Escherichia coli. As expected, RRF depletion leads to enrichment of post-termination 70S complexes in 3′-UTRs. We also observe that elongating ribosomes are unable to complete translation because they are blocked by non-recycled ribosomes at stop codons. Previous studies have suggested a role for recycling in translational coupling within operons; if a ribosome remains bound to an mRNA after termination, it may re-initiate downstream. We found, however, that RRF depletion did not significantly affect coupling efficiency in reporter assays or in ribosome density genome-wide. These findings argue that re-initiation is not a major mechanism of translational coupling in E. coli. Finally, RRF depletion has dramatic effects on the activity of ribosome rescue factors tmRNA and ArfA. Our results provide a global view of the effects of the loss of ribosome recycling on protein synthesis in E. coli.We used ribosome profiling to characterize the biological role of ribosome recycling factor (RRF) in  After the synthesis of a protein is complete, the ribosomal subunits are separated from each other and from mRNA to be reused in the next round of translation, a process known as ribosome recycling . AlthougSaccharomyces cerevisiae using ribosome profiling to study recycling factors in vivo revealed that depletion of the 80S recycling factor Rli1 results in an accumulation of ribosome density at stop codons, consistent with a build-up of post-TCs that fail to be recycled, and an associated queue of elongating ribosomes that collide with these post-TCs at defined distances upstream of stop codons  ribosomes translating the upstream gene may unwind secondary structures that otherwise block de novo initiation at the downstream gene  or (2) f for RRF . No cleaE. coli. We used ribosome profiling (deep sequencing of ribosome-protected mRNA fragments)   to deteraraBAD promoter so that cells cultured in media with arabinose express RRF, but following the switch to media with glucose, they strongly repress its transcription. In addition, we added a FLAG-epitope to the C-terminus of RRF to facilitate its detection, and the short peptide tag YALAA to target the protein for degradation by ClpXP  whose abundance drops significantly at elevated temperatures (43°C) . Given tby ClpXP . In mediby ClpXP . To measby ClpXP . Even atUsing this strategy, we performed ribosome profiling and RNA-seq on the wild-type (WT) and RRF knockdown (KD) strains over an extended time course  after the second media switch . In addirpsB gene map almost exclusively to the coding sequence, after 60 min of RRF depletion, there is a clear accumulation of RPFs in the 3′-UTR  from the e 3′-UTR . These ee 3′-UTR . The nume 3′-UTR . BecauseS. cerevisiae upon depletion of Rli1 . Again, the fact that 3′-UTR ribosomes are reduced by the high-salt lysis buffer argues that most of them are not translating but have moved into the 3′-UTR by diffusion. In contrast, the peaks corresponding to stacked ribosomes are not reduced by the high-salt conditions consistent with the idea that they represent elongating ribosomes whose intact peptidyl-tRNA stabilizes the subunits against dissociation in high salt.Prior studies using the ts-RRF allele in isrupted . We therisrupted ; we experevisiae . In addirevisiae . The redarcA and stpA) that show high levels of ribosome density in the 3′-UTR when RRF is depleted. To detect re-initiation events, we inserted the GFP coding sequence into the 3′-UTR upstream of the first stop codon in every frame, creating three reporters, each in a different reading frame with respect to the upstream ORF   . If postarcA, we observe the GFP protein in the GFP-only construct using antibodies against GFP . These data show that because highly translated genes have more ribosomes per mRNA, there is an increased chance for collisions of elongating ribosomes with post-TCs trapped at stop codons.As shown above, when RRF is depleted, elongating ribosomes form a queue behind the stop codon (spaced one ribosome footprint apart) because they are sterically blocked by post-TCs that have failed to be recycled. We hypothesized that the number of stacked ribosomes would be particularly high on messages with high ribosome density. Using ribosome profiling and RNA-seq, we can estimate the ribosome occupancy of each transcript in the cell . We selected two sets of genes corresponding to the top 20% and bottom 40% of ribosome occupancy (RO) values in the WT sample. For these two sets of genes, we calculated the average ribosome density aligned at stop codons after RRF depletion . As antiUnexpectedly, genes with high RO have fewer ribosome footprints in the 3′-UTR relative to the CDS than genes with low RO . To quanOne of the distinctive characteristics of bacterial genomes is that many genes are organized in polycistronic operons. For translationally coupled genes, translation of a downstream gene in an operon is dependent on the translation of an upstream gene . FollowitrpB-trpA  is very close to one both before and after RRF depletion. We then expanded this analysis to all pairs of upstream/downstream genes in polycistronic messages, as shown in To look for effects of RRF depletion on the translation of neighboring genes genome-wide using ribosome profiling, we calculated the ratio of ribosome density on pairs of upstream and downstream genes, comparing the ratios in the WT and RRF KD strain. As shown for the Despite this general lack of impact of RRF on translational coupling genome-wide, it is possible that this analysis misses strong effects on certain subsets of genes. We identified those sets of gene pairs that are most affected by RRF knockdown, showing either a twofold increase or decrease in their ratio of ribosome density . We thenE. coli, more than 30% of gene pairs have overlapping stop and start sites. We plotted the ratio of upstream and downstream ribosome density for these pairs and again observed that the WT and RRF-depleted strains are highly correlated . Ribosome stalling at the 3′-end of this truncated arfA transcript is resolved by the primary ribosome rescue factor, tmRNA, which tags the truncated ArfA protein to target it for degradation. tmRNA also recruits RNase R so that the arfA mRNA is rapidly degraded  at stop codons across the transcriptome upon depletion of RRF. Although we did not see differences in stop codon peaks, we did observe indirect evidence that post-TCs are in fact present at stop codons. Elongating ribosomes form a queue behind stop codons, spaced one ribosome footprint apart. We infer that post-TCs accumulate at stop codons, blocking elongation and trapping upstream ribosomes in an inactive state that prevents them from completing protein synthesis. We speculate that post-TCs retain some affinity for the site where termination occurred due to base-pairing between the deacyl-tRNA in the P-site and the last sense codon in the ORF. These findings are consistent with prior observations of stacked ribosomes that appear upon knockdown of the yeast 80S recycling factor Rli1  and the Our second main observation is the accumulation of non-translating 3′-UTR ribosomes upon RRF depletion. Unlike the stacked ribosomes upstream of stop codons, which contain peptidyl-tRNA, ribosomes in the 3′-UTR are lost in high-salt concentrations, arguing that they are post-TCs and not elongating 70S ribosomes. How did they get into the 3′-UTR? We argue that over time, post-TCs, only weakly retained by codon-anticodon pairing with the tRNA, eventually slide along the mRNA past the stop codon into the 3′ UTR. The dramatic enrichment of ribosome density downstream of stop codons highlights the importance of RRF in maintaining ribosome homeostasis by regenerating ribosome subunits to initiate translation. When RRF is depleted, many ribosomes are trapped in an inactive state in the 3′-UTR, as seen in earlier studies .S. cerevisiae, loss of Dom34 leads to the accumulation of non-translating ribosomes in the 3′-UTR, indicating that Dom34 normally helps remove empty 80S ribosomes from 3′-UTRs  delivers Dom34 to the ribosomal A site, where it works together with the canonical recycling factor Rli1 to promote subunit splitting . In S. c 3′-UTRs . Because 3′-UTRs . In contE. coli reflects post-TCs that are not translating, as evidenced by their sensitivity to the high-salt buffer , and cultured at 37°C for 10 min. Cells were collected by centrifugation, resuspended in 500 mL of LB + glucose (0.2% w/v) a second time and cultured at 37°C for 5, 15, or 60 min.For ribosome profiling and RNA-seq libraries, cells were cultured at 37°C in 500 mL of LB + arabinose (0.2% w/v) to OD2HPO4,22 mM KH2PO4,8.6 mM NaCl, 18.7 mM NH4Cl), 100 μM CaCl2, 1 mM MgSO4, 1x minimal essential medium (MEM) vitamin solution, 1x methionine biosynthesis inhibition amino acids (L-lysine (100 μg/ml), L-threonine (100 μg/ml), L-phenylalanine (100 μg/ml), L-isoleucine (50 μg/ml), L-leucine (50 μg/ml), and L-valine (50 μg/ml)), and 0.2% carbon source. To monitor bulk translation rate in arabinose media, HPG (50 μg/ml) was added to a culture in arabinose media at OD600 = 0.5, and cells were incubated for 10 min. To determine the background level of the Alexa Fluor 488 signal, methionine (50 μg/ml) was added (instead of HPG) in arabinose media and the cells were incubated for 10 min. For RRF depletion, cells were transferred to glucose media at OD600 = 0.5, and after another 10 min, a second change to fresh glucose media was performed. At the indicated time points after the second media change , HPG (50 μg/ml) was added and the cells were further incubated for 10 min. Cells were lysed with Click-iT lysis buffer . Total protein concentration was measured with the DC protein assay (Bio-Rad). 15 μg of lysate in 30 μl Click-iT lysis buffer was first mixed with 15 μl of 4 × Click master mix , then with 15 μl of 400 uM Alexa Fluor 488 Azide (Thermo Fisher) in Click-iT lysis buffer. The Click-iT reaction mixture was incubated in the dark for 2 hr. Unreacted Alexa Fluor 488 Azide was removed by CHCl3/MeOH precipitation. Samples were resolved by SDS–PAGE and the Alexa Fluor 488 signal was detected by Typhoon FLA9500 . Total protein was visualized by Coomassie brilliant blue. The signals were quantified by ImageJ  or 1 M NaCl lysis buffer  using a Spex 6870 freezer mill with 5 cycles of 1 min grinding at 5 Hz and 1 min cooling. Lysates were centrifuged at 20,000 x g for 30 min at 4°C to pellet cell debris. To exchange the buffer prior to nuclease digestion, samples in the 1 M NaCl lysis buffer were layered on a 1 mL sucrose cushion , centrifuged by a TLA 100.3 rotor at 65,000 rpm for 2 hr, and resuspended in the standard lysis buffer.Cells were harvested by filtration using a Kontes 99 mm filtration apparatus and 0.45 μm nitrocellulose filter (Whatman) and then flash frozen in liquid nitrogen. Cells were lysed in lysis buffer . 5–20 AU of E. coli lysate was loaded on top of sucrose gradient and centrifuged in a SW41 rotor at 35,000 rpm for 2.5 hr at 4°C. Fractionation was performed on a Piston Gradient Fractionator (Biocomp). Libraries for ribosome profiling and RNA-seq are prepared as in 10–54% sucrose density gradients were prepared using the Gradient Master 108 (Biocomp) in the gradient buffer . After centrifugation, cells were resuspended in 12.5 mM Tris pH 6.8, 4% SDS and heated to 90°C for 10 min. 5x loading dye  was added and lysate was heated to 90°C for 10 min. Protein was separated by 4–12% Criterion XT Bis-Tris protein gel (Bio-Rad) and XT MES buffer and transferred to PVDF membrane by Trans-Blot Turbo Transfer system (Bio-Rad). Membranes were blocked in 5% milk for 1 hr at room temperature. The membranes were probed by antibodies diluted in TBS-tween. FLAG-tagged proteins were detected by anti-FLAG-HRP in 1:10,000 dilution (Sigma). GFP was detected by anti-GFP in 1:2000 (Takara) and anti-mouse-HRP in 1:10,000 (Thermo Fisher). Cysteinyl-tRNA synthetase (CysRS) was detected by anti-CysRS in 1:2000 (from Dr. Ya-Ming Hou) and anti-rabbit-HRP in 1:4000 (from Dr. Ya-Ming Hou). Chemiluminescent signals of HRP were developed by SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher), and exposed on Amersham Hyperfilm ECL (GE).2HPO4 pH 7.8 and 20 mM EDTA) and frozen on dry-ice. Frozen samples were mixed with 150 µl of Luciferase assay lysis buffer (Cell Culture Lysis Reagent (Promega), 1.25 mg/ml lysozyme, and 2.5 mg/ml BSA) and incubated for 10 min at room temperature. Nanoluc and firefly luciferase activity were detected by Nano-Glo Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. Chemiluminescence was monitored by G:BOX (Syngene).Cells were grown as described for the ribosome profiling and RNA-seq libraries. At the second media change to fresh LB + glucose, anhydrotetracycline (1 nM) was added and the cells were harvested after another 60 min. Of cultured samples, 45 µl were mixed with 5 µl of phosphate buffer .Thank you for submitting your article "RRF is critical for ribosome recycling and homeostasis in The reviewers have discussed the reviews with one another, and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The reviewers found the work to be of high quality, and agreed that it represents an important advance in our understanding of both RRF function and translational coupling. Our one significant concern is that the bulk translation rate after RRF depletion was not measured. Interpretation of data from several experiments looking at translation coupling relies on the assumption that translation is ongoing in the RRF-depleted cells. The reviewers have a number of other suggestions that can all be addressed without the need for further experiments.Essential revisions:1) Measure the bulk translation rate across the time-course of RRF depletion. The current interpretation of Figure 3, Figure 5 and Figure 6 is based on the assumption that translation is ongoing in the RRF-depleted cells. If translation levels are greatly reduced, the conclusions about translational coupling might not be valid.2) Acknowledge the fact that residual recycling is likely occurring in the RRF-depleted cells. If it is possible to measure this residual activity, that would be great, but such an experiment is not required.3) Include some discussion of the potential impact of pleiotropic effects of RRF depletion on the observed phenotypes. Given that most of the phenotypes were observed at the 5 minute time-point, the reviewers did not see this as a critical problem.4) Change the title to focus solely on translational coupling, which in the opinion of the reviewers is the most important aspect of the paper.The reviewers have additional comments, detailed below, that you should respond to with changes to the text where appropriate.Reviewer #1:Summary:E. coli, it depends upon the correctness of two fundamental assumptions that are not examined critically in the text. The first assumption is that effects resulting from the absence of RRF protein, which is almost complete at 15' and complete at 60' , can be solely attributed to the loss of RRF protein. Given that rrf is an essential gene, RRF removal could well have pleiotropic effects not considered by the authors. The second, and related, assumption is that ribosome occupancy is a faithful reporter of protein translation rates when RRF is absent. Missing are any direct measures of protein synthesis and cell viability that would test these assumptions.Although this MS addresses the interesting question of RRF function in the translation of coupled transcripts in Essential revisions:1) The present Discussion section is overlong and very speculative. It would be improved by substantial condensation. On the other hand, a critical analysis of the shortcomings of prior results on translational coupling and RRF would be valuable.2) Subsection “Loss of RRF does not affect translational coupling in polycistronic messages”. First paragraph. The last sentence is unclear. Also, it would be helpful to the reader to include a description of the essence of the Yamamoto et al., proposed mechanism. This might be better placed in the Discussion section.3) Figure 7F should be redrawn. At present, it is very reader unfriendlyReviewer #2:E. coli using ribosome profiling and a series of ad hoc expression reporters. The major (somewhat surprising) findings are that RRF KD has relatively little effect on translational coupling and that RRF KD triggers upregulation of ArfA ribosome rescue pathway via inactivation of tmRNA by unrecycled ribosomes. This work is of an outstanding technical quality and is very important for our understanding of ribosome recycling in bacteria and of the mechanism of translational coupling.In this study, Saito et al., explored the role of RRF by examining how mRNA translation responses to RRF knockdown in Essential revisions:1) In the absence of direct evidence, the authors are reasonably cautious not to state that translation re-initiation does not take place in bacteria, instead they cautiously state that it is not a major factor in translation coupling. Indeed, it is likely that some level of recycling occurs in RRF KD even after 60 minutes. The splitting could be spontaneous, assisted by backup pathways or by residual levels of RRF. Therefore, we cannot exclude a possibility that reinitiation makes some contribution to the coupling and still occur in RRF KD. Did author try to estimate what is the level of recycling in RRF KD?2) RO differences between WT and KD in Figure 5B seems larger than what would be normally described as "little or no effect", though it is hard to understand the significance of these differences without a reference for comparison. Assuming that translation initiation should affect expression of downstream cistrons more than upstream, perhaps in addition to the analysis in Figure 6, a comparison of upstream and downstream cistrons should be carried out separately. If RRF KD has no affect on coupling we should expect the correlation between WT and KD for upstream cistrons not significantly exceed that for downstream. Further, the ratios of upstream/downstream cistrons RO vary up to 2 orders of magnitude . Are they true coupled pairs within the same operons? How the same analysis would look like if done for RNA-seq reads? Would it make sense to do this with footprint densities instead of RO? Perhaps Ribosome Occupancy and Footprint Density  should be explicitly defined with a mathematical formula in the Materials and methods section.3) Presumably for binning mRNAs into highly and lowly translated, RO was calculated using WT, because otherwise collisions in KD would affect RO and collisions would be overrepresented in the high bin resulting in a circular argument. This is not clear. Also, it would be interesting to see a relationship between collision scores and 3'UTR scores to support the statement about their inverse correlation. Could stacking be induced by inability of "unrecycled" ribosomes to progress to 3'UTRs?4) The manuscript provides a very good overview of the literature related to translational coupling and reinitiation in bacteria, however, I think one relevant work is missing – Osterman et al., 2013. I think it would be good to add it to the Discussion section since it addresses many related questions and some of their observations are not in agreement with this work.Reviewer #3:E. coli. The authors argue that depleting RRF leads to piling up of ribosomes at the ends of ORFs, and association of non-translating ribosomes in 3' UTRs. Depleting RRF has no effect on the translational coupling of closely spaced ORFs, arguing against both of the two major models for this process. Lastly, the authors show that RRF depletion leads to induction of arfA expression, although the evidence for the proposed mechanism involving tmRNA is a little weak. The data are of high quality throughout, and the work is beautifully presented.This is a very clearly written manuscript that describes the consequences of depleting ribosome recycling factor (RRF) in The authors use the piling up of ribosomes upstream of stop codons to infer that a ribosome is stalled at the stop codon in RRF-depleted cells. However, the signal from the ribosome at the stop codon is unchanged. Either the model is wrong, or ribosome profiling data cannot capture this stalled state. In the latter case, there may be other ribosome states that are not being captured, meaning that the ribosome profiling data may give a misleading view of where ribosomes are located in RRF-depleted cells. I think it's important to figure out why the stalled terminating ribosomes are not detected, including the possibility that there are no stalled terminating ribosomes.- Figure 7F. The authors speculate that increased ribosome occupancy in tmRNA downstream of the ORF is due to translationally inactive ribosomes. This should be possible to assess using the high salt datasets.- Figure 7F. Assuming the ORF within tmRNA is the degradation tag, the ribosome occupancy represents ribosomes translating tagged non-stop mRNAs. A simpler model for arfA induction could be that free tmRNA levels are depleted due to ribosomes getting stuck at the end of the ORF in tmRNAs rescuing non-stop mRNAs. Summary:The reviewers found the work to be of high quality, and agreed that it represents an important advance in our understanding of both RRF function and translational coupling. Our one significant concern is that the bulk translation rate after RRF depletion was not measured. Interpretation of data from several experiments looking at translation coupling relies on the assumption that translation is ongoing in the RRF-depleted cells. The reviewers have a number of other suggestions that can all be addressed without the need for further experiments.Essential revisions:1) Measure the bulk translation rate across the time-course of RRF depletion. The current interpretation of Figure 3, Figure 5 and Figure 6 is based on the assumption that translation is ongoing in the RRF-depleted cells. If translation levels are greatly reduced, the conclusions about translational coupling might not be valid.Thanks for this suggestion. We measured bulk translation rates 5, 15, and 60 minutes after the second media change (the same time points as the ribosome profiling experiments). We find roughly 40% of protein synthesis levels remain after RRF depletion, a significant level that gives us confidence in our conclusions regarding translational coupling. These data are added as Figure 1—figure supplement 1. We also note that the nanoLuc reporters in Figure 5 used to measure translational coupling are induced by addition of anhydrotetracycline at the second media change. Their transcription and translation therefore takes place under conditions of RRF depletion, further arguing that we can detect translational coupling over this time period.2) Acknowledge the fact that residual recycling is likely occurring in the RRF-depleted cells. If it is possible to measure this residual activity, that would be great, but such an experiment is not required.This caveat was added to the first paragraph of the Discussion section.3) Include some discussion of the potential impact of pleiotropic effects of RRF depletion on the observed phenotypes. Given that most of the phenotypes were observed at the 5 minute time-point, the reviewers did not see this as a critical problem.This caveat was added to the first paragraph of the Discussion section.4) Change the title to focus solely on translational coupling, which in the opinion of the reviewers is the most important aspect of the paper.The title was changed to focus on translational coupling as suggested.The reviewers have additional comments, detailed below, that you should respond to with changes to the text where appropriate.Reviewer #1:Summary:E. coli, it depends upon the correctness of two fundamental assumptions that are not examined critically in the text. The first assumption is that effects resulting from the absence of RRF protein, which is almost complete at 15' and complete at 60' , can be solely attributed to the loss of RRF protein. Given that rrf is an essential gene, RRF removal could well have pleiotropic effects not considered by the authors. The second, and related, assumption is that ribosome occupancy is a faithful reporter of protein translation rates when RRF is absent. Missing are any direct measures of protein synthesis and cell viability that would test these assumptions.Although this MS addresses the interesting question of RRF function in the translation of coupled transcripts in We included the caveat about indirect effects in the Discussion section. To minimize these effects, we focus our analyses on phenomena directly related to recycling. We also note that most of these effects are seen even at the shortest time point (5 min). As described above, we directly measured protein synthesis levels over the course of the experiment to validate our use of ribosome density in the translational coupling assays.Essential revisions:1) The present Discussion section is overlong and very speculative. It would be improved by substantial condensation. On the other hand, a critical analysis of the shortcomings of prior results on translational coupling and RRF would be valuable.The Discussion section was edited to emphasize translational coupling. We removed two paragraphs containing speculative comments about recycling in general. With respect to prior studies with RRF, we improved the discussion of these studies in the introduction. One problem was that studies focusing on recycling did not use known translationally coupled pairs in their native context .2) Subsection “Loss of RRF does not affect translational coupling in polycistronic messages” -. First paragraph. The last sentence is unclear. Also, it would be helpful to the reader to include a description of the essence of the Yamamoto et al., proposed mechanism. This might be better placed in the Discussion section.This sentence was deleted. The mechanism proposed by Yamamoto et al., was fleshed out more in the Discussion section.3) Figure 7F should be redrawn. At present, it is very reader unfriendlyWe redrew Figure 7F so that the traces are no longer overlapping and the cartoon of the tmRNA sequence is more informative.Reviewer #2:E. coli using ribosome profiling and a series of ad hoc expression reporters. The major (somewhat surprising) findings are that RRF KD has relatively little effect on translational coupling and that RRF KD triggers upregulation of ArfA ribosome rescue pathway via inactivation of tmRNA by unrecycled ribosomes. This work is of an outstanding technical quality and is very important for our understanding of ribosome recycling in bacteria and of the mechanism of translational coupling.In this study, Saito et al. explored the role of RRF by examining how mRNA translation responses to RRF knockdown in Essential revisions:1) In the absence of direct evidence, the authors are reasonably cautious not to state that translation re-initiation does not take place in bacteria, instead they cautiously state that it is not a major factor in translation coupling. Indeed, it is likely that some level of recycling occurs in RRF KD even after 60 minutes. The splitting could be spontaneous, assisted by backup pathways or by residual levels of RRF. Therefore, we cannot exclude a possibility that reinitiation makes some contribution to the coupling and still occur in RRF KD. Did author try to estimate what is the level of recycling in RRF KD?See above in Essential revisions, point 2.2) RO differences between WT and KD in Figure 5B seems larger than what would be normally described as "little or no effect", though it is hard to understand the significance of these differences without a reference for comparison.We removed these data from Figure 5B.Assuming that translation initiation should affect expression of downstream cistrons more than upstream, perhaps in addition to the analysis in Figure 6, a comparison of upstream and downstream cistrons should be carried out separately. If RRF KD has no affect on coupling we should expect the correlation between WT and KD for upstream cistrons not significantly exceed that for downstream.As suggested, we selected pairs of genes and compared the upstream gene ribosome density in the WT and KD strains. Separately, we compared the downstream gene density in the WT and KD strains. These data have the same correlation, as expected, and consistent with our conclusions in Figure 6.Further, the ratios of upstream/downstream cistrons RO vary up to 2 orders of magnitude . Are they true coupled pairs within the same operons? How the same analysis would look like if done for RNA-seq reads?We agree that the ratios vary more than one would expect for ORFs in the same mRNA. We were conservative in our operon definitions, using the Wanner et al., 2014 definitions of transcriptional units as curated in RegulonDB. We have now applied an additional criterion for inclusion: less than a five-fold difference in RNAseq levels between the upstream and downstream gene, because we agree that greater differences in RNA level were likely to arise from differential transcription. Figure 6 now includes these updated data but the conclusion remains the same.Would it make sense to do this with footprint densities instead of RO? Perhaps Ribosome Occupancy and Footprint Density  should be explicitly defined with a mathematical formula in Materials and methods section.The data shown in Figure 6B are ratios of footprint densities, not RO. We have included more description of these metrics in the Materials and methods section.3) Presumably for binning mRNAs into highly and lowly translated, RO was calculated using WT, because otherwise collisions in KD would affect RO and collisions would be overrepresented in the high bin resulting in a circular argument. This is not clear. Also, it would be interesting to see a relationship between collision scores and 3'UTR scores to support the statement about their inverse correlation. Could stacking be induced by inability of "unrecycled" ribosomes to progress to 3'UTRs?mRNAs were binned using RO values from the WT sample, as you say, and then the density from the KD is shown in Figure 4. The text and caption were altered to make this clear. Regarding the last point, we agree that stacking is caused by post-TCs stalled at stop codons. But whether or not the post-TC remains there or slides into the 3’-UTR depends on the ribosome occupancy of the gene (which is at least in part determined by its initiation rate). The simplest way to explain the correlation between RO and stacking (and lower 3’-UTR density) is to postulate that stacking prevents post-TCs from progressing into the 3’-UTR.4) The manuscript provides a very good overview of the literature related to translational coupling and reinitiation in bacteria, however, I think one relevant work is missing – Osterman et al., 2013. I think it would be good to add it to the Discussion section since it addresses many related questions and some of their observations are not in agreement with this work.We have decided not to include this reference because although it describes experiments aimed at understanding the mechanism of translational coupling, it does not specifically address recycling or RRF. Like many papers on coupling, it cannot distinguish between models of re-initiation and the effects of RNA structure on de novo initiation.Reviewer #3:E. coli. The authors argue that depleting RRF leads to piling up of ribosomes at the ends of ORFs, and association of non-translating ribosomes in 3' UTRs. Depleting RRF has no effect on the translational coupling of closely spaced ORFs, arguing against both of the two major models for this process. Lastly, the authors show that RRF depletion leads to induction of arfA expression, although the evidence for the proposed mechanism involving tmRNA is a little weak. The data are of high quality throughout, and the work is beautifully presented.This is a very clearly written manuscript that describes the consequences of depleting ribosome recycling factor (RRF) in The authors use the piling up of ribosomes upstream of stop codons to infer that a ribosome is stalled at the stop codon in RRF-depleted cells. However, the signal from the ribosome at the stop codon is unchanged. Either the model is wrong, or ribosome profiling data cannot capture this stalled state. In the latter case, there may be other ribosome states that are not being captured, meaning that the ribosome profiling data may give a misleading view of where ribosomes are located in RRF-depleted cells. I think it's important to figure out why the stalled terminating ribosomes are not detected, including the possibility that there are no stalled terminating ribosomes.We recognize that it is unsatisfying that we cannot directly observe a strong increase in post-TCs at stop codons. We looked for stop codon peaks in reads of all sizes, thinking that perhaps these reads were shorter or longer than normal, but did not find an increase in stop peaks with any footprint size. It may be that collisions with upstream elongating ribosomes prevent us from isolating the relevant ribosome footprints. Nevertheless, we are confident that the queuing of elongating ribosomes upstream of stop codons is strong indirect evidence of post-TCs stalled at stop codons.- Figure 7F. The authors speculate that increased ribosome occupancy in tmRNA downstream of the ORF is due to translationally inactive ribosomes. This should be possible to assess using the high salt datasets.This is a good suggestion. We observe fewer ribosome footprints on tmRNA in the high salt datasets. A sentence was added to this effect in the Results section.- Figure 7F. Assuming the ORF within tmRNA is the degradation tag, the ribosome occupancy represents ribosomes translating tagged non-stop mRNAs. A simpler model for arfA induction could be that free tmRNA levels are depleted due to ribosomes getting stuck at the end of the ORF in tmRNAs rescuing non-stop mRNAs.Yes, this is our model, that ribosomes stuck on tmRNA inhibit its activity, leading to activation of ArfA.


The majority of births in the United Kingdom happen in hospital or at stand-alone midwife led centres, or with the support of midwives in a planned fashion outside of hospital. The unplanned birth of a baby in the pre-hospital setting is a rare event which may result in an ambulance being called, so attendance at a birth is a rare event for ambulance clinicians. A service evaluation was conducted to report which clinical observations were recorded on babies born in the pre-hospital setting who were attended by ambulance clinicians from the North East Ambulance Service (NEAS) over a one-year period.A retrospective service evaluation was conducted using routinely collected data. All electronic patient care records covering a one-year period between 1 October 2017 and 30 September 2018 with a primary impression of ‘childbirth’ were examined.This evaluation identified 168 individual pre-hospital childbirth cases attended by NEAS clinicians during the evaluation timeframe. The majority (85%) of babies were born to multiparous mothers with a median gestation of 39 weeks. Very few clinical observations were recorded on the babies  and no babies had all five of these observations documented. Only 5% of babies had any complications documented.This study showed that NEAS ambulance clinicians rarely attend babies born in the pre-hospital setting and that complications were infrequently recorded. There was a lack of observations recorded on the babies, which is an issue due to the clear link between easily measurable characteristics such as temperature and mortality and morbidity. The majority of births in the United Kingdom occur in hospital or at stand-alone midwife led centres, or with the pre-planned support of midwives outside of hospital. The birth of a baby in the pre-hospital setting is often unplanned and attendance at a birth is an uncommon event for ambulance clinicians. The rate of babies born before arrival (BBA) at hospital in the United Kingdom was reported as 0.5% . The ratBabies born in an unplanned fashion in the pre-hospital setting have an increased risk of complications such as hypothermia, perinatal mortality, low birth weight and admission to neonatal intensive care . DespiteA recent update to the UK Ambulance Service Clinical Practice Guidelines  revised The aim of this service evaluation was to report if these clinical observations were being fully recorded on babies born in the pre-hospital setting who were attended by ambulance clinicians from the North East Ambulance Service (NEAS). This evaluation was conducted as the data describing the pre-hospital care of these patients are scarce, to identify areas for improvement and to support future research.A retrospective service evaluation was conducted using routinely collected data to determine what clinical observations were recorded on babies born in the pre-hospital setting who were attended by NEAS clinicians over a one-year period.NEAS is the regional ambulance provider for around 2.5 million people in north east England, covering Northumberland, Tyne and Wear, County Durham, Darlington and Teesside. NEAS employs around 1200 ambulance clinicians  who work out of 56 stations across the region .All electronic patient care records (ePCRs) covering a one-year period between 1 October 2017 and 30 September 2018 with an ambulance clinician recorded primary impression of ‘childbirth’ were requested from the NEAS informatics team.The ePCRs were then filtered based on the following inclusion and exclusion criteria:Inclusion:Baby born in the pre-hospital settingExclusion:Baby born in hospitalMidwife in attendance at pre-hospital birthMiscarriage or still birthThe following data were manually extracted from the ePCRs by the authors:Relevant timings Destination hospitalAge of motherParity (baby number)GestationClinical observations recorded on the baby Complications and interventions performedThese items were selected by the authors as being relevant to care of the newborn, available in the pre-hospital setting and included in national ambulance guidelines .All data are presented in a descriptive fashion along with the number of cases where data were available. Summary statistics are presented using median, interquartile range (IQR) and range. Complications and interventions were identified and extracted from the narrative and treatment sections of the ePCR and are presented in an aggregate fashion due to the small number of patients involved.There were 236 ePCRs with ‘childbirth’ recorded as the impression between 1 October 2017 and 30 September 2018. The ePCRs were filtered using the inclusion and exclusion criteria, which resulted in 168 individual childbirth cases being included in this service evaluation.From the original 236 ePCRs identified, 68 cases were excluded for the following reasons: baby transported prior to crew’s arrival (n = 1); babies born and cared for by midwives (n = 2); duplicate cases (n = 24); miscarriages (n = 6); not childbirth (n = 5); still birth (n = 1); transported to hospital for birth (n = 29).The median call to hospital time (n = 146) was 65 minutes . The median on scene time (n = 149) was 30 minutes . The median birth to hospital time (n = 138) was 55 minutes .The times when the calls leading to the pre-hospital birth were received by the ambulance service were examined (n = 154), with 23% of calls in office hours (0900–1700) and 63% overnight (2000–0800).One hundred and sixty-six babies were transported to 10 different hospitals across the north east; two babies were not transported as midwives arrived after the babies were born and stood the ambulance crews down.The median age of the mother (n = 166) was 28 years .Parity was recorded in 110 cases, with the median value of second baby  was 39 weeks .Eighteen (10%) babies had a temperature recorded. In 14 of these cases a single temperature was recorded and in four cases two temperatures were recorded. The median temperature was 36.3°C . The location where the temperature was taken from was recorded in 13 cases: axillary (n = 2); temporal (n = 2); tympanic (n = 9).APGAR was recorded for 13 (8%) babies, with one baby scoring eight, one baby scoring nine and 11 babies scoring 10.Two (1%) babies had a BM recorded (3.1 and 5.9).Thirty-five (21%) babies had a heart rate recorded. In 28 cases a single heart rate was recorded, in five cases two heart rates were recorded and two cases had four heart rates recorded. The median heart rate was 150 .Thirty-eight (23%) babies had a respiratory rate recorded. In 30 cases a single respiratory rate was recorded, in six cases two respiratory rates were recorded and in two cases four respiratory rates were recorded. The median respiratory rate was 38 .The total number of observations included in the basic minimum set defined earlier  documented on each baby is displayed in Ninety-seven (58%) babies had some form of warming recorded involving combinations of blankets, clothing and hats, towels and foil/blizzard blankets.Nine (5%) cases had complications documented including issues with the umbilical cord and concerns around breathing. Six (4%) cases involved inflation breaths and/or oxygen administration to the baby. Three (2%) cases had CPR or resuscitation documented.Eight (5%) mothers received syntometrine, with one patient receiving tranexamic acid.This service evaluation describes the clinical data recorded on babies born in the pre-hospital setting attended by NEAS and shows that very few observations, including those recommended in national guidelines, were documented. No babies had all five basic observations documented and 71% had none of these observations documented. Of pre-hospital births attended by ambulance clinicians, 93% had no complications or interventions recorded, so the omission of basic observations is difficult to explain.The mothers described in this study were slightly younger than the national average which was reported as 30.5 in 2017 , but werPrevious research reporting babies’ BBA  describeThere was a low rate of recording of all of the clinical observations reported. Only 10% of babies had a temperature recorded and 72% (n = 13) of these were below 36.5°C, which is the lower limit recommended by the World Health Organization  and the The maintenance of normothermia (36.5–37.5°C) has been shown to be very important to newborns, and deviations from this range, particularly hypothermia, are linked to increased mortality and morbidity . EvidencThis evaluation is limited by the retrospective nature of the data and the potential for observations and interventions to not have been documented. The small number of births could also be a limitation of this study. However, the total number of live births in the north east in 2016 was 28,574 . ApplyinThere is no reason to believe that pre-hospital births in the north east differ significantly from pre-hospital births across the United Kingdom; therefore, the data from this service evaluation should be generalisable to other areas. However, a national audit or database of pre-hospital births, which would be beneficial for research and development in this area, would be needed to prove or disprove this assertion. NEAS clinicians are trained in a similar fashion and to a similar standard to other UK pre-hospital clinicians, and ambulance protocols across the United Kingdom are based on national guidelines such as JRCALC ; howeverThe low rate of recording of basic observations is worrying and the reasons for this need to be explored. Possible explanations for this lack of recorded observations could be: a lack of confidence in taking neonatal observations; thinking taking and/or recording the observations was unnecessary in babies that appear well; lack of equipment (e.g. saturation probes and blood pressure cuffs) designed for neonates; and other reasons.The number of pre-hospital births can be used to estimate the exposure of ambulance clinicians to these events. If 168 births are evenly distributed between approximately 1200 clinicians , then thAmbulance clinician education and training may need to reinforce the reasons why taking, recording and acting on observations such as temperature in a newborn baby are important in order to change practice going forward. In order to enable good practice, equipment suitable for taking observations on newborn babies needs to be available.Further research is needed to more comprehensively describe the clinical characteristics of babies born in the pre-hospital setting. These data could potentially be used to explore any links between pre-hospital birth, the initial clinical presentation of the baby, the need for treatment in or out of hospital and longer-term outcomes.This study shows that NEAS ambulance clinicians rarely attend babies born in the pre-hospital setting, and when they are present the births are often uncomplicated and require few interventions. There was a lack of observations recorded on the babies which is an issue due to the clear link between easily measurable characteristics such as temperature and mortality and morbidity. More research is needed to establish the barriers and facilitators to taking and recording observations on babies. Consistent recording of these data would allow areas for improvement to be identified and linking the pre-hospital data to hospital data would allow longer-term implications and outcomes to be explored.All authors contributed to the study and read and approved the final manuscript.None declared.Not required.None.


The long-term risks and time trends of subsequent primary neoplasms (SPNs) among Ewing (ES) and osteosarcoma (OS) survivors are not fully understood.We performed a nationwide study of all ES and OS patients identified in the Swedish Cancer Registry from 1958 to 2015 with up to 58 years of follow-up. The risk of SPN was compared with that of the general population using standardised incidence ratios (SIRs) and absolute excess risks (AERs).2000-2015 2.0; 95% CI, 1.1–3.5). The highest absolute excess risks (AER) was due to breast cancer  followed by female genital malignancies . The excess breast cancer risk among ES survivors was noted also after 30 years of follow-up with 127 extra breast cancers/10,000 person-years .One hundred and fifteen SPNs were diagnosed among 1779 patients with ES or OS, yielding an overall SIR of 2.3 , 1.9–2.7). The risk remained significantly increased in the latest treatment era (SIRBreast- and female genital malignancies contribute most to the excess risk of SPN among ES and OS survivors. Importantly, excess risks did not decline over calendar time or long-term follow-up. The lower risk was clearly associated with a reduction in the use of radiotherapy (RT) and radiation dose.6 Heritable retinoblastoma, Hodgkin lymphoma and bone sarcoma are the three primary childhood malignancies with the highest excess risk for SPNs.6 The increased risk can be attributed to genetic-, disease- or treatment related factors. Heritable retinoblastoma survivors are at increased risk due to germline inactivation of the retinoblastoma gene.7 Hodgkin lymphoma survivors have an increased treatment related cancer risk mainly driven by RT, but also by treatment with alkylating agents.9 Osteosarcoma (OS) and Ewing sarcoma (ES), the two most common childhood bone sarcomas, may have different aetiologies contributing to the excess cancer risk, yet they are commonly reported as one entity.3 Treatment with high dose multi-agent chemotherapy for OS and ES has hardly changed since the 1970s; however, only ES patients commonly receive RT, perhaps the most significant driver of excess cancer risk. Importantly, if a lower risk in recent treatment eras can be confirmed it may reflect a positive effect of a change in primary treatment such as decreased use of RT. Whether the risk persists with extended follow-up has important implications for recommendations on how long bone sarcoma survivors should be followed. Furthermore, as bone sarcoma survivors reach the age at which the cancer incidence in the general population increases significantly, it is important to identify which neoplasms contribute most to the excess risk. The aims of this nationwide population-based study were to; (1) estimate the risk of SPNs among ES and OS survivors; (2) investigate whether the absolute excess risk persists with long-term follow-up; (3) identify which subsequent neoplasms that contribute most to the excess risk; and (4) analyse the risk of SPNs among patients treated during more recent treatment eras.Compared with the general population the increased risk for subsequent primary neoplasms (SPNs) in childhood cancer survivors has been clearly demonstrated in large studies in the U.S. and Europe.10 In addition, histologically benign tumours of the central nervous system (CNS) are also included in the register. All tumours of the lower urinary tract with cellular atypia, including papilloma of the bladder, are included in the register because of the difficulties in classifying the malignant potential of bladder tumours.3 Basal cell skin carcinoma is not included in the register. Reporting from multiple sources is required and the completeness of the register is over 96%.12 The methods of data collection in the register have been described elsewhere.12This was a nationwide population-based register study. The cohort consisted of all ES and OS patients identified in the Swedish Cancer Registry during the period 1 January 1958–31 December 2015. All cancers occurring after the primary bone sarcoma diagnosis were included as outcomes in the analysis. The ES and OS patients were identified through International Classification of Diseases, 7th edition (ICD-7) codes in the Swedish Cancer Registry. Reporting with newer versions of the International Classification of Diseases (ICD-9 and ICD-10) was automatically converted to ICD-7 codes. The register includes data corresponding to a specific ICD-7 and morphology code of all new malignant tumours diagnosed in Sweden since 1958. Acceptance of a new cancer to the registry in a patient with an already known cancer follows the rules of the International Association of Cancer Registries (IACR).13 Before 1993 tumour morphology and tumour behaviour were coded according to the World Health Organization C24 code.14The ICD-7 codes we used to identify the ES or OS cases by histology and anatomical location were: central location ; extremity ; unknown primary skeletal location (96.9); and soft tissue origin . The morphological tumour type was ascertained from code 92603 (ES) or 91803 (OS) using the ICD-O WHO  morphological classification of cancers, which has been used in Sweden since 1993.15 It is controversial whether multiple skeletal OS are two distinct clonally unrelated tumours or if they represent one primary tumour with skeletal metastasis.16 For this reason, the SPNs in these four cases were regarded as metastatic disease rather than new primaries.Time at risk for a SPN started at the time of the first diagnosis of ES or OS and continued until death, end of follow-up (31 December 2015) or until a diagnosis of a SPN was made, whichever came first. If an individual had two SPNs, only the first SPN was included in the analysis of overall risk. However, for the analysis of specific SPNs, subsequent primary neoplasms beyond the second neoplasm were also assessed. Four individuals had the same morphology code for the primary and subsequent neoplasms. Three of these individuals were diagnosed with a primary OS and the subsequent malignancies, all of which occurred within 24 months, were also osteosarcomas. There was also one ES patient with a subsequent tumour classified as a new ES. Synchronous (at time of diagnosis) or metachronous (separated in time) OS, occasionally seen among patients with a predisposing syndrome, are rare manifestations of OS, where two or more skeletal lesions are seen without the presence of metastasis in the lungs or other non-skeletal foci.17 The number of expected cancer cases was calculated by multiplying the Swedish incidence rates by the total person-time at risk for each corresponding age, sex, and calendar year stratum in our cohort. Overall and cancer type-specific standardised incidence ratios (SIRs) were estimated by dividing the observed number of cancer cases by the number of expected cases. SIRs were calculated stratified by sex, age at diagnosis, years since ES or OS diagnosis, calendar year at ES or OS diagnosis and site of primary tumour. The absolute excess risk (AER) was defined as the observed minus expected number of cancers divided by person-years at risk multiplied by 10,000. Exact 95% confidence intervals for SIR and AER estimates were calculated assuming that the number of observed cases followed a Poisson distribution. In analyses limited to our cohort we estimated the cumulative incidence of overall cancer stratified by ES or OS diagnosis. Here, death was considered as a competing event.Data regarding age- (five-year classes), sex- and calendar year-specific incidence rates relative to the Swedish population for overall and specific cancer types were retrieved from the National Board of Health and Welfare.Patients with a malignancy prior to ES or OS diagnosis were believed to represent a high-risk group for SPN. A sensitivity analysis was therefore conducted by excluding patients with a previous history of cancer prior to their primary bone tumour malignancy (Supplementary Table n = 18) followed by genitourinary cancer (n = 17), skin cancer (melanoma or squamous cell carcinoma) (n = 13), CNS neoplasm (n = 11), sarcoma (other than the primary bone sarcoma type) (n = 10) and female genital malignancy (n = 10).Through December 2015, 115 SPNs were diagnosed in 104 out of the 1779 patients in the cohort. Fourteen patients had two SPNs and two patients had three SPNs. The total follow-up time was 16,170 person-years . The most commonly observed SPNs were breast cancer (n = 18), skin cancer , genitourinary malignancies (n = 16) and female genital malignancies (n = 15). Two patients were diagnosed with retinoblastoma and three patients with a sarcoma (two soft tissue sarcomas and one chondrosarcoma) prior to the primary bone sarcoma diagnosis. The median time from the previous malignancy to the primary bone sarcoma diagnosis was 6 years (range 0–42 years).Eighty-six patients (5%) had suffered a malignancy prior to their primary diagnosis of OS or ES: seventy-nine (7%) of the OS patients and 7 1%) of the ES patients  compared with earlier treatment eras   and OS survivors 1.9  times more likely to develop a SPN than expected. By age at primary tumour diagnosis, the highest SIR among ES survivors was evident in the age group 10–19 years at diagnosis, with a 7.2-fold increased risk for a SPN compared to the general population . For OS survivors, the highest SIR was in patients aged 0–9 years at diagnosis who were 6.7 times more likely to develop a SPN than expected . By anatomic site, the highest SIR was observed for pelvic ES . The risk was not lower for patients treated in later eras (SIR6) Table . The oveWith regard to specific SPNs, the overall SIR for subsequent female genital malignancies was 3.3 times more than expected  with an AER of 9.7 cases per 10,000 person-years . Only OS survivors demonstrated increased risks for CNS neoplasms  and skin cancer . The overall SIR  for digestive tract malignancies was not higher than expected Table .Table 3SES 4.7; 95% CI, 1.7–10.2 and SIROS 2.1; 95% CI 1.1–3.6) . The highest SIR was evident for ES survivors with a centrally but non-pelvic primary ES location . No breast cancers were recorded among pelvic ES survivors. Due to small numbers, any trend in risk over calendar period was difficult to evaluate. The median time from primary diagnosis to the development of a breast cancer was 27 (1–49) years for ES- and 23 (3–46) years for OS survivors.With respect to primary tumour type, ES survivors were 4.7 and OS survivors 2.1 times more likely than the general population to develop a subsequent breast cancer . Eight subsequent soft tissue sarcomas developed after a median of 18 and 19 years for ES and OS survivors, respectively. Two secondary osteosarcomas were observed in 2 ES survivors and no secondary bone sarcomas were seen among the OS patients. The median time to development of a subsequent bone sarcoma was 8 years.The overall risk for developing a secondary soft tissue sarcoma was nearly 21-fold that of the general population  Table . The risThe rate of a subsequent haematological malignancy was 5.6  and 1.6  times that expected among ES and OS survivors respectively  population-based cancer registry in the US, but this study had a smaller sample size and fewer SPNs than in the current study.4This large-scale population-based study is the first of its kind to demonstrate that even after 30 years of follow-up, ES survivors are at a significantly higher risk than the general population for developing a subsequent breast cancer. Due to the high incidence of breast cancer in the general population the excess risk after 30 years of follow-up is especially noteworthy. This result stands in contrast to the British Childhood Cancer Survivor Study (BCCSS), which did not demonstrate an increased risk for a subsequent breast or any other SPN among ES and OS survivors after 30 years of follow-up, perhaps due to smaller sample size. Significantly elevated risks for subsequent breast cancers in the present study were most apparent for patients treated at age 10–19 years and for centrally located tumours. Although not consistent in different paediatric cancer cohorts these data support the notion that females treated at pubertal age, when breast tissue proliferates, have the highest risk for a subsequent breast cancer.6 In the latter study, there was a reduction in SPNs among childhood cancer survivors treated in the 1990s compared with those diagnosed in the 1970s; a reduction that correlated clearly with a reduction in radiation dose.6 However, when stratified into individual malignancies, the risk was only significantly reduced for Hodgkin lymphoma. The use of RT was reduced from 77% to 34% of patients during the study period. More restricted use of RT was also noted in a Children’s Oncology Group study, including ES patients treated from 1996-98 in which 65% of the patients were treated without RT.23 The reason that the risk for SPNs remains high for the ES survivors in the current study is unknown. An explanation could be that the use of RT has not changed substantially over the time period studied and that RT is still a significant cause of SPNs. Lastly, there may also be other factors that drive the high risk of SPNs among ES and OS survivors.Another important finding in this study is that the enhanced risk for SPNs remains elevated for patients treated during recent years, a finding that was also noted in the CCSS cohort.24 The BCCSS is a population-based national childhood cancer registry study encompassing 664 individuals diagnosed with bone cancer between 1940 and 1991.24 Although not population-based but unique due to the size, long follow-up and detailed treatment information, the CCSS study included 1205 OS and 714 ES patients diagnosed before the age of 21 years.6 Another population-based Swedish study performed more than 10 years ago estimated SIRs of 1.5 and 5.6 for OS and ES survivors, respectively.25 The SEER study (overlapping cohorts with the CCSS) reported SIRs of 12.9 and 5.6 for ES and OS survivors, respectively, although the AERs were lower than in the present study.4 Studies of cumulative incidence of SPNs in ES cohorts have reported great variations, with the risk varying from 0.9% at five years to 42.8% at 20 years, but the competing risk of death has not always been accounted for.33 The cumulative incidences of SPN at 30 years, available in the aforementioned CCSS and BCCSS cohorts,24 were strikingly similar to the results in the present study.The ES- and OS-specific SIRs and AERs for patients younger than 20 years in the present study correspond well to the results reported by the North American CCSS and the aforementioned BCCSS.37 To our knowledge there is no such genetic predisposition associated with ES. Moreover, nearly 7% of the OS patients in this cohort were diagnosed with another malignancy prior to OS diagnosis, whereas this was only the case for 1.2% of the ES patients. This indicates that a number of OS patients in our cohort harbour a cancer predisposition syndrome, or have received treatment prior to primary OS diagnosis with alkylating agents, anthracyclines or RT, all of which may cause OS.39 Apart from genetic predisposition in OS patients, differences in the use of RT may be a major reason for the differences observed in SPNs in this study. Radiotherapy has traditionally been considered mainstay of local treatment of ES. OS on the contrary, is regarded as a RT- resistant malignancy, and therefore RT has been restricted to non-resectable or incompletely resected tumours associated with poor prognosis,  where few patients will survive long enough to acquire a SPN. The use of RT in the treatment of primary ES is debated and has led to different local treatment strategies in the US and Europe.The increased risk for SPNs among OS and ES survivors is likely to have different aetiologies. For OS survivors the excess risk can be partly attributed to a genetic predisposition. Li Fraumeni, hereditary retinoblastoma and Rothmund-Thompson syndrome are associated with OS and other malignancies including breast and skin cancer.40 Another concern associated with the length of the study period is the changes in treatment that have occurred over time. However, the risk was not lower for patients treated later during the study period, after which conformal radiation techniques such as intensity modulated- and image guided techniques were introduced. Regarding chemotherapy, the regimes have been unchanged for the past 40 years.This study is limited by the lack of treatment information. The Swedish Cancer Registry does not encompass information about treatment and there is no clinical registry available for linkage that spans as far back as the beginning of this study period. One of the strengths of this study includes the solid quality of the Registry and the length of the study period. The quality of the data relies on the unique personal identification number available for all Swedish citizens, and the multiple sources for reporting to the register. Adding treatment variables such as type and dose of chemotherapy and type and site of RT would be very useful but would likely be at the expense of the quality of the Registry. Assessing individual patient records would be a possibility, but due to the long study period it is likely that detailed treatment information still would be lacking to a large extent. Other studies on ES survivors with information on treatment have still had difficulties comparing patients treated with and without RT since the majority (75–90%) of ES survivors have been treated with RT, thus rendering the non-RT group too small for meaningful analysis.3 However, basal cell carcinomas are not registered in the Swedish Cancer Registry and all SPNs except for 3 meningiomas observed in this study were invasive malignancies unlikely to be missed. Furthermore, brain imaging is not part of routine follow-up of OS patients.Some of the excess risk observed in this study may be due to surveillance bias. Basal cell carcinomas and non-glioma CNS tumours are known to be underestimated in the general population due to their indolent course.41 The Children’s Oncology Group in the United States have developed comprehensive exposure related guidelines (COG-LTFU guidelines) aiming at early detection and timely intervention of late effects such as SPNs.42 However, as stated by the ESMO-PaedCan-EUROCAN guidelines, there is a lack of consensus among experts on optimal follow-up recommendations.43 We believe the current study provides important data which will improve current guidelines for surveillance among patients who have survived primary bone sarcoma.As the number of childhood cancer survivors increases, so does the need for guidelines for long-term follow-up of treatment related side-effects. Two out of three survivors will develop at least one late onset therapy related complication.In conclusion, this study shows that ES and OS survivors are at increased risk for developing specific SPNs, a risk that remains elevated with continued long-term follow-up, especially for female ES patients who have substantial risk for subsequent breast cancer even after 30 years. Early and continued screening for breast cancer among females is motivated as ES and OS survivors reach the age at which the breast cancer incidence becomes significant in the general population. Furthermore, an association between primary bone sarcoma and subsequent female genital malignancies was shown in this study. The significant contribution of breast cancer and female genital malignancies to the excess cancer risk suggests a possible role of BRCA-associated genetic aberrations among female bone sarcoma patients. Lastly, this study demonstrates that the increased cancer risk that bone sarcoma patients face, remains high even with modern treatment regimens.Supplementary tables


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes the coronavirus disease 2019 (COVID-19), is having a deep impact across the globe.As of 4 February 2021, over 103 million confirmed cases and 2.25 million deaths due to COVID-19 had been reported.,On 30 January 2020, the World Health Organization (WHO) declared a Public Health Emergency of International Concern; in early February 2020, it launched its strategic preparedness and response plan and the accompanying operational guidelines. This plan outlined key public health measures to guide efforts of national and international partners.The platform has facilitated actions within these initial nine strategic pillars and continues to do so.As of 4 February 2021, the platform has tracked 9.3 billion United States dollars (US$) of requested resources and approximately US$ 8 billion in donor contributions.Coordination is at the centre of the platform and critical to an effective response. In January 2021, a tenth pillar was added to the platform to support national deployment of COVID-19 vaccines when available.,,,Vaccines are currently being provided to several countries, mostly high-income, for mass vaccination programmes.With the coordinated sharing of expertise and resources at national, regional and international levels, the novel digital platform is a new way of responding to emergencies: a way that can decrease disparities in readiness among countries that are struggling in their response to mitigate the spread and impact of the virus.


A comparative study was made to investigate the effect of average power by varying frequency (1.2–1.6 kHz) and pulse width (20–60 μs) on the deposition rate, microstructure, crystal orientation, and current waveforms of Cr2AlC MAX phase coatings. X-ray diffraction (XRD), scanning electron microscopy (SEM), and atomic force microscopy (AFM) were used to characterize the deposited coatings. The influence of pulse width was more profound than the frequency in increasing the average power of HiPIMS. The XRD results showed that ex situ annealing converted amorphous Cr-Al-C coatings into polycrystalline Cr2AlC MAX phase. It was noticed that the deposition rate, gas temperature, and roughness of Cr2AlC coatings depend on the average power, and the deposition rate increased from 16.5 to 56.3 nm/min. Moreover, the Cr2AlC MAX phase coatings produced by HiPIMS exhibits the improved hardness and modulus of 19.7 GPa and 286 GPa, with excellent fracture toughness and wear resistance because of dense and column-free morphology as the main characteristic.The high-power impulse magnetron sputtering (HiPIMS) technique is widely used owing to the high degree of ionization and the ability to synthesize high-quality coatings with a dense structure and smooth morphology. However, limited efforts have been made in the deposition of MAX phase coatings through HiPIMS compared with direct current magnetron sputtering (DCMS), and tailoring of the coatings’ properties by process parameters such as pulse width and frequency is lacking. In this study, the Cr It may be because of the depletion of deposited Cr and Al, because of high ionization in HiPIMS, and will affect the Cr/Al ratio .Similarly, GIXRD patterns of as-deposited and annealed coatings prepared by HiPIMS at different average power are shown in DCMS see a. It is morphous . It is ke energy . Experime energy ,46,47. He energy ,43. The hkl from Bragg’s law  according to our previous work [2AlC coatings are in good agreement with the theoretical and experimental results for the Cr2AlC bulk and coatings. Moreover, the grain size of the coatings obtained at different average power ranges from 12 to 15 nm, which is smaller than that of bulk material, as expected. It is also observed that there is some degree of lattice strain in the coatings deposited by HiPIMS and DCMS, which is the possible reason for the peak shift towards the lower angle in the XRD patterns.It is noted that MAX phase peaks after annealing shift towards a lower Bragg’s diffraction angle as compared with that of the target material, which can have many origins, such as atomic ordering, change in chemical composition, change in crystallize size, lattice strain, and stresses arising due to mismatching at the interface of coating and substrate or due to thermal annealing. Therefore, lattice parameters, dislocation density, and lattice strain are calculated by calculating the dous work . It is o2AlC MAX phase coatings deposited by conventional DCMS and HiPIMS is shown in 2AlC MAX coatings without a Ti buffer layer deposited by DCMS. It is observed that the coating surface has a granular structure, which is a typical structural morphology of Cr2AlC coatings prepared by DCMS [2AlC coatings is affected by the bias voltage applied to the substrate during the DCMS. It is found that, with the increase in bias voltage, the column growth angle decreases from the right angle [2AlC MAX phase coatings are required because the columnar grain of Cr2AlC coatings has poor oxidation resistance due to inward diffusion of oxygen through the columnar grain boundaries, which cause the internal oxidation of the Cr2AlC coatings [2AlC coatings could be enhanced by changing the coating’s morphology from columnar to equiaxed [2AlC MAX phase coatings have been improved by increasing the columnar grain boundaries [The surface and cross-sectional morphology of the Cr by DCMS ,27. The equiaxed . Furtherundaries . Thus, t2AlC MAX phase coatings with a Ti buffer layer deposited by HiPIMS. The surface and cross-sectional morphology of coatings is significantly different from that of DCMS. It is observed that the coating surface possesses a smooth and crack-free morphology with finer grain size compared with that deposited by DCMS. Similarly, the cross-section microstructure of Cr2AlC MAX phase coatings deposited by HiPIMS possesses a smooth and glassy morphology with a fully dense structure without columns. The coatings prepared by both DCMS and HiPIMS without or with a Ti buffer layer are crack-free and well adherent to the substrate material, and interfaces between the Cr2AlC/Si(111) by DCMS and Cr2AlC/Ti/Si(111) by HiPIMS are clear and distinct. Our results are consistent with the Cr2AlC coatings deposited by DCMS without a buffer layer [One of the main characteristics of HiPIMS includes high ionization of target material that can be achieved at target peak power. The high ionization of target material can modify the deposited layer microstructure, and hence their properties. It is reported that modulating the ion-to-atom ratio (by changing pulse width and/or frequency) can change the microstructure of CrN and TiN films from columnar to fully dense along with improved hardness and roughness with excellent adhesion ,54. Figuer layer .2AlC MAX phase coatings deposited by HiPIMS is exclusively different from the structures reported previously [2AlC MAX phase coatings is responsible for enhanced hardness and modulus (discussed later). According to the structural zone model (SZM) by Thornton, the deposited Cr2AlC coating structure belongs to zone-T [2AlC is 2. It is also observed that the deposited Cr in HiPIMS is depleted as compared with deposited Cr in DCMS in coatings owing to the high degree of ionization. Moreover, the elemental mapping was enriched in Cr, Al, and C elements and is equally distributed throughout the coatings’ surface with a small amount of O element, which could be from the impurity of the target or due to exposure of samples to ambient for ex situ annealing , as expected. The duplex morphology consists of outer Cr2AlC-layer/Ti-layer and Ti-layer/substrate interfaces that are clear, distinct, and well bonded to each other. Featured grains are non-distinguishable at this scale and are believed to be polycrystalline Cr2AlC MAX phase, as shown in GIXRD spectra in 2AlC coating was according to the deposition parameters, and the deposition rate was measured from SEM micrographs by taking an average of ten measurements. The purpose of the diffusion barrier (buffer layer) between the coating and substrate proved to be helpful to prone elemental interdiffusion  from the coating to substrate [2AlC and Al diffusion into the Zr substrate up to 1000 °C [2AlC/Ti/substrate interfaces are distinct and clear for all coatings, which indicates that there is no interdiffusion of Al occurring between the Cr2AlC coating and substrate along the Ti diffusion barrier during annealing. Furthermore, all the coatings deposited at different average power in HiPIMS show a similar morphology. The microstructure of TiBw/Ti6Al4V substrate consists of the darkish and white phase corresponding to equiaxed eviously ,32,58. Iubstrate . A doubl 1000 °C . The Cr22AlC Max phase. The Cr/Al ratio for as-deposited and annealed coatings grown by DCMS The Cr/Al atomic ratio of the coatings is presented in  process . Moreove2AlC coatings deposited by DCMS is also presented. Although the deposition rate of DCMS is almost double that of HiPIMS at the same level of power input, there is a limitation in the increase in deposition power, which leads to overheating and, consequently, damage of sputter magnetron. This drawback is overcome by HiPIMS, in which much higher power densities are applied to the target during the short pulse on-time, and overheating or damage to sputter magnetron is avoided. The average power increased by increasing pulse width and/or frequency; consequently, the deposition rate increases linearly. With the increase in pulse width and/or frequency, high ionization of target material occurred due to substantial ion bombardment on the target surface. This leads to more ejection of target atoms from the target surface, resulting in a higher deposition rate. According to the equation The deposition rate and gas temperature at different average power are shown in ing gas) . Moreovegas) [μs . However2AlC coating was examined by AFM. The coating roughness significantly affects the tribological performance and corrosion resistance of the coatings under service [2AlC coatings deposited on Si wafer by HiPIMS at different average power is presented in a) observed is 0.49 nm at an average power of 440 W. The energy of the ion of the sputtered material plays an essential role in the surface roughness and morphology of film [2AlC coatings with relatively high roughness are observed.Further, the surface morphology of the surface of the Cr service ,65. From service . In HiPI of film . When av2AlC coatings deposited on TiBw/Ti6Al4V composite by HiPIMS is shown in HiPIMS is a relatively new addition to the class of PVD techniques for thin-film technology compared with DCMS. The major difference between HiPIMS and conventional DCMS is the operation mode and more control over deposition parameters. There is a high degree of plasma density and a high degree of ionization of sputtered material in HiPIMS, while in DCMS, sputtered material mainly consists of neutral species. Therefore, HiPIMS can produce a better quality of coatings with improved hardness, roughness, and adhesion ,69. It h surface b,c. ThesThe formation of these craters might be due to the occasionally arcing when a sudden increase in current and decrease in discharge voltage occurred during the deposition process. Because of this, microscopic debris target material was introduced on the surface of the substrate, which may produce an explosive eruption on the films during sputtering deposition. Although the unipolar HiPIMS power supply used in this study is equipped with arc detection and its suppression  technology, but there has always been a delay in suppression after detection of an arc, which leads to plasma instability. The discharge plasma stability could be achieved by utilizing bipolar-pulse in which a small positive potential pulse was applied to the target during the pulse-off time to prone the arcing .2AlC MAX phase coatings before and after annealing deposited by DCMS and HiPIMS are illustrated in 2AlC MAX coatings deposited by HiPIMS compared with that of DCMS is the focus. It is noted that all the as-deposited coatings prepared either by DCMS or HiPIMS show relatively low indentation hardness (11.5–15.5 GPa) deposited at different average power. The low hardness is because of the amorphous nature of coatings before annealing. There is significant improvement in hardness and modulus observed after ex situ annealing due to crystallization of the Cr2AlC MAX phase. At different deposition power, the crystallized Cr2AlC MAX coatings show almost identical hardness ranging from 18 GPa to 20 GPa and elastic moduli between 270 GPa and 285 GPa, respectively. However, the obtained hardness of coatings is nearly double that of the TiBw/Ti6Al4V composite (5–8 GPa) used as a substrate [3/E2 ratio of the Cr2AlC coatings in function with the deposition power in HiPIMS. The H/E ratio represents the resistance of coating against elastic strain to failure, and the H3/E2 ratio shows the resistance to plastic deformation [3/E2 ratio is increased monotonously after annealing the coatings. All the coatings showed a nearly identical H/E and H3/E2 ratio, and the maximum value of the H3/E2 ratio is 0.098 for the coatings deposited at a power of 240 W. It is known that the high value of H/E can delay elastic to failure and a high H3/E2 indicates an excellent resistance to crack deformation and its propagation. The coatings produced by HiPIMS showed better wear resistance compared with DCMS. The column-free and dense microstructure of Cr2AlC MAX phase coatings benefits the excellent hardness and modulus along with high resistance to plastic deformation as compared with coatings prepared by conventional DCMS.The hardness (H) and elastic modulus (E) of Crubstrate . Figure ormation ,74. It i2AlC coatings deposited in this study and other references. It is observed that the hardness and modulus of coatings prepared by HiPIMS show the highest values as compared with the Cr2AlC coatings prepared through DCMS and other PVD techniques , according to the Hall–Patch equation. This fact could be understood from the findings of Kooi et al. [2AlC coatings as compared with coatings deposited by DCMS. Furthermore, the obtained elastic modulus value is in good agreement with the value of the reported Cr2AlC thin-film [i et al.  and Emmei et al. , which phin-film .2AlC coatings, the Vickers indentation was performed. Fracture toughness is one of the essential mechanical properties of the material, representing the ability to resist crack propagation during deformation up to fracture [2AlC MAX phase coating determined by the Vickers indentation technique. 1/2) at different average power of the deposition process. These values correspond with the previous H/E ratio of coatings because all of the coatings showed similar morphology.In order to gain insight into the fracture toughness of the Crfracture . Figure 2AlC MAX phase coating on the TiBw/Ti6Al4V composite deposited by HiPIMS, the scratch test was performed, and a critical load (Lc) of 12.6 N was observed, which is relatively better than that of coatings prepared by DCMS [2AlC coatings showed a relatively low friction coefficient before forming a through-thickness crack . The Cr2AlC coatings with column-free morphology showed better hardness and modulus, and values of H/E and H3/E2 implied an excellent toughness and wear resistance compared with the DCMS and other PVD techniques, and the coatings exhibited maximum hardness and modulus of 19.7 GPa and 286 GPa, respectively. The fracture toughness and scratch test showed the good mechanical integrity of Cr2AlC coatings on TiBw/Ti6Al4V composite, and the critical load of 12.6 N was observed. Hence, the plasma density, temperature around the substrate, and average power can be controlled in HiPIMS easily, which is beneficial for achieving coatings with desirable properties. The dense and column-free microstructure of Cr2AlC coatings prepared by HiPIMS yielded the combined high hardness, good fracture toughness, and superior wear resistance, which will benefit the potential application of TiBw/Ti6Al4V composite in abrasive wear conditions. Moreover, it is believed that the denser morphology of Cr2AlC coating will exhibit enhanced oxidation and corrosion resistance, which will be investigated in our future work.In this study, Cr


Background and purpose — Pain catastrophizing contributes to acute and long-term pain after total knee arthroplasty (TKA) but currently there are only limited treatment options. This study investigates the effectiveness of patient education in pain coping among patients with moderate to high pain catastrophizing score before TKA. Secondary outcomes were physical function, quality of life, self-efficacy, and pain catastrophizing.Patients and methods — The study was a parallel-group randomized controlled trial including patients with moderate to high levels of pain catastrophizing. 60 patients were recruited from December 2015 to June 2018. The mean age of the patients was 66 (47–82) years and 40 were women. The patients were randomized to either cognitive-behavioral therapy (CBT) based pain education or usual care. The primary outcome measure was pain under activity measured with the Visual Analog Scale (VAS). All outcomes were measured preoperatively, at 3 months, and at 1 year after surgery.Results — We found no difference in the primary outcome measure, VAS during activity, between the 2 groups but both groups had large reductions over time. The CBT-based pain education group reduced their VAS score by 37 mm (95% CI 27–46) and the control group by 40 mm (CI 31–49). We found no statistically significantly differences between the 2 groups in any of the secondary outcomes.Interpretation — Future research is warranted to identify predictors of persistent pain and interventions for the approximately 20% of patients with persisting pain after a TKA. Up to 20% of patients report persistent pain after a total knee arthroplasty (TKA) , proficiency in written and spoken Danish, and informed written consent.The patients were excluded if they had severe depression as diagnosed with the Major Depression Inventory (MDI) or were planning to have a contralateral knee arthroplasty within 1 year after the operation.The patients were included consecutively and randomized 1:1 to either the intervention group or usual care in 7 blocks of 8 persons. Because of high withdrawal rate, we decided to continue including patients and randomized another 16 patients. At the predefined deadline of June 1, 2018 we stopped the inclusion of patients. The physiotherapists and nurses who did the baseline and follow-up testing where blinded for the randomization. It was not possible to blind the patients and the 2 physiotherapists who delivered the patient education.Patients in the control group received usual care. Preoperatively this consisted of a multidisciplinary information meeting for patients and their relatives. Postoperative patients in the usual care group were offered a phone call after 1 week and 3 control visits at the hospital after 2, 4, and 12 weeks.In addition to usual care the intervention group participated in patient education based on CBT. The patient education consisted of 7 individual sessions of approximately 45 minutes, 3 sessions preoperatively and 4 sessions postoperatively. The 1st session was delivered approximately 2 weeks before the operation and the last session was given 3 months after the operation. The patient education was delivered by 2 physiotherapists who followed a manual describing in detail the content of each of the 7 sessions. This ensured that each session was standardized although there was room to discuss individual needs. The physiotherapists observed each other on a regular basis to make sure that they delivered the intervention in the same way. As far as possible the same physiotherapist followed the patient through all the sessions. The patient education covered 3 main components: (1) education in pain and the interaction between cognition and pain perception; (2) training in cognitive and behavioral pain coping skills; and (3) training in how to apply the learned coping skills in real-life situations. For details see the protocol : 0 indicating “no pain,” to 100 indicating “worst pain imaginable” on a mm scale. Pain during activity was measured right after the 6-minute walk test. Secondary outcomes included physical function, quality of life, physiological factors, and pain during rest. The Oxford Knee Score (OKS) was used to measure knee-related pain and physical function. Overall scores run from 0 to 48 with 48 being the best outcome , and data not normally distributed by median (range). In the analysis the intention-to-treat principle was used including all randomized participants. For all outcomes, between-group mean differences, changes over time, and 95% confidence intervals (CI) from baseline to 3 and 12 months were analyzed by a mixed-effects linear regression model with a random person level and systematic effects of time, group, and the interaction between time and group. In the analysis of change in the primary outcome from baseline to 12 months we included VAS during activity at baseline as a covariate. Model validation was performed by comparing observed and expected within-subject standard deviations and correlations and by inspecting QQ plots. We tried to include morphine intake as a covariate, but this did not change the results or conclusions.We filled missing values with mean values as described in the manuals if less than half of the answers were missing in the SF-36 (PF) and if 2 or fewer of the answers were missing in the OKS, the PSEQ, and the PCS. In each questionnaire missing values were filled with mean values between 0 and 5 times.The significance level was set at < 0.05. The statistical analyses were performed using STATA 15  software package.The study was conducted in accordance with the Declaration of Helsinki and the CONSORT statement, and registered in the Danish Data Protection Agency (j.nr. 1-16-02-245-15). All patients gave informed written consent. The protocol was approved by the Central Denmark Regions Committee on Biomedical Research Ethics  and registered at ClinicalTrials.gov (NCT02587429). The study was supported by the Tryg Foundation (grant number: 113944) and the Danish Rheumatism Association (grant number: A3622). None of the funders have any role in the study other than providing funding. The authors have no competing interests. Data is available from the corresponding author on reasonable request.From December 2015 to June 2018, 324 patients were considered for inclusion. 283 patients were screened and 105 of these had a PCS score > 22. We randomized 67 patients but shortly after the randomization 7 patients decided not to participate and 60 patients were measured at baseline and included in the analysis . Of the We found a mean difference of 3 (CI –6 to 11) mm in the primary outcome measure VAS during activity 12 months after TKA between the pain education group and the usual care group (p = 0.6). Mean VAS during activity 12 months postoperatively was 12 mm (CI 5–18) for the pain education group and 9 mm (CI 3–15) for the usual care group . Both grWe found no statistically significantly differences between the 2 groups in any of the secondary outcomes . In bothParticipation in patient education in addition to usual care for patients with a high pain catastrophizing score did not result in better outcomes 12 months after TKA. However, for both groups OKS and VAS during activity improved statistically significantly over time indicating that knee function improves and knee pain reduces after a TKA.Recent research on the effect of CBT on pain severity has shown mixed results and meta analyses show that the effect size on pain is small (Dixon et al. We found no additive effect of pain education based on CBT on pain, physical function, quality of life, self-efficacy, or pain catastrophizing. There may be several reasons for this. 1st, most of the former studies reporting an effect of a CBT intervention on these factors have included patients with knee OA and only a few studies have included patients scheduled for TKA. Cai et al.  found th2nd, based on existing research suggesting that pain catastrophizing is a predictor of persistent pain after a TKA, we decided to use PCS as a screening tool for inclusion of patients. However, recent research has suggested that patients with elevated scores on more than 1 risk factor are more likely to develop prolonged pain and disability, and that multiple psychological factors need to be considered with respect to pain and physical disability in knee OA (Sinikallio et al. We wanted to include the one-third of our TKA population with the highest PCS score. Based on data collected in our department in the years 2011–2013 we defined a cut-off for inclusion at PCS > 22 in this study (Birch et al. Our intervention consisted of only CBT-based pain education and consistent with the study of Riddle et al.  we foundFor the psychological outcomes we expected a larger reduction in PCS and an increase in PSEQ among the patients in the intervention group compared with the usual care group because these issues were emphasized in the pain education. However, this was not the case and both groups achieved similar improvements after 12 months. 3 months after the operation both groups have a PCS score far under 21 points, which is the cuff point for moderate PCS.There are some limitations too. 1st we had to change the number of sessions from 7 to 6 approximately 1 year after we started including patients. This means that not all patients have received the same number of sessions, but they all received the same content and we found no difference in the primary outcome between patients who received 7 sessions and patients who received 6 sessions. Furthermore, because we wanted to design a method of patient education that fits into a hospital setting, it consisted of only 7 sessions contrary to previous studies where the CBT intervention ranged from 10 to 12 sessions (Keefe et al. This study showed that pain education based on CBT was not superior to usual care after TKA in terms of reducing pain or improving physical function, quality of life, self-efficacy, and pain catastrophizing. Future research is warranted to identify predictors of persistent pain and interventions for the approximately 20% of patients with persisting pain after a TKA.The authors would like to thank all the participating patients, nurses, physiotherapists, and secretaries at the University Clinic for Hand, Hip, and Knee surgery and the Department of Physiotherapy and Occupational Therapy, Holstebro Regional Hospital, Hospital Unit West, Denmark.SB, MS, IM, and TBH participated in the conception and design of the study and helped to revise the manuscript. All authors read and approved the final manuscript.Acta thanks Johan Creutzfeldt and Kristian Kjaer Petersen for help with peer review of this study.