. 3B bimodal, as would be expected from a single causal variant? It is difficult to assess this as presented. Please provide a histogram or something to that effect.2. The images in Fig. 6C are rather small and difficult to assess. I’d suggest larger, high-quality images that give the reader a more complete view of sites of expression. In addition, providing a well-labeled image for each of the promoter regions used would be helpful for future mechanistic studies of rcan-1 function.Reviewer #3: In this manuscript, the authors investigate the genetic basis of increased fitness in a single C. elegans outlier recombinant inbred line (RIL) that has higher fitness than its parental strains. This higher fitness is correlated with increased exploration of a bacterial lawn. The authors provide compelling data that show that the major causative allele contributing to these phenotypes is a de novo complex structural rearrangement of the rcan-1 gene involving a series of tandem inversions/duplications that ultimately leads to reduced expression of rcan-1 due primarily to truncations of the rcan-1 5’ noncoding region. Though the paper does not explore the specific cellular mechanisms by which this rearrangement and rcan-1 affect fitness/exploration, it is a very interesting and clearly-written study based on solid data and is appropriate for PLOS Genetics. In particular, the genetic and long-read sequencing data to map the rearrangement and determine its structure are very strong. We suggest a few experiments that could strengthen the paper, but do not feel that these are critical for publication.Considerations:1. One of the interesting conclusions of the paper is that even though the rearrangement creates two full-length copies of the rcan-1 coding sequence, there is actually reduced rcan-1 expression . This is supported by solid RNA-seq data and expression reporters . Given that the 5’ regulatory regions of both intact rcan-1 ORFs are truncated, reduced expression makes sense. The simplest model is that the truncations remove important enhancers and these enhancers no longer function as well when present more distantly in the remaining intact 5’ regulatory region. However, the authors push the data further to argue that there are tissue-specific effects on expression and that the two orientations of the inverted and truncated 5’region have different effects on expression. These latter two conclusions are not as well supported by the existing data that are based on expression reporters of the truncated/inverted 5’ regulatory region in the two orientations . The problem is that these expression reporters are extrachromosomal arrays that are overexpressed and are often variable in structure and expression levels from line to line or even different animals of the same line and thus should not be used for the kinds of fine quantitative inferences the authors make, such as the head expression being more affected than the body, especially as these inferences are based on a very small number of lines. To be able to make such fine comparisons the authors should generate single-copy transgenes that are not prone to variability. Additionally, the images in Fig 6C&D do not clearly show the distinct and tissue-specific effects the authors claim, which could again be the result of analysis of a small number of selected lines. Higher magnification images with better quantitation of expression in specific cells of interest (such as the unaffected head neurons) could also help.We suggest that the authors either repeat these expression experiments with single-copy transgenes or tone-down their conclusions . For the data in Fig. 6B, they should specify whether the ~30 animals analyzed come from one or more transgenic lines. Three lines of each are shown in the strain list, but it isn’t clear whether the data come from all three lines. In the raw data (Table S1), please list the strain and allele # of each animal analyzed as well.2. The authors provide good evidence that the residual expression of rcan-1 in the rearrangement is important for increased fitness by showing that near-isogenic lines (NILs) carrying this rearrangement outcompete a rcan-1 null mutant . The rcan-1 null has increased exploration  compared to a WT control, but it is unclear whether the null mutant also has increased fitness. To test this, a good experiment would be to compete the rcan-1 null vs. the WT control. This experiment would further address the argument that reduced expression of rcan-1 increases fitness and whether fitness is indeed tightly correlated with exploration.It is clear that some of the complexity of the rearrangement is necessary for its fitness advantage, but it is unclear how much of the complexity is necessary. The simplest model is that the two intact rcan-1 ORFs with their altered upstream regulatory regions would be sufficient – that reduced rcan-1 expression plus some residual expression (possibly tissue-specific) is sufficient. To test this, a strain could be generated with the two altered 5’ regions R1 and R2 driving the rcan-1 ORF (as single-copy transgenes to control expression levels) in the background of the rcan-1 null mutant. Would this strain fully recapitulate the phenotypes of the more complex rearrangement lines?Other simple experiments could further probe the relationship between rcan-1 dosage and exploration/fitness and strengthen the conclusions. The rearrangement has about 25% normal total expression of rcan-1, though this may be higher in some cells and lower in others. What is the phenotype of a rcan-1(lf)/+ heterozygote that would be predicted to have 50% rcan-1 levels in all cells? What is the phenotype of the rearrangement when heterozygous? Is the rearrangement rescued (i.e. fitness and exploration decreased) by injection of WT rcan-1(+)?3. The NILs are shown to be sufficient for increased exploration and fitness . But do they fully account for the increased fitness of the original RILhf strain? To test this, a good experiment would be to compete the NILs with RILhf.4. On several occasions, it is suggested that variation on chromosome V contributes to the exploration phenotype of RILhf . Though the LSJ2/RILhf RILS clearly show an effect of chromosome V , it seems that variation on this chromosome can contribute to exploratory behavior, but it seems unlikely that such variation is contributing to the phenotype of RILhf since it has no LSJ2 alleles on chromosome V . More likely, alleles on LSJ2 chromosome V have increased exploration independently of the rest of the RILhf genetic background, a possibility that could be easily tested if desired. However, this is really a fairly minor point and tangential to the paper. We suggest just making it clear in the writing that chromosome V variation may affect exploration, but not necessarily the exploration phenotype of RILhf.5. Some simple investigations into the modified regulatory sequence would be interesting. Are there known transcription factor binding sequences or conserved sequences in the 5’ noncoding region that are lost in the truncations?6. A deeper discussion of worm exploration behavior could enrich this manuscript. What is known about the circuitry controlling this behavior? Is rcan-1 expressed in specific relevant neurons (and expression lost in the R1 and R2 constructs)? Additionally, it is ultimately unclear how exploration relates to fitness and whether this correlation is causal or coincidental. Throughout the paper, we are led to believe that the altered exploration could lead to increased fitness by changing how worms feed . But late in the discussion (line 405), we learn that there is no difference in food consumption in strains carrying the rcan-1 rearrangement. An interpretation of this surprising result seems warranted. Does this mean that exploration is unrelated to food consumption (and fitness) or might there be other ways that exploration is related to fitness? The authors don’t need to have the answers or take a side, but it would be nice to have some conclusion since it was just confusing to provide this result with no further comment. .7. Is the known functional connection of rcan-1 to tax-6/calcineurin important? If rcan-1 inhibition of calcineurin is relevant to its exploration and fitness phenotypes, then it might be expected that there is increased calcineurin activity in strains with the rearrangement, and a tax-6 mutation would suppress the increased exploration and fitness phenotypes of the rearrangement.8. There’s a fair bit of discussion of the rcan-1 rearrangement being formed as an adaptation in response to selective pressures . Though the rearrangement clearly increases fitness, it does not seem that it was selected for increased fitness, but rather was likely just formed and fixed accidentally. In fact, the way the RILs were made, there did not seem to be any obvious selection for fitness since about half of the lines have low fitness like their parent LSJ2 . We recommend a more conservative discussion of these points.Minor points:1. The authors are to be commended for using box plots rather than bar graphs in the figures , but the figure legends should give n values for quick reference.2. It is unclear why the authors measured animal length . This is the only time we hear of this phenotype in the paper and it is unclear if it is considered to be an important contributor to increased fitness. As presented, the experiment doesn’t really seem to fit in the paper, but is just a random piece of data thrown in.3. For Fig. 6A and S4, it would be nice to know which other genes have significantly altered expression (the green and red dots), and whether the same genes were affected in both NILs. This information is not easily derived from the raw data in Table S3.4. The way the Fig 6A legend is written, it is unclear whether this figure shows combined data from the two NILs (“differences between the NIL and N2* strains”). Given that the figure itself is labeled NIL1, we presume that Fig 6A shows the data from NIL1 and Fig S4 shows the data from NIL2. Please make this clear.5. The Fig 6A legend should say what the green and red dots mean. The Fig 6C&D legend should say what the arrowheads indicate. It would be best if all the images in Figs 6C and 6D are shown with the same anterior-posterior orientation and mention this orientation in the legend. It seems that the zoomed-in images in 6D show different animals than in 6C, but this should be made explicit.6. In Figure 4A, the schematic for the gene position is difficult to interpret. It would be good to color code rcan-1 differently from pst-2 so it can be seen where these genes start and end, and show the direction of transcription of each gene . In fact, we recommend using the same schematic in 4C and 4A so that they can be directly compared, especially for the 5’ noncoding region. A scale bar for Fig 4A would also help so we can more easily see how much of the 5’ region has increased coverage.7. In Figure 6B, the significance bars for statistical comparisons are difficult to interpret, especially the top two levels of these comparisons. It is unclear which data sets are indicated as significantly different from each other. For instance, what do the two *** values at the top refer to? The head of WT compared to head of R1 and R2, the body of WT compared to the body of R1 and R2, etc? Or head, body, and total of WT compared as a combined group to head, body, and total of R1 and R2?8. It would be interesting to see if there are differences in movement speed between the N2*, RILhf , and rcan-1 NILs. This may be linked to exploratory behavior.9. The description of the structural changes in the rcan-1 rearrangement is useful but could be condensed. The paragraph at the bottom of page 9/top of page 10 repeats many of the same things said in the opening paragraph of page 9 where the rearrangement is first described.10. There should be explanation for what the different colors mean in Figures S1 and S3.11. The acronym “CNV” should be defined when first used in the abstract.12. In Table S3, rcan-1 is written as rcn-1. Please correct this to facilitate searching for the data.13. Line 93 should read: “in wild strains of C. elegans” instead of “in wild strains C. elegans.”14. There is inconsistency on whether the rearrangement carries five or six tandem inversions (e.g. lines 25 and 213).15. Line 366. The rearrangement is described as “similar” to a previously published rearrangement that causes another phenotype in C. elegans. However, it’s not clear what is meant by “similar” and this other rearrangement is in fact quite different. It consists of several duplications and a triplication, but no inversions, and causes a phenotype through increased gene dosage rather than decreased expression as reported here. Additionally, the other rearrangement seems to be formed by chromoanasynthesis in which the rearrangement is made by templated synthesis and the breakpoint junctions have short microhomologies. This seems quite different from the rearrangement described here, which does not seem to have any microhomologies at the breakpoints (though this could be explicitly mentioned). The only similarity seems to be that complex rearrangements can cause phenotypes, but we would argue that the rearrangements themselves are not similar at all.16. Lines 383-385: the point of this sentence is unclear.17. The paragraph in lines 407-412 seems overly speculative.18. References 56 and 58 are the same (and can be updated now that the paper is published).Reviewed (and signed) by Michael Ailion and Lews Caro**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  and your responses to reviewer comments. If eligible, we will contact you to opt in or out. *Dear Dr McGrath,Thank you very much for submitting your Research Article entitled 'A spontaneous complex structural variant in rcan-1 increases exploratory behavior and laboratory fitness of C. elegans' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. You will see that all three reviewers are happy with your revisions. However, I hope you will take their comments about the writing in some of the 'new' parts to heart- I agree with Reviewer 3 that some of the changes you have made are not as nicely written as the original pieces. This is your opportunity to smooth those rough edges out before your article is published. I hope you will take that opportunity to do so. 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You will be contacted if needed following the screening process.PLOS has incorporated To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.[LINK]Please let us know if you have any questions while making these revisions.Yours sincerely,Harmit S. MalikAssociate EditorPLOS GeneticsKirsten BombliesSection Editor: EvolutionPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: The authors have identified an interesting laboratory-derived strain (RILhf) with a spontaneous mutation that causes increased fitness and roaming behavior. My primary concern with the manuscript was that the claim that the rcan-1 rearrangement is causal for the increased fitness and roaming behavior needed to be strengthened. The authors have attempted to address this concern, and the revised Table S3 is excellent and addresses my questions about the backcrossed NILs.However it is disappointing that rescue of RILhf with wildtype rcan-1 did not work, with respect to foraging behavior . In the author’s “response to reviewers” this experiment is interpreted clearly, but I have some concerns with the authors interpretation in the paper (lines 300-302). My concerns are as follows:“potentially this indicates that the rearranged region causes ectopic expression necessary for the changes in foraging behavior…” – this is inconsistent with the rcan-1 CRISPR KO allele promoting foraging behavior.“…or overexpression of rcan-1 decreases foraging behavior” – this is the opposite of what the data is showing, so perhaps a typo? Do the authors mean overexpression of rcan-1 might increase foraging behavior? Otherwise you would have expected the construct to rescue, since RILhf displays increased foraging behavior.I think more likely is this particular transgenic experiment didn’t work, either for technical reasons, or because additional variations in RILhf promote foraging behavior independent of the rcan-1 rearrangement. Nevertheless, the manuscript deserves to be published in PLoS Genetics, as the rcan-1 rearrangement is a very complex mutation, and fully dissecting the genetics of the various rcan-1 duplications is outside the scope of this paper. Additionally, the findings are interesting and novel, and the experiments are performed carefully and not over-interpreted.Typo in line 94: “in certain context”Typo in lines 238-239: should delete “enhancers and other r promoter regions are probably present in the truncated promoters”Reviewer #2: The authors have addressed all of my concerns.Reviewer #3: I support publication of this paper, which has been significantly strengthened in this revision. The authors have done a very good job of responding to and addressing the majority of the critiques. Though I do not have major concerns, a few of my original points have not been fully addressed and I repeat them here. Also, many of the parts of the manuscript that were changed upon revision are not written with the same quality as the rest of the manuscript. I point out a number of clear grammar mistakes and typos, but suggest the authors look again at the writing in all their new or edited sections and work more carefully on these parts.Points:1. The authors have done a better job of toning down their conclusions about tissue-specific effects of the altered rcan-1 promoter regions in some places and pointing out some of the caveats of extrachromosomal arrays, but there are other places where they still explicitly state that there are tissue-specific effects: Fig 6 title (line 456), line 116, lines 277-279. I am fine with the authors suggesting there might be tissue-specific effects, but think they should not flat out state it as if it were demonstrably proven because the evidence is quite weak.2. I strongly recommend that the authors not use the term “foraging behavior” for the exploration phenotype and not equate increased exploration with “increased foraging activity.” It isn’t exactly clear what the increased exploration of rcan-1 variants indicates and whether it is causally related to the increased fitness, but it does not seem clearly related to foraging for the rcan-1 variants as their increased exploration does not lead to increased food consumption. Thus, it would be more conservative to just call it “exploration” as that is what is being assayed. The word foraging means “searching for or obtaining food.” The evidence given is that rcan-1 does not affect “obtaining food” and I don’t think it is addressed whether the exploration of rcan-1 variants indicates “searching for food.” An alternative unexplored possibility is that the rcan-1 variants may simply have increased locomotory activity or decreased reversals. Though increased exploration may be a foraging strategy for other mutants, the evidence in this paper does not suggest it is for the rcan-1 variants. Thus, continued use of the term foraging to describe this behavior is both potentially confusing and misleading. Examples include: the short title of the paper, line 51, lines 168-172, line 254, lines 293-308, lines 315-316, lines 349-353, and several Figure legends, but there may be others.3. Related to point 2, I think the food consumption experiment would fit better in the Results, in the same section with the experiment using uniform bacterial lawns, as a general consideration of the question of how exploration relates to foraging.4. Though the authors now state that the rcan-1 rearrangement was unlikely to have been selected for its higher fitness, the sentences in lines 38-41 and 346-348 still make it sound that this rearrangement formed as an adaptation in response to selective pressures. I would tone these down.5. I previously suggested that the paragraph in lines 371-376 is overly speculative and the authors responded by saying that they have toned this paragraph down, yet it reappears exactly the same as in the original.6. I did not realize that body size is considered a fitness proximal trait, and am surprised that a smaller size would be associated with increased fitness . Thus, the experiment on body length could be better explained and motivated for naïve readers like me. For me, it still seems to come out of the blue, and the immediate segue from short body to increased fitness (line 230) is not intuitive at all.7. In Figure 5D (body length), the y-axis is unclear. It shows “normalized body length” but the units are unclear and it is unclear what it is being normalized to – the legend says “controls” but it is unclear what these controls are and there are no apparent data for any control strains in the Table S1 source data file.8. Figure 6C&D: please indicate anterior-posterior direction of these worms.9. The rcan-1 rearrangements suggests a simple and interesting model for the evolution of gene duplicates that wasn’t clearly presented in the discussion of this phenomenon (lines 321-338). How do gene duplicates evolve from redundancy to become paralogs? The rcan-1 rearrangement suggests a model that by imperfectly duplicating or rearranging the regulatory regions of the duplicated genes, two new genes can be created in a single step that aren’t functionally redundant because they may have differential expression levels and differential tissue-specific expression. Thus, this mechanism might create paralogs instantaneously without having to first evolve through redundancy.Typos/grammar:line 49: understandingline 167: causalline 230: these data line 237: should be “upstream sequence”lines 237-242: run-on sentence line 238: r at end of lineline 281: fluorescenceline 307: solelylines 317-318: I think the cited paper has just one rearrangement, so shouldn’t be pluralline 319: unclear antecedent of “this.” Just say: “The rcan-1 rearrangement”line 335: grammarline 337: RILhfline 342: “single single-nucleotide” is awkwardline 378: describedline 378: should be “often” instead of “common”line 466: comparedlines 467-468, 470-471: grammar problems. Also, say what the white arrows in the R2 panels are showing.lines 460 & 1045: Table S5lines 1053 & 1076: grammar problemsTable S3 title: says Table S2 by mistakeFigure S4: rearrangedFigure S7: “deletion” is written on top of “region”Reviewed (and signed) by Michael Ailion.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********what does this mean?). 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Three decades of extensive work in the HIV field have revealed key viral and host cell factors controlling proviral transcription. Various models of transcriptional regulation have emerged based on the collective information from in vitro assays and work in both immortalized and primary cell-based models. Here, we provide a recount of the past and current literature, highlight key regulatory aspects, and further describe potential limitations of previous studies. We particularly delve into critical steps of HIV gene expression including the role of the integration site, nucleosome positioning and epigenomics, and the transition from initiation to pausing and pause release. We also discuss open questions in the field concerning the generality of previous regulatory models to the control of HIV transcription in patients under suppressive therapy, including the role of the heterogeneous integration landscape, clonal expansion, and bottlenecks to eradicate viral persistence. Finally, we propose that building upon previous discoveries and improved or yet-to-be discovered technologies will unravel molecular mechanisms of latency establishment and reactivation in a “new era”. The development of combination anti-retroviral therapy (ART), which targets several steps of the viral life cycle, decreased patient viral titers to below the limit of detection using contemporary methods ,2. This + T cells  using Micrococcal nuclease (MNase) digestion followed by enzyme restriction analysis , which hAlthough the underlying sequence of nuc-1 is thermodynamically unfavorable for nucleosome formation, the position of nuc-1 in the latent proviral state (in Jurkat models of latency) is maintained by the switch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complex , which d+ T cell agonists in both immortalized (Jurkat) and primary models of latency, as well as in aviremic patient samples [Interestingly, both SWI/SNF complexes temporally occupy the proviral promoter with BAF in the basal phase and PBAF during the viral, Tat-dependent activation phase . These o samples ,208, it The BAF complex appears to be recruited to the proviral genome to maintain latency by one member of the BET family, specifically the shorter isoform of BRD4 . Upon TaIn addition to the chromatin-modifying and chromatin-remodeling complexes required to switch “on” or “off” provirus transcription, chromatin reassembly factors (CRFs) like SPT6, CHD1, FACT, and histone H3 chaperones (ASF1a and HIRA) are involved in transcription interference ,219, thu+ T) models of latency by interfering with P-TEFb binding to the proviral promoter [The FACT (facilitates chromatin transcription) complex, a heterodimer of SPT16 and SSRP1, removes and then reinstates the histone H2A/H2B dimer after Pol II elongation ,221. Twopromoter . These cpromoter  and not promoter , or to uNucleosome positioning is typically concerted with the deposition and/or removal of histone tail PTMs C. For exHistone tails are post-translationally modified to establish regulatory codes that facilitate or prevent factor-chromatin interactions . Specifi+ T cells) models of latency [Since HDACs prevent histone tail acetylation, thereby promoting chromatin compaction, it came to no surprise that HDAC deposition at the proviral genome was linked to, and strongly correlated with, latency maintenance and establishment ,43. Thus latency ,230,231. latency .The HDAC family is composed of five classes  based on function and sequence similarity (reviewed in ). SpecifBesides histone acetylation/deacetylation, histone methylation of nucleosomes, by histone methyltransferases (HMTs), surrounding the proviral genome is another epigenetic mechanism contributing to nucleosome positioning, thereby regulating proviral fate. Multiple HMTs  target distinct chromatin domains with different degrees of histone tail Lys methylation  . For insXenopus oocytes showed that HP1 recruitment to chromatin requires not only the presence of H3K9 methylation but also a direct interaction of HP1 with HMTs. Thus, while both SUV39H1 and G9a have H3K9 methyltransferase activity, only SUV39H1 is able to recruit all isoforms of HP1 to chromatin through direct interaction [Despite previous evidence that these three HMTs regulate proviral transcription, it is still unclear how they target proviruses integrated in diverse chromatin states. Since HMT have varying mechanisms of action at different chromatin domains , it is peraction .The multiple HP1 isoforms and their different chromatin distribution profiles opened the question as to which HP1s regulate HIV proviral transcription, and what are the underlying molecular mechanisms. Surprisingly, distinct HP1 isoforms were found to associate with the proviral genome in various models and under different contexts, thus questioning the central mechanism.In 2007, Benkirane and colleagues ascribed HP1-γ function, but not HP1-α and HP1-β, in maintaining provirus latency in both immortalized (HeLa and Jurkat) models of latency and PBMCs isolated from aviremic patients . In 2008Another histone methylation (H3K27me3) has long been known to have a repressive role (reviewed in ) in tran+ T helper (Th17)) and in resting memory T cells isolated from aviremic patients [Consistent with the deposition of H3K27me3 at the proviral genome in immortalized models of latency (Jurkat), EZH2  responsible for H3K27 di- and tri-methylation) was also found in the basal state; however, its levels rapidly decreased during the transition to the host-viral phases of the program in response to stimulation (TNF-α) , in agrepatients .Together, these initial observations indicate that site-specific H3 Lys methylation (both H3K9me3 and H3K27me3) contributes to proviral latency, potentially by maintaining nucleosomes in a repressed state at the 5′-LTR. Although both H3K9me2/3 and H3K27me3 are involved in gene silencing and can act in a concerted manner for the establishment of heterochromatin loci , the fun+ T) models of latency [More recently, another HMT (SMYD2: SET and MYND domain-containing protein-2) was identified using an RNAi-based screening approach . SMYD2 b latency . Given t+ T) models of latency were proposed [+ T) models of latency, thereby promoting latency establishment [+ T cells under suppressive therapy conferred resistance to provirus reactivation in contrast to hypomethylated HIV 5′-LTR in viremic patients [+ T cells under suppressive therapy [+ T cells harbor defective proviruses [Besides histone PTMs, DNA methylation of the proviral promoter is another epigenetic silencing mechanism implicated in transcriptional regulation. Two CpG islands (at positions ‒194 to ‒94 and +180 to +368 respective to TSS) flanking the proviral TSS in immortalized (Jurkat) and primary , and ne+ T cells as well as in patient samples regulate nucleosome positioning at the proviral genome? Can the initial results obtained in the above immortalized models be informative of the physiologic mechanism of chromatin control at integrated proviruses? Are those results generalizable? What is/are the role/s of the integration site, nuclear topology, sub-compartments and chromatin states to histone methylation, nucleosome positioning and proviral fate? Does the precise position and magnitude of H3 and H4 site-specific  methylation matter in terms of restricting, and or allowing future, proviral activation and latency-reversal potential? These are some of the many questions that remain to be answered to fully understand how histone site-specific PTMs shape proviral responses. Certainly, we face an exceeding limitation due to the scarcity of, not only the latent reservoir but importantly, the “intact” latent reservoir  and low-resolution tools, compared to chemical cleavage techniques, such as MNase digestion followed by foot printing analysis. Despite these pioneer discoveries, it remains unclear how applicable these early models are for intact proviruses persisting in aviremic patients. Is the current dogma in the field concerning nucleosome positioning still valid for intact proviruses in patients and/or for models that can better interrogate and explain disease progression? How does the integration site influence the position of nucleosomes in both defective and intact proviruses? How does the orientation and direction of proviruses respective to nearby regulatory elements in the 1D and 3D dimensions  influenceservoir A. Togeth+ T cell survival  and homeostatic proliferation . Im. Im+ T clls too  during EMT  gradual+ T cells latently infected with HIV in vitro, co-culturing with CD8+ T cells inhibited N-803–mediated latent provirus reactivation [+ T cells in vivo resulted in a robust SIV increase. Together, these studies illuminate how animal models can be used not only to test compound efficacy but also to develop latency reversing strategies that take into account physiologic variables.The strongest indications of LRA’s efficacy are in vivo studies using animal models. Garcia and colleagues showed how treatment with the compound AZD5582 in HIV infected BLT mice and SIV infected rhesus macaques under ART induced HIV and SIV RNA expression in several tissues including lymph node of the macaques and lymph node, thymus, bone marrow, liver, and lung of BLT mice . Though tivation . This waOverall, the importance of cross-validation using different models of latency, with emphasis on physiologically relevant models, is becoming increasingly apparent. Additionally, a careful analysis of hundreds or even thousands of single proviruses integrated in unique positions and bearing unique genetic variants, and not population-based studies, will hone our understanding on the role of integration landscape and cell state on latency reversal potential and other therapeutic schemes.Characterizing the latent reservoir composition was the first core discovery spurring an entire field dedicated to understanding the mechanisms underlying its establishment and maintenance. Though initially capturing the precise reservoir size and composition were low resolution estimates , improved and combined methodologies have continuously revised the latent reservoir profile, unveiling that only a miniscule proportion is intact and inducible A. By surBeyond fixed provirus physical location and compartmentalization, other influences of latency establishment and maintenance are much more fluid. Cell status, as illustrated by large fluctuations of nuclear transcriptional regulators and coregulators, can galvanize transcriptional responses when transitioning from resting to active status. The factor fluctuations may also be small enough only to generate heterogenized threshold within a state (at rest for example) due to stochasticity. Or the fluctuations may be at the local gene level where deposition of epigenetic marks, Pol II pausing, and/or nucleosome positioning can influence the inducibility of an otherwise intact provirus. Again, many conflicting results are often attributed to differences in models used for their respective studies given the multiple layers of variability B.+ T cells contain proviruses integrated in unique regions in addition to the clonally expanded pool.Because of the multifactorial aspects contributing to latency , it is obvious the era of singular cause is over. From a therapeutic perspective, the failure of LRAs has always been attributed to the same reason: inability of a single drug, which often target a single molecular rule of latency , to reactivate the entire latent reservoir. This extends to screening for new drug compounds. In the “new era”, it will be imperative to integrate multiple approaches  followed by deep learning  to prediAs physiological relevance is vital to remove distracting or inapplicable models/mechanisms from the discussion, preference must be given to use patient samples or primary cell systems. Yet, with the limitation of the number of intact proviruses within a patient to collect data from, alternative models may need to be developed. In this context, can primary models of latency be created in the presence of ART, without the ectopic expression of anti-apoptotic factors but allowing their long-term culturing without causing T cell exhaustion? With the advent of genome editing tools , can we With our efforts to discuss a surmountable number of key discoveries and situate them in the context of previously published literature, we hope to provoke future work that will advance our understanding of this fascinating biomedical research challenge. New data supporting or refuting the models discussed herein will undoubtedly increase our knowledge and generate discussions to help with future clinical applications rather than engendering unproductive controversy. Even the more skeptical researchers should be attracted to challenge these models and bring their own points of view to fuel progress in the field.


Pisum sativum). Application of TMTD can negatively affect nodulation; nevertheless, its effect on the histological and ultrastructural organization of nodules has not previously been investigated. In this study, the effect of TMTD at three concentrations  on nodule development in three pea genotypes  was examined. In SGE, TMTD at 0.4 g/kg reduced the nodule number and shoot and root fresh weights. Treatment with TMTD at 8 g/kg changed the nodule color from pink to green, indicative of nodule senescence. Light and transmission electron microscopy analyses revealed negative effects of TMTD on nodule structure in each genotype. ‘Finale’ was the most sensitive cultivar to TMTD and Sprint-2 was the most tolerant. The negative effects of TMTD on nodules included the appearance of a senescence zone, starch accumulation, swelling of cell walls accompanied by a loss of electron density, thickening of the infection thread walls, symbiosome fusion, and bacteroid degradation. These results demonstrate how TMTD adversely affects nodules in the pea and will be useful for developing strategies to optimize fungicide use on legume crops.In Russia, tetramethylthiuram disulfide (TMTD) is a fungicide widely used in the cultivation of legumes, including the pea ( Pisum sativum L.), are important food and feed crops. In 2018, the largest pea producers in the world were Canada (3.6 million tonnes), Russia (2.3 million tonnes), and China (1.5 million tonnes) [Legumes, including peas . It is a protective contact fungicide that does not penetrate plant tissues or seeds and inhibits spore germination or the initial growth of surface pathogenic mycelium ,11. ThisSimilar to other legumes, the garden pea enters symbiotic relationships with rhizobial soil bacteria to form symbiotic nodules, allowing it to fix nitrogen from the atmosphere. Fungicides have the strongest inhibitory effect on soil microorganisms , and mosBradyrhizobium japonicum cultures [Rhizobium leguminosarum bv phaseoli [Previous studies have explored the diverse impacts of pesticides on the legume–rhizobial symbiosis. These effects vary depending on the selected pesticide and its concentration ,16,17, acultures ,22,23. Wcultures . HoweverR. meliloti [R. phaseoli [B. japonicum [Previous attempts to isolate fungicide-insensitive rhizobia strains for seed inoculation have resulted in the isolation of meliloti , R. phasphaseoli , and B. aponicum . HoweverTrifolium repens L.), pea, and soybean (Glycine max (L.) Merr.) revealed that TMTD and rhizoctol are harmful to clover and the pea [Lens culinaris Medik.) seeds with TMTD reduces the incidence of seedling damping-off caused by Pythium sp., improves plant growth, and increases seed yield [Cicer arietinum L.), low concentrations of TMTD increase yields [Mesorhizobium ciceri strain, leading to decreased root and shoot biomass and seed yield of chickpea [Phaseolus vulgaris L.) is unaffected by TMTD treatment [The effect of TMTD on nodule formation and nitrogen fixation depends not only on the rhizobial species, but also on the plant species. Different fungicides vary in their effects depending on the symbiotic system involved. An evaluation of the effect of a variety of fungicides applied as a seed treatment to white clover  and the infection zone F. At a TUnder treatment with 0.4 g/kg of TMTD, some bacteroids in the nitrogen-fixation zone of infected cells were unaffected A and somIn the SGE line treated with TMTD at 0.4 g/kg, cells in the nodule meristem often had an irregular shape with electron-translucent cell walls (data not shown). In response to TMTD at 4 g/kg, the meristematic cells became irregularly shaped with a folded cell wall. In addition, these cell walls were electron translucent and swollen. Numerous small vacuoles characteristic of meristematic cells occasionally merged to form a large vacuole A. The plIn the infection zone, numerous vesicles proximal to the infection thread wall were engaged in deposition of polysaccharide material at the cell walls through exocytosis D. In resIn response to the lowest TMTD concentration (0.4 g/kg), protrusions of the symbiosome membranes developed, and spherical inclusions of moderate electron density formed in juvenile bacteroids in the infection zone A. In theIn the cultivar ‘Finale’ treated with TMTD at 0.4 and 4 g/kg, the meristematic cells often had an irregular shape with an electron-translucent cell wall (data not shown). Treatment with TMTD at 8 g/kg resulted in cell wall disorders A. ElectrElectron micrographs revealed striking differences in infection threads between untreated and treated nodules, especially in the 8 g/kg of TMTD treatment. The walls of the infection threads in TMTD-treated plants were swollen and became thickened. The thick walls of the infection threads consisted of many layers of fibrous material E. Some iTreatment with TMTD at 0.4 g/kg resulted in marked changes in the nitrogen-fixation zone of infected cells. In some cells, as a result of symbiosome membrane fusion, symbiosomes contained several bacteroids at different stages of degeneration A,B. In aTreatment with TMTD affected the plant cell walls and walls of infection threads in each of the studied pea genotypes. However, TMTD did not affect the structure of plastids and mitochondria B. ApplicTreatment of legume seeds with fungicides prevents fungal spore germination and reduces mycelial growth, thereby promoting seed germination and seedling growth ,29,30,322-fixing bacterial community [Rhizobium-infected cells in pea symbiotic nodules.Numerous studies have demonstrated the toxicity of diverse fungicides, including TMTD, to rhizobia ,18,19,20ommunity ,39. In tDithiocarbamic acid and its derivatives, including TMTD, have indiscriminate (multifarious) effects and disrupt a variety of biochemical processes that involve enzymes containing copper or sulfhydryl groups in pathogenic organisms ,40,41. TMedicago sativa L.) cultivar ‘Apollo’ showed a different reaction to various TMTD concentrations. Treatment with TMTD at low concentrations (3–20 μg/mL) increased nodulation and nitrogenase activity, whereas higher concentrations inhibited these processes [In another study, the alfalfa  was sufficient to impair the development of symbiotic nodules. This was accompanied by abnormalities in the infection process and premature degeneration of infected cells in nodules. The most obvious structural changes in nodules of ‘Finale’ were impaired growth of infection threads, modification of cell walls and walls of infection threads, and degradation of bacteroids A,B. TreaIn this study, treatment with TMTD modified the walls of plant cells and infection threads B–F. DespP. sativum symbiotic mutants [M. truncatula nodules formed by Ensifer meliloti mutants with succinoglucan deficiency (EPSI) [P. sativum [M. sativa nodules [In the present study, the infection threads containing a fibrillar matrix had thicker walls in TMTD-treated plants than in control plants E,F. Simi mutants ,60,61,62y (EPSI) , and P.  sativum  and M. s nodules  formed bThe thickening of cell walls has been described as a response to the action of heavy metals on plants ,67,68 anIn the current study, the walls of plant cells and infection threads showed different changes in response to TMTD treatment B–F. The Pythium ultimum Trow from entering the vascular stele of cucumber plants [Phyllanthus tenellus under copper stress [Lotus corniculatus L. growing on ultramafic soils contaminated with nickel, cobalt, and chromium, vacuoles in the nodule endoderm and some cortical and parenchymal cells were filled with dark phenolic substances [M. sativa plants was observed in the presence of arsenic [M. sativa, a gene encoding phenylalanine-ammonia lyase, which catalyzes the initial step in lignin synthesis, was expressed in nodules [–-2 harboring the allele sym33-3 of the gene PsCYCLOPS/PsIPD3, the release of rhizobia from infection droplets stimulated cell wall formation and suberin accumulation in the vacuole [Light and electron microscopy analyses revealed inclusions, presumably consisting of phenolic compounds, in vacuoles of meristematic cells in the nodules of the TMTD-sensitive pea cultivar ‘Finale’ A. Phenolr plants . Elevater plants , corn unr plants , faba ber stress . In Lotubstances . Inducti arsenic . Similar nodules . Previou vacuole .In this study, we observed accumulation of PHB in bacteroids A,B. Othe–-3 (sym26), which is characterized by an early nodule senescence phenotype [M. lupulina nodules [Treatment with TMTD also caused the appearance of large spherical inclusions G–I with henotype . In the  nodules ,90 and c nodules ,50. In t–-5 harboring the allele sym33-2 of the gene PsCYCLOPS/PsIPD3 [The degradation of bacteria inside infection threads was an additional negative effect observed in nodules of ‘Finale’ plants treated with TMTD F. PrevioS/PsIPD3 . The deg3) treatment [As a result of exposure to TMTD, the cytoplasm of infected cells became less electron dense and showed clear signs of plasmolysis, including nuclear pyknosis I, but threatment .P. sativum L.) laboratory lines SGE [R. leguminosarum bv. viciae strain 3841 was used for inoculation [The pea . The plants (15 plants per variety) were grown in a growth chamber  under the following conditions: 21 °C, light intensity 280 μmol m−2 s−1, photoperiod 16 h light/8 h dark, and relative humidity 75%. Plants and nodules were harvested at 3 weeks after inoculation.Seeds were sterilized with concentrated sulfuric acid for 30 min and then washed 10 times with sterile water. The seeds were germinated in Petri dishes in an incubator  at 24 °C. For the TMTD treatments, TMTD was added to the vermiculite at a concentration of 0.4, 4, or 8 g/kg, and mixed thoroughly into the vermiculite. Seedlings were transferred to plastic pots containing 150–200 g vermiculite and 250 mL nitrogen-free Fåhraeus nutrient solution  and eachGrowth and nodulation parameters were analyzed for the SGE line only. Experiments were performed with 15 TMTD-treated plants and 15 untreated (control) plants. For weight measurements, shoots and roots were separated and cotyledons were removed. Excess moisture was removed from the plants with filter paper. All nodules on the root system were removed with tweezers and counted. Pea nodules were photographed under a SteREO Lumar.V12 stereomicroscope equipped with an AxioCam MRc 5 video camera . Object visualization was performed using AxioVision Rel. 4.8 software (Carl Zeiss).p ≤ 0.05) and the least significant difference test.Statistical analysis of the data was performed using the software STATISTICA version 10 . Statistically significant differences were evaluated using one-way ANOVA . Then, the samples were placed under vacuum to remove air from the intercellular spaces and were left in fixative overnight at 4 °C.Nodule ultrastructure was analyzed for all genotypes. About 10–15 nodules were harvested from different plants of each variety and placed directly into fixative. A lateral cut was made on each nodule to improve penetration of the fixative. The nodules were fixed in 2.5% glutaraldehyde  in 0.01% PBS (2.48 g/L NaHThe nodules were washed in buffer (four times for 15 min each) and postfixed in 2% osmium tetroxide in 0.1 M phosphate buffer for 2 h. The samples were dehydrated as described previously . DehydraFor light microscopy, the embedded material was cut into semi-thin sections (1 μm) using a Leica EM UC7 ultramicrotome . Sections were placed on glass slides (SuperFrost)  and stained with methylene blue-azure II for 20 min at 65 °C . The secFor transmission electron microscopy, ultrathin sections (90–100 nm thick) were cut with a Leica EM UC7 ultramicrotome (Leica Microsystems) and counterstained as described previously . Nodule Our results show that pea symbiotic nodules are highly sensitive to the phytotoxic effect of the fungicide TMTD. High concentrations of TMTD in the substrate cause structural changes in the plant cell walls and infection threads, and premature degradation of bacteroids. The plant cell walls and infection threads show different responses to TMTD. Therefore, further investigations should include studies of the mechanism of cell wall modification, and antioxidant defense systems at the morphological, biochemical, and molecular levels. Such analyses will reveal the mechanism of fungicide toxicity in legumes. To the best of our knowledge, this is the first report on the effects of TMTD on the ultrastructure of symbiotic nodules. An enhanced understanding of the adverse effects of agricultural pesticides on legume crops will be useful for developing strategies to use pesticides optimally in agricultural systems. Notably, TMTD has been banned in the European Union because of health and environmental risks.


Here, we describe a detailed practical protocol of spleen transplantation and its evaluation for long-term graft survival. Unlike implantation of splenic morsels in the great omentum, our approach uses arterial and venous anastomoses which rapidly restores blood flow and facilitates long-term survival of the graft. The use of congenic mouse strains permits the use of immunofluorescence and flow cytometry-based methodologies to unambiguously track the migration of spleen-derived cells to peripheral tissues.The spleen is a large lymphoid organ located in the abdomen that filters blood and regulates the immune system. The extent of mobilization of splenic immune cells to peripheral tissues in health and disease, however, remains poorly understood. This is due, in large part, to a lack of  Numerous immune cells reside within the organ, different subsets of T and B lymphocytes and myeloid cells among them4. Due to lack of in vivo cell labeling strategies, the extent of mobilization and function of splenic immune cells in peripheral tissues remains poorly understood. The most common loss-of-function approach is surgical removal of the organ, a procedure that indiscriminately eliminates all splenic cells. Splenectomy has additional effects including increased circulating white blood cell and platelet counts and heightened susceptibility to bacterial and protozoal infections7. To circumvent these issues, we developed a murine model of spleen transplantation with arterial and venous anastomosis. Surgical transplantation between congenic mouse strains  not only preserved tissue integrity but allowed unambiguous and quantitative tracking of the movement of immune cells between the spleen and peripheral tissues.Located within the abdomen of most vertebrates, the spleen is a large lymphoid organ known for its function in filtering the blood (ie. recycling iron and removing damaged and senescent red blood cells) and regulating the immune systemThis study was approved by the Institutional Animal Care and Use Committee (IACUC) at the Massachusetts General Hospital under the protocol number 2009N000022. The protocol as conforms to the USDA Animal Welfare Act, PHS Policy on Humane Care and Use of Laboratory Animals, the “ILAR Guide for the Care and Use of Laboratory Animals” and other applicable laws and regulations.+ C57BL6/J) with an intraperitoneal (i.p.) injection of ketamine (90 mg/kg)/xylazine (10 mg/kg) using a 30-G needle. Once the donor mouse is anesthetized , secure the animal on an acrylic movable surgical table using surgical tape. Shave and disinfect the anterior abdominal and thoracic walls using a combination of iodine and alcohol prep pads. To access the heart for whole body perfusion, make a transversal incision on the left thoracic wall at the 4th intercostal space with surgical scissors. Retract the ribs, open the pericardium, and cut a small hole in the right atrium. Using a 23-G needle and 5 ml syringe, slowly inject 5 ml of cold saline solution containing 300 units of heparin into the left ventricle. CRITICAL STEP: When perfusing, do not flush all blood. Leave behind a small volume of heparinized blood for better visualization of donor vasculature. To visualize the donor spleen, pancreas, and abdominal vasculature in the epigastric region, make a longitudinal ‘V’ incision on the anterior abdominal wall extending from the pelvis to the inferior ribs. CRITICAL STEP: To prevent donor tissue from drying out, place gauze soaked in cold saline solution over the intestines, pancreas and spleen. Keep gauze in place until the spleen isolation is completed. Using 6–0 silk suture, ligate the gastric artery as well as the small vessels between the pancreas, duodenum and colon . CRITICAL STEP: Be sure to handle the pancreas gently, since pancreatic trauma negatively affects transplantation outcome. Using 10–0 nylon suture, ligate the hepatic and superior mesenteric artery. Make double ligatures of the distal aorta directly below the mesenteric artery and above the right renal artery using 10–0 nylon suture . Ligate the 4 small posterior spinal-aortic arteries with 10–0 nylon suture. Cut the proximal aorta below the diaphragm at an internal angle of 45 degrees toward the right kidney, placing a 10–0 nylon notch suture on the external angle at the tip of the open aorta . Cut the distal aorta below the double ligatures, above the right renal artery. Isolate and ligate the common bile duct and the pancreatic-biliary vessels using 10–0 nylon suture. Cut the portal vein below the first bifurcation at an internal angle of 45 degrees towards the right hepatic lobe and place a 10–0 nylon notch suture on the external angle at the tip of the open portal vein . Using cotton tip applicators, gently remove the organ package containing the spleen, pancreas, and vascular connections from the abdominal cavity and place it in a small petri dish containing ice cold saline while the recipient mouse is being prepared.Anesthetize the donor mouse  with isoflurane supplemented with oxygen (2–3 Vol%). Once anesthetized, remove the animal from the induction box and secure to a small, movable cork surgical table by the tail with surgical tape. Do NOT tape arms and legs. Maintain anesthesia during surgery using an inhalation apparatus. Administer preemptive analgesia via subcutaneous injection (5 mg/kg Meloxicam). Shave the anterior abdominal wall and disinfect the skin using a combination of iodine and alcohol prep pads. Cover the operating field with a sterile fenestrated surgical drape. Make a mid-line incision on the abdominal wall and open the peritoneal cavity. Retract both sides of the abdominal wall with a sterilized modified paper clip. Pin the clips down on the cork table with bent 23-G needles. To visualize the recipient’s spleen, gently mobilize the intestines to the right side of the anterior abdominal wall using cotton tip applicators and cover them with saline solution soaked gauze. Remove the orthotopic CD45.2+ spleen by ligating the splenic vessels with 6–0 silk suture and excising the organ. Cover the liver, stomach, and pancreas with gauze soaked with ice cold saline solution. Cut the avascular tissue that connects the descendent colon to the pancreas with fine micro-scissors and gently release the infra-renal aorta and inferior vena cava (IVC) from surrounding tissues using fine forceps and cotton tips. CRITICAL STEP: Be careful not to damage the sciatic nerves that run parallel to the aorta and IVC. Place a microvascular clamp on the aorta-IVC below the renal vessels . CRITICAL STEP: Be sure not to clamp sciatic nerves. CRITICAL STEP: To have enough space to perform anastomosis, make sure at least 10 mm of vessel is pinched within the clamp. Using a 30 G needle, puncture the proximal area of the isolated aorta. Using this opening as a starting point, make a 2 mm incision of the clamped aorta using fine micro-scissors. Flush out any micro-clots with ice cold saline . Position the donor spleen and pancreas on the left side of the abdomen and make an anchorage notch on the inferior angle of recipient aorta using 10–0 nylon suture. Using 10–0 nylon suture, begin a running terminal-lateral anastomosis (joining donor and recipient aortas) with one stop from the superior angle of opened right side of aorta. Gently move the donor spleen and pancreas to the right side of the abdomen using cotton applicator tips and continue the running anastomosis on the left side of aorta . CRITICAL STEP: Do not over-tighten the running aortic anastomosis. Using cotton applicator tips, move the spleen carefully to the left side of the abdomen. Then, using a 30-G needle, puncture the distal end of clamped IVC. Using this opening as a starting point, make a 2 mm incision of the clamped IVC using fine micro-scissors . Flush out any micro-clots with ice cold saline. Make an anchorage notch on the inferior angle of recipient opened IVC with 10–0 nylon suture. Start a running terminal-lateral anastomosis  with one stop from the superior angle of the opened right side of the IVC. Move the spleen carefully to the right side of the abdomen and continue the running anastomosis on the left side of IVC . CRITICAL STEP: Do not over-tighten the running vein anastomosis. Once the anastomoses are completed, open the vascular clamp slowly to perfuse the spleen. If minor bleeding in the splenic and/or pancreatic vessels occurs, ligate leaky vessels with 10–0 nylon suture. Once certain all bleeding has stopped, gently place intestines back inside the peritoneal cavity and close the abdominal wall using absorbable 6–0 vicryl suture. Place the recipient mouse in a clean cage, under a heat lamp until the animal regains consciousness.Anesthetize the transplant recipient mouse . Once anesthetized, animals were removed from the induction box and placed in dorsal position on a heated imaging platform and remained anesthetized via a nose cone. Prior to start imaging, the tail vein was catheterized, using a 27-gauge needle connected to a modified P10 tubing catheter, and the anterior abdominal wall was shaved. Aquasonic-100 ultrasound transmission gel  was applied over the abdominal wall and a 40-MHz imaging transducer was placed longitudinally over the left region of the abdomen. B-mode and color doppler imaging were taken before and after a bolus tail vein injection of 100-μL of microbubbles contrast agent  and continue microbubbles injection at a constant rate of 9 μL/min through the duration of spleen imaging.Short and long term blood perfusion of transplanted spleens was confirmed by high resolution ultrasonography using Vevo 2100 system .CD45.2+ hematopoietic spleen cells were identified as: CD11b+IAb+CD11c+ , CD11b+IAb+CD11c−Ly6C+/− (monocytes), Lineage−CD11b−F4/80+CD115low (red pulp macrophages), CD11b+IAb−CD11c−Ly6Cintermediate (neutrophils), CD19+IgMlowCD23+ (B2/Follicular B cells), CD19+IgMhighCD23− , CD3+CD4+ (CD4 T cells), CD3+CD8+ (CD8 T cells), and CD3−NK1.1+ (NK1.1+ cells). Data were acquired on a LSR II (BD Biosciences) and analyzed using FlowJo software . Positive and negative gating strategies were determined using isotype controls unless otherwise stated.Single cell preparations were stained for 30 minutes on ice unless otherwise noted before being fixed with Cytofix buffer (BD Biosciences). CD45To assess splenic architecture, spleens were resected and fixed in 4% paraformaldehyde, cryoprotected in 20% sucrose in PBS before being embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek), and flash frozen in isopentane on dry ice. 5-μm transverse sections were made and stained with hematoxylin and and eosin (H&E).. In previous studies we demonstrated perfusion of the transplanted spleen using a combination of Angiosense-680 injection, a fluorescent blood pool agent, and near infrared imaging (NIR)8. Here, we confirm long term blood perfusion of transplanted spleens by contrast ultrasonography  at 1 week . Similarly, transplantation had little effect on the distribution of several subsets of B and T lymphocytes and natural killer (NK) cells . Histological analysis of transplanted spleens revealed normal architecture of the red and white pulp structures . Collectively, the data show that our approach preserves tissue integrity and is associated with long term survival of the transplanted organ.Spleens from 12 week old donor C57BL6/J mice were transplanted into 12 week old recipient C57/BL6/J mice  and recipient (CD45.2+) cells was assessed in several organs of the transplant recipient including the blood, bone marrow, liver, lung, thoracic lymph nodes and aorta. Our data demonstrate that steady state trafficking of immune cells in and out of the spleen is remarkably dynamic . Moreover, CD45.1 spleen-derived leukocytes migrated to all of the peripheral tissues analyzed . How splenic leukocyte migration is altered in the context of different pathological conditions remains to be determined.To demonstrate how our approach can be used to track the migration of splenic immune cells, spleens from 12 week old donor CD45.1 C57BL6/J mice were transplanted into 12 week old recipient CD45.2 C57BL6/J mice. Two days later, the proportion of donor  mouse strains which are identical but for one molecule expressed on the surface of white blood cells that can be identified by antibody detection. Therefore, immunofluorescence and flow cytometry methodologies can be used to distinguish hematopoietic cells derived from the CD45.1 donor spleen and CD45.2 recipient. A key aspect of our approach is the creation of arterial and venous anastomoses between donor and host, resulting in rapid restoration of blood flow and long-term survival of the spleen graft. Vascular anastomosis to the recipients’ circulation is facilitated by transplanting the donor splenic artery and vein with a section of the abdominal aorta and portal vein, respectively. The CD45.1 donor organ package, which includes the spleen, a portion of the pancreas, and vascular connections is then transplanted into the peritoneal cavity of the CD45.2 recipient using end-to-side anastomoses of the donor aortic cuff to the recipient descending aorta, and of the donor portal vein to the recipient inferior vena cava. To achieve spleen exchange rather than supplementation, the endogenous CD45.2 spleen is removed.et al. demonstrated that the spleen is a monocyte reservoir9. In this study, our spleen transplantation protocol revealed splenic monocytes mobilize in response to myocardial infarction, accumulate in the injured heart, and participate in wound healing. Using the approach, we have also showed that the spleen is an active site of extramedullary hematopoiesis during atherogenesis8. We determined that monocytes produced in the spleen contribute to disease progression by infiltrating atherosclerotic lesions and differentiating to foamy macrophages, secreting inflammatory cytokines, reactive oxygen species and proteolytic enzymes. In a genetic mouse model of lung adenocarcinoma, Cortez-Retamozo and colleagues used the transplantation protocol to establish that neutrophils and macrophages borne in a splenic niche relocate to the site of the tumor and promote cancer growth10. Collectively, these studies demonstrate that our spleen transplantation method is an effective in vivo approach for labeling spleen cells and assessing their function in a remote tissue context. Of note, our previous studies followed graft survival to ~1.5 weeks. Here, we report graft survival, perfusion, and normal distribution of immune cell subsets in the transplanted spleen out to one year post transplant, demonstrating the utility of the approach for long-term studies. The procedure can be used in different pathological settings and possible modifications include transplantation of spleens from donors in which certain genes are either knocked out or over expressed.The approach has broad applications—it can be used to determine the fate and function of various cell types migrating in and out of the spleen. The use of congenic mouse strains allows clear identification of donor and host cells in any tissue of interest. The method is therefore of interest to all scientists studying inflammation and immunity in health and disease. In 2009, Swirski 11. However, the approach does not incorporate vascular anastomoses which leads to prolonged ischemia and eventual resorption of the transplanted tissue. Furthermore, lack of an adequate blood supply precludes the study of physiologic cell trafficking. Transplantation of the intact spleen with arterial and venous anastomosis, therefore, represents a major technical advance, not simply an extension of previous implantation strategies. A recent video publication of spleen transplantation with venous and arterial anastomosis in mice utilized cauterization of the short gastric vein attached to the spleen12. In many jurisdictions, the use of vessel cauterization when harvesting organs for transplant in animals is prohibited. Moreover, cauterization often leads to inflammation, injuring the vessels that are required to be ligated to the stomach, pancreas, colon and duodenum. Here, we demonstrate that vessel ligation leads to durable survival of the graft long-term.Implantation of spleen morsels into the great omentum of mice has previously been reportedall cell populations. Injection of single cell suspensions from spleen homogenates removes cells from their physiological tissue context. Our spleen transplantation protocol avoids these issues, allowing spleen cells to be unambiguously marked without disturbing the tissue environment in which they reside.The most commonly used approaches to study immunological processes in the spleen include splenectomy and/or adoptive transfer of single cell suspensions prepared from tissue homogenates. Splenectomy restricts controlled investigation of specific subsets of immune cells since removal of the organ at once eliminates Supplementary Information.


This systematic review synthesized evidence from European neck and low back pain (NLBP) clinical practice guidelines (CPGs) to identify recommended treatment options for use across Europe.Comprehensive searches of thirteen databases were conducted, from 1st January 2013 to 4th May 2020 to identify up‐to‐date evidence‐based European CPGs for primary care management of NLBP, issued by professional bodies/organizations. Data extracted included; aim and target population, methods for development and implementation and treatment recommendations. The AGREE II checklist was used to critically appraise guidelines. Criteria were devised to summarize and synthesize the direction and strength of recommendations across guidelines.neck pain, there were consistent weak or moderate strength recommendations for: reassurance, advice and education, manual therapy, referral for exercise therapy/programme, oral analgesics and topical medications, plus psychological therapies or multidisciplinary treatment for specific subgroups. Notable recommendation differences between back and neck pain included, i) analgesics for neck pain (not for back pain); ii) options for back pain‐specific subgroups—work‐based interventions, return to work advice/programmes and surgical interventions (but not for neck pain) and iii) a greater strength of recommendations  for back pain than those for neck pain.Seventeen CPGs  from eight European countries were identified, of which seven were high quality. For This review of European CPGs identified a range of mainly non‐pharmacological recommended treatment options for NLBP that have broad consensus for use across Europe.Consensus regarding evidence‐based treatment recommendations for patients with neck and low back pain (NLBP) from recent European clinical practice guidelines identifies a wide range of predominantly non‐pharmacological treatment options. This includes options potentially applicable to all patients with NLBP and those applicable to only specific patient subgroups. Future work within our Back‐UP research team will transfer these evidence‐based treatment options to an accessible clinician decision support tool for first contact clinicians. Relevant outcomes also included evidence‐based recommendations regarding factors  that may be associated with the effectiveness of treatment, and recommendations regarding clinical prediction rules or decision tools supporting the selection of treatment for specific patient subgroups (where mentioned in the guideline).2.3.2Non‐European guidelines;All publications that are not evidence‐based clinical practice guidelines, including guidelines based solely on consensus or without an explicit literature search, and other publication types: systematic reviews, randomized trials, cohort studies, case series, editorials, protocols, letters;Paediatric only populations (under 18 years);NLBP as a result of severe trauma, for example fracture and spinal cord injury, inflammatory arthritis including spondyloarthropathies, and those that focused on broader conditions, for example (chronic) pain that may encompass spinal pain;Guidelines focused on patients managed in secondary care with an established diagnosis of radiculopathy;Guidelines focused specifically on surgical treatment options/comparisons or on specific interventions not limited to spinal pain, for example analgesics in older adults;Guidelines that involved populations admitted to hospital (not ambulatory care);Guidelines for which translations could not be obtained.2.4https://endnote.com/) and duplicates removed. Unique citations were then imported into Covidence  to manage the screening process.Results from all searches were imported into EndNote X9  independently screened all titles and abstracts for relevance against eligibility criteria and excluded ineligible publications by agreement. Full texts were independently assessed for inclusion by pairs of independent reviewers . Disagreements were noted and resolved between pairs of reviewers and where necessary the involvement of a third reviewer. Reasons for exclusion at the full‐text stage were recorded.2.5A data extraction form was purposively designed in Excel to record relevant information from each of the clinical practice guidelines included in the review. Complementary documents were sourced where relevant, such as methodological reports and evidence syntheses. Information was extracted regarding general guideline information ; methods regarding guideline development and implementation  and intervention recommendations, specifically only those that can be offered in primary care, and guidance for referral .One reviewer extracted data from each guideline; in the case of guidelines in English, this was independently checked by a second reviewer with any disagreements resolved through discussion. For non‐English guidelines, no independent check with a second experienced reviewer was feasible within the timeline of conducting this review.2.6The AGREE II  reporting checklist was used to critically appraise guidelines  was used to complete appraisals online, based on the user manual. Each item is presented for scoring alongside detailed guidance on how to score the item, including where to find relevant information and what to consider when deciding on the score for each item.The web‐based platform My AGREE PLUS . One reviewer appraised each guideline; in the case of guidelines in English, this was independently checked by a second reviewer with any disagreements resolved through discussion. For non‐English guidelines no independent check was feasible.No set thresholds exist for determining high‐/low‐quality guidelines, however, AGREE II guidance suggests users decide these according to their specific context , country and target population/diagnostic classification). Members of the review team, which included researchers with academic and clinical expertise in musculoskeletal pain, were presented with these tables for review. Following discussion of the many very specific intervention options, for example different forms of exercise, nuanced and/or inconsistently used terms and translation anomalies/country‐specific terminology (often reported in only 1 or 2 guidelines), interventions were merged by treatment type/modality. A general practitioner (physician) was invited to review the recommendations relating to medications specifically and undertook a similar process of refining the grouping of treatment options.i.e. for, against or open) and strength of recommendations were harmonized, taking into consideration the array of methods and terminologies used across included guidelines : consistent recommendations in at least two high‐quality guidelines from different countries (at least one guideline of which reports a 'strong' i.e.// or XX recommendation).Moderate FOR/AGAINST recommendation (could do/could not do): consistent recommendations in at least one high quality (where the recommendation is not based on expert opinion i.e. O + or O‐) and if only one high quality, then one or more low‐quality guidelines.Weak FOR/AGAINST recommendation: recommendations from high‐quality guidelines but based on expert opinion only and/or recommendations from multiple low‐quality guidelines.Inconsistent: inconsistent recommendations from guidelines of high quality (for/against).Inconclusive: open/uncertain recommendations only, or recommendations from low‐quality guidelines are inconsistent.33.1The systematic search resulted in 3,941 unique citations, from which 17 clinical practice guidelines (CPGs) were identified Figure .3.2n = 12; 71%), whereas six (35%) are concerned with neck pain and/or radicular pain. Five guidelines (29%) focus specifically on patients presenting with symptoms of radiculopathy. Three of these guidelines , employed formal grading of evidence and/or recommendations . Just over half the guidelines detailed timeframes for future revisions , whereas just under half detailed or undertook a consensus process .A large majority of CPGs were developed by multidisciplinary groups , diagnostic assessment , evaluation of red  and/or yellow  flags. Conversely, less than half the guidelines detailed the evaluation of blue/black flags, that is blue: individuals’ perceptions of work‐related factors and the relationship between work and health, black: system‐level factors  , practitioner education  or organization and policy implications .In addition to treatment recommendation most guidelines addressed planning of care  achieved high ratings for Domain 5 (applicability). Domains 2 (stakeholder involvement) and 6 , together with overall assessment score, were each reported to a high quality in a large majority of studies . Domain 3 (rigour of development) with its higher cut‐off point of 70% determining high quality was achieved by just over half the CPGs .The AGREE II domain scores for each guideline are presented in Table Seven CPGs (41%) were considered high‐quality overall: 2 focused on neck pain, both Danish  recommendations from high quality  interventions were recommended for specific subgroups of patients with neck pain, with either psychosocial risk factors or for those with more persistent neck pain or disability.Recommendations were inconsistent or inconclusive regarding manual therapies ; traction; electrotherapies; thermotherapies; cervical orthoses; acupuncture/dry needling and referral for imaging.3.5Twelve guidelines provided treatment recommendations for back pain  were only mentioned in one guideline, and these were not considered further. Positive (weak to strong) recommendations from high quality  cancer, neurological emergencies including cauda equina syndrome, aortic aneurysm or systemic inflammatory arthritis ; epidural steroid and other injections; acupuncture and manual, postural and thermotherapies.3.6In order to examine the consistency of CPG recommendations across neck and low back pain, overall strengths of recommendation for each identified intervention . Eleven of them addressed low back pain, five neck pain and one both LBP and neck pain. Data extraction showed considerable variation in guideline development processes with seven guidelines  considered as high quality, based on their development rigour, strong stakeholder involvement and the applicability of their recommendations.neck pain, high‐quality guidelines consistently recommended the following evidence‐based treatment options: reassurance, advice and education (including to remain active and exercise), manual therapy in combination with other treatment, referral for exercise therapy/programme and a range of oral analgesics and topical medications, plus psychological therapies or multidisciplinary treatment for specific subgroups of patients. There was no strong evidence for use across Europe  and it should be noted that these medications are no longer consistently recommended for low back pain within the recent European guidelines. In fact, for low back pain the guidelines recommended entirely non‐pharmacological treatments, additionally including work‐based interventions, advice/programmes to return to work and surgical intervention for specific subgroups. These recommendations were based on stronger evidence than those for neck pain.For In relation to previous literature, the findings of this review summarising the consensus from European guidelines, are consistent with recommendations in The Lancet back pain series . However, it was notable that clear assessment criteria to facilitate clinician decision making about when to use these treatment options for specific patient subgroups were largely lacking. Similar to Lin et al. who highlighted that guidelines for patients with thoracic pain are lacking , which can be considered a limitation. This may be partly explained by fewer guidelines being produced in southern or eastern Europe, but also by the fact that we only included guidelines published in the past 7 years. While focusing on contemporary guidelines (2013 onwards) ensured that we identified the most relevant treatment options for current practice, we acknowledge that this meant that some earlier European guidelines, were not included. However, for the purposes of this review, we felt it was important to exclude guidelines that may not be based on up‐to‐date evidence of effectiveness. Although we included guidelines written in any European language, one limitation was that we were not able to carry out independent data extraction and quality appraisal by a second reviewer for guidelines not available in English. However, for most of these guidelines, the reviewer had the advantage of being involved in data extraction for English language guidelines, which helped to ensure consistency of data extraction and interpretation.Only seven CPGs (41%) were considered to be of overall high quality, with limitations mainly related to rigour of development ; and to applicability with few guidelines providing guidance on how to apply recommendations or taking into account practical and financial implications of their recommendations. Variation in the methods used to grade evidence and agree the strength of recommendations may potentially explain some of the variability in treatment recommendations across guidelines. We tried to incorporate quality as well as consistency in our synthesis of CPGs, aiming to arrive at a transparent and systematic approach for summarizing and grading recommendations across guidelines.Future work within the Back‐UP research project will embed these evidence‐based treatment options in an accessible clinician decision‐support tool for first contact clinicians, aiming to offer patients with NLBP treatment options better matched to the risk of persistent pain and disability.5In conclusion, this systematic review identified seventeen contemporary clinical guidelines regarding NLBP  from 8 European countries, of which seven were considered high quality. Recommendations were notably consistent for neck and low back pain, despite the larger evidence base and more guidelines for the latter. The implications of this review are that clinicians have a broad range of mostly non‐pharmacological evidence‐based treatment options to consider for their patients with NLBP. These include some treatments which are a) potentially applicable to all patients such as advice and education and b) those applicable only to certain patient subgroups .The authors have no conflicts of interest to declare.GM, DvdW and JH designed the study protocol with input from NC. NC designed the literature search with input from DvdW. NC, GM and DvdW performed the study selection with input from LM. NC, GM, LM, SS and GW‐J carried out data extraction and quality assessment. NC, JH, SS and DvdW carried out the analysis and interpretation of the data. NC and JH drafted the manuscript. All authors critically revised the manuscript for intellectual content and read and approved the final manuscript.Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.Appendix S3Click here for additional data file.Appendix S4Click here for additional data file.Appendix S5Click here for additional data file.Appendix S6Click here for additional data file.Appendix S7Click here for additional data file.