Plasmodium falciparum. Using a chemical bypass method, we deleted four of the FeS pathway proteins involved in sulfur acquisition and cluster assembly and demonstrated that they are all essential for parasite survival. However, the effect that these deletions had on the apicoplast organelle differed. Deletion of the cysteine desulfurase SufS led to disruption of the apicoplast organelle and loss of the organellar genome, whereas the other deletions did not affect organelle maintenance. Ultimately, we discovered that the requirement of SufS for organelle maintenance is not driven by its role in FeS biosynthesis, but rather, by its function in generating sulfur for use by MnmA, a tRNA modifying enzyme that we localized to the apicoplast. Complementation of MnmA and SufS activity with a bacterial MnmA and its cognate cysteine desulfurase strongly suggests that the parasite SufS provides sulfur for both FeS biosynthesis and tRNA modification in the apicoplast. The dual role of parasite SufS is likely to be found in other plastid-containing organisms and highlights the central role of this enzyme in plastid biology.Iron-sulfur clusters (FeS) are ancient and ubiquitous protein cofactors that play fundamental roles in many aspects of cell biology. These cofactors cannot be scavenged or trafficked within a cell and thus must be synthesized in any subcellular compartment where they are required. We examined the FeS synthesis proteins found in the relict plastid organelle, called the apicoplast, of the human malaria parasite  Malaria parasites contain a relict plastid organelle called the apicoplast that is required for its survival . The essPlasmodium falciparum, FeS cofactors are formed within the apicoplast by the sulfur utilization factor (SUF) pathway, for use by FeS-dependent proteins within the organelle , a product of the apicoplast isoprenoid biosynthetic pathway, is exogenously provided to the parasite  under continuous mevalonate supplementation , which suggests that there may be some redundancy between complex proteins , along with its partner SufE (PF3D7_0206100), mobilize sulfur from L-cysteine, with SufE transferring the sulfur to the SufBC2D complex  is currently annotated as a tRNA methyltransferase  (2U34) of tRNALysUUU, tRNAGluUUC, and tRNAGlnUUG  . MnmA inNAGlnUUG . E. coli/B/C/D/E , which uoli SufS . We coularasites .P. falciparum MnmA (Pf MnmA) with orthologs from Synechococcus sp., B. subtilis, E. coli, Arabidopsis thaliana, and Saccharomyces cerevisiae reveal that it has a 421 amino acid (aa) N-terminal extension  (Toxoplasma gondii (ToxoDB ID: TGME49_309110) has recently been described (Pf MnmA (residues 422–1084) and the corresponding regions of other apicomplexan MnmA orthologs predicts a MnmA-like fold  of the putative xtension , which ixtension . MSA als(SGGXDS) . This PP(SGGXDS) . The fir(SGGXDS) . Other p(SGGXDS)  and the escribed . In siliike fold .Pf MnmA, we generated a parasite line with two C-terminal FLAG tags in tandem appended to the endogenous Pf MnmA  of mnmA to use with the TetR-DOZI system  by immunofluorescence , confirming Pf MnmA localization to the apicoplast  to MnmA  MnmA in PfMevattB parasites    using thbsmnmA and bsmnmA-yrvO expression cassettes encode a conditional localization domain (CLD) at the protein N-terminus for inducible control over protein localization . Taken together, these results show that Bs MnmA and Bs MnmA-YrvO can be expressed in the apicoplast of P. falciparum.The lization  and contlization . The CLD vacuole . An apta vacuole . We succ vacuole . Live ep+bsmnmA-yrvO parasites to investigate whether loss of Pf MnmA can be complemented by the Bs MnmA-YrvO fusion protein. We employed the same gRNA and repair construct used in the experiment described in pfmnmA in the +bsmnmA-yrvO line. In repeated independent transfection experiments, we successfully generated +bsmnmA-yrvO ΔmnmA parasites  and non-permissive conditions  for 8 days. Under the non-permissive condition, the +bsmnmA-yrvO ΔmnmA parasites showed a significant growth defect from day 4 onward  blocks further sulfur utilization. Alternatively, dominant negative SufC could lead to dysfunctional iron homeostasis. ATP binding to SufC elicits a conformational change in the SufBC2D complex, exposing sites required for iron binding and enabling the formation of nascent clusters  is too low in the absence of SufE to support FeS synthesis. The fact that SufE is not required for apicoplast maintenance (and presumably tRNA thiolation) further suggests that SufS desulfurase activity in the absence of SufE is adequate for tRNA thiolation and that SufS may transfer sulfur directly to MnmA.Gene deletion studies exposed different roles for the two proteins (SufE and SufS) involved in sulfur acquisition. We found that both SufE and SufS are required for parasite survival, however, only SufS is required for apicoplast maintenance . These preported . SufE pring SufE . The reqP. falciparum reported 28 different tRNA modifications, including s2U and mcm5s2U  is predicted to catalyze s2U modification of nuclear tRNAs while MnmA is predicted to make this modification in the apicoplast. To investigate the biological function of P. falciparum MnmA, we complemented parasites lacking endogenous MnmA by expressing the well-studied MnmA ortholog from B. subtilis (Bs MnmA) with and without its cognate cysteine desulfurase (Bs YrvO) partner in the apicoplast. The role of Bs MnmA in the s2U modification of target tRNAs has been demonstrated by both genetic and biochemical analysis  function together to produce essential tRNA s2U modifications  is an IscS ortholog and accomplishes the same feat through four sulfur transferases  transfers sulfur directly to MnmA  able to transfer sulfur directly.The dual activity of ynthesis . Thiolat to MnmA . Similarsferases . While hl domain . This pu to MnmA . The uniiolation . We coululfurase . FurtherT. gondii. Deletion of T. gondii SufS or MnmA results in apicoplast defects and parasite death . The RPMI 1640 medium was supplemented with 20 mM HEPES, 0.2% sodium bicarbonate, 12.5 μg/mL hypoxanthine, 5 g/L Albumax II , and 25 μg/mL gentamicin. Cultures were incubated at 37°C and maintained in 25 cm2 gassed flasks .Asexual stage We employed Cas9-mediated gene editing to delete genes of interest . For genP. falciparum NF54 genomic DNA with HA1 and HA2 forward and reverse primers  ligation independent cloning (LIC). The gRNA sequences  were amplified from  primers d. The HAequences d were inPf MnmA, we created the pKD-mnmA-2xFLAG-10xapt plasmid. This plasmid also allowed us to tag Pf MnmA C-terminally with 2xFLAG. To make this plasmid, ~300–400 bp HA1 and HA2 of the pfmnmA gene were PCR amplified with MnmAKD.HA1F+MnmAKD .HA1R and MnmAKD.HA2F+MnmAKD .HA2R, respectively from P. falciparum NF54 genomic DNA  with a redesigned 10x-aptamer array (10xapt) that prevents aptamer loss -sufC plasmids as described above. Synchronized ∆sufD parasites were added to the transfected RBCs and cultured in medium with 50 µM mevalonate. The transfectants were selected with 2.5 µg/mL blasticidin , 1.5 μM DSM1, 2.5 nM WR99210, and 50 µM mevalonate, after which the culture was maintained in complete medium containing 2.5 nM WR99210 and 50 µM mevalonate until parasites appeared. Upon parasite appearance, the culture was maintained in complete medium with 2.5 µg/mL blasticidin, 2.5 nM WR99210, and 50 µM mevalonate.For generating the ∆mnmA-flag parasite lines, the linearized pKD-mnmA-2xFLAG-10xapt and pCasG-mnmAKD plasmids were co-transfected into RBCs as mentioned above. Following transfection, these RBCs were mixed with PfMevattB parasites  and native gene loci  were amplified with corresponding primers d. Expectg for 10 min at 4°C and washed three times with cold PBS. These parasites were used immediately or were snap-frozen in liquid N2 and saved at –80°C for later use.Asynchronous parasite cultures were washed three times with cold-complete medium. The cultures were treated with 0.15% (wt/vol) saponin in cold phosphate-buffered saline  for 10 min on ice for permeabilization of the RBC and parasitophorous vacuolar membranes. Saponin-isolated parasites were centrifuged at 1940 × Saponin-isolated parasites were resuspended in 1× NuPAGE LDS sample buffer  containing 2% β-mercaptoethanol and boiled for 5 min. Proteins were resolved on 4–12% gradient reducing gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% milk in PBS containing 0.1% Tween 20 (milk/PBST) for 1 hr at room temperature and incubated overnight at 4°C with mouse anti-FLAG monoclonal antibody  diluted 1:1000 in milk/PBST. The membrane was then incubated for 1 hr at room temperature with sheep anti-mouse horseradish peroxidase (HRP)-conjugated antibody  diluted 1:10,000 in milk/PBST. Chemiluminescent signal was developed with SuperSignal West Pico chemiluminescent substrate  according to the manufacturer’s protocol and detected on autoradiography film. For loading controls, the membrane was stripped of antibodies with 200 mM glycine (pH 2.0) for 5 min at room temperature. After blocking the membrane with 5% milk/PBST, the membrane was re-probed with 1:25,000 mouse anti-aldolase monoclonal antibody  and 1:10,000 sheep anti-mouse HRP-conjugated antibody, using the methods described above.sufB gene (PF3D7_API04700) was used as a proxy for the presence of the apicoplast genome and was amplified by PCR with a primer pair listed in ldh, PF3D7_1324900) and the mitochondrial genome  were amplified with corresponding primer pairs  in PBS for 10 min to reduce free aldehyde groups. After a 2 hr blocking step with 3% bovine serum albumin (BSA) in PBS, cells were incubated overnight at 4°C with 1:500 rabbit anti-ACP antibody  and 30 nM MitoTracker CMX-Ros  for 30 min at 37°C. The stained cells were then washed three times with complete medium and incubated for 5 min at 37°C after each wash. After the final wash, the cells were resuspended in 20 µL of complete medium, placed on a slide and sealed under a cover slip with wax. For live cell imaging of the +, bsmnmA-yrvO+, bsmnmA+bsmnmA ∆mnmA, +bsmnmA-yrvO ∆mnmA, and +bsmnmA-yrvO ∆sufS parasite lines, cells were stained with 1 µg/mL DAPI only.For live cell imaging of the ∆All images were taken with a Zeiss AxioImager M2 microscope  equipped with a Hamamatsu ORCA-R2 camera  using a 100×/1.4 NA lens. A series of images were taken spanning 5 µm along the z-plane with 0.2 µm spacing. An iterative restoration algorithm using the Volocity software  was used to deconvolve the images to report a single image in the z-plane.The degree of colocalization between the red channel (anti-ACP or mCherry) and green channel (anti-FLAG or api-SFG) signals was determined with Volocity software. The fluorescent intensity thresholds were set using the region of interest tool using Volocity. To set the thresholds, the fluorescence intensity of a region of the cell with no staining for either signal (background) was used. To measure the degree of colocalization, the Manders’ coefficient (M1)  was deteShield1  (permissive condition) or in the absence of aTc and presence of 0.5 µM Shield1 (non-permissive condition). In all the experiments presented in Parasite growth was monitored using an Attune Nxt Flow Cytometer  as previously described . For detP. falciparum , Synechococcus sp. , B. subtilis , E. coli MnmA , A. thaliana , S. cerevisiae , B. microti (PiroplasmaDB ID: BMR1_03g03155), T. annulata (PiroplasmaDB ID: TA12620), T. gondii (ToxoDB ID: TGME49_309110), E. tenella (ToxoDB ID: ETH_00027940), P. falciparum , B. subtilis , E. coli , A. thaliana , and S. cerevisiae .All of the MSA were generated with the ClustalW program in MEGA11 . The MSASynechococcus sp. (Sy MnmA), B. subtilis (Bs MnmA), E. coli MnmA (Ec MnmA), A. thaliana (At MnmA), S. cerevisiae (Sc Mtu1), P. falciparum MnmA , B. microti , T. annulata , T. gondii , and E. tenella . For E. coli SufE , P. falciparum SufE , P. falciparum MnmA , B. microti , T. annulata , T. gondii , and E. tenella .Structural alignments presented in E. coli SufE (PDB id: 1MZG) and E. coli MnmA (PDB id: 2DER) were obtained from RCSB PDB (https://www.rcsb.org/)  , Pf MnmA N-terminal domain (Ile146-Tyr380), and Pf SufE (Leu105-Ile243). The crystal structures and the predicted models were visualized with UCSF ChimeraX  molecular visualization program   and used/phyre2)  using th program . Structuh MEGA11  using thh MEGA11 . In all P. falciparum SufS (PF3D7_0716600) was PCR amplified in three overlapping fragments with Phusion DNA polymerase  using the primers in blastn’ tool of the Basic local alignment search tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (The gene encoding ast.cgi) . Plasmodium falciparum. The work is based on elegant and robust genetic approaches, and not only confirms the essentiality of the plastid-hosted Suf iron-sulfur cluster synthesis pathway, but also highlights an important additional role for the cysteine desulfurase SufS in apicoplast maintenance via tRNA modification. The work provides compelling evidence for a dual function of parasite SufS, although impact on tRNA has not been established directly. These findings reveal a potential new target for metabolic intervention and will be of interest to researchers studying apicomplexan parasites, and more broadly, in the field of plastid biology.This study provides important new insights into iron sulfur biosynthesis in the human malaria parasite  public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:Plasmodium falciparum apicoplast cysteine desulfurase provides sulfur for both iron sulfur cluster assembly and tRNA modification" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Anna Akhmanova as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "The The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:All three reviewers appreciated that this is an important and elegant study that substantially advances our understanding of the metabolic functions of Plasmodium apicoplast. The study as it stands provides compelling evidence that SufS plays a dual role in the FeS synthesis pathway and apicoplast maintenance. However, there are a few points that should be addressed in a revised version of the manuscript:1) The claim that SufS role in apicoplast maintenance is linked with tRNA modification is inferred from the genetic complementation experiments and the assumption that SufS plays a similar enzymatic role as the bacterial enzymes. However, this is not directly supported by data. In the absence of analysis of tRNA thiolation levels in the parasite (which would strengthen the study and make it complete), the authors should modify the text (and title) to clarify that the effect on tRNA remains a hypothesis that will require confirmation in follow-up work.2) A more detailed phylogenetic analysis and sequence comparisons not only of MnmA but also of SufS should be included (see comments from reviewers #1 and #2).3) The authors need to clarify the experimental conditions for the experiments shown in Figure 5F and 6F (see reviewer 1 point #2), if necessary through additional experiments.Further suggestions to improve the manuscript are listed below in the reviewers' recommendations for the authors.Reviewer #1 (Recommendations for the authors):P. falciparum as YrvO in Bacillus (i.e. sulfur transfer to MnmA for tRNA thiolation). However this is not formally demonstrated, and the impact on apicoplast integrity could reflect other functions of the protein. Experiments directly showing the alteration of apicoplast tRNA thiolation would make the story even more complete. Furthermore, the authors show comparisons between Pf and Bs MnmA, but it would be useful to also include information on the sequence/structural similarities between SufS and YrvO. In particular, what are the common features of SufS and YrvO that would not be observed in other bacterial cysteine desulfurases (E. coli…), which do not transfer sulfur to MnmA?1) This work is elegant and robust. However, the claim that SufS (via MnmA) mediates tRNA modification remains a hypothesis but is not supported by data. The observation that BsYrvO and BsMnmA can complement the function of PfSufS and PfMnmA, respectively, strongly suggests that SufS plays a similar enzymatic role in 2) In the genetic complementation experiments in Figure 5F and 6F, the authors show that under non-permissive conditions (at day 8) there is disruption of the apicoplast. However, these experiments were apparently performed in the absence of mevalonate rescue, in conditions where a severe growth defect is observed. Therefore the effect on the apicoplast could be related to the parasite death. A more convincing approach would be to document the disruption of the apicoplast in a well-growing parasite in the presence of mevalonate. This would strengthen the conclusion that BsMnmA complements PfMnmA specific function in apicoplast maintenance.3) In Figure 3F, how do the authors explain that knockdown has no effect on growth during the 4 first days, despite efficient protein depletion as shown in Figure S3A? Is it due to the inertia of the system for metabolite depletion?4) While the paper is very well written with a clear narrative, some aspects of the presentation of data or experiments could be improved:– In Figure 1D, it would be useful to show a normal apicoplast in the parental line– In Fig3E, the apicoplast looks different  than in other parasites, is it representative of the normal apicoplast morphology?– Line 338-340: The rationale for the experiment in Figure 7 could be explained in a more explicit way. The experiment is designed to test if YrvO is capable of complementing PfSufS functions, especially for apicoplast maintenance.Reviewer #2 (Recommendations for the authors):This is a very complete study, presenting an already considerable amount of data that adequately support the conclusions drawn by the authors. However, I feel that the tRNA modification part of the work  could have been explored further. The complementation assays with the bacterial BsMnmA strongly suggest that PfSufS would be able to directly transfer sulfur to the bacterial protein, however, tRNA analysis in the PfSufS and PfMnmA (and its complemented cell line) would provide compelling evidence that the key impact of depleting these proteins is indeed on thiolation of tRNAs. tRNAs can be purified and then identified and quantified by performing size exclusion chromatography and mass spectrometry analyses (see PubmedID 30287681 for a recent example in Plasmodium).In the same line of thoughts, several bacterial Mnma seem to show strong specificity to their partner desulfurase and in some bacterial lineages there is even indirect sulfur transfer to the tRNA via sulfur-carrier protein, so it is in fact quite unexpected to see that PfSufS could be a direct sulfur donor to a procaryotic MnmA, which in turn is potentially able to perform sulfur transfer directly on the parasite tRNA . Recent work has shown that some bacterial sulfur transferases may contain specific features linked to the ability to directly incorporate sulfur into tRNAs (see PubmedID 31801798). Given the cyanobacterial origin of the apicoplast, would it be possible that PfMnma is more closely related to procaryotic Mnma enzymes that are able to perform direct sulfur transfer? To complement the protein sequence alignments currently provided in Figure 3 supplements 1&2, it may be quite informative to include a small phylogenetic analysis coupled with a comparison of the sequence features of apicomplexan MnmA with procaryotic MnmA .Finally, in complement to the data presented on Figure 7, depleting PfSufS in the bsmnmA+ (thus not expressing the YrvO desulfurase) cell line is likely to lead to both a loss of the apicoplast and a loss of parasite viability. This would be strengthening the idea that SufS acts by transferring sulfur to BsMnmA and would rule out an unrelated effect of MnmA alone.Reviewer #3 (Recommendations for the authors):1) The authors could elaborate some more on the result of the Mev-dependent phenotype of δ mnmA construct . This genetic construct presumably would contain an intact Fe-S biosynthetic pathway; however, loss of the apicoplast genome encoding sufB, ultimately leads to defects in Fe-S biogenesis, explaining the result.2) The authors commented on the experimental challenge of generating a bsmnmA+ on a deltamnmA parasite line. Did the authors sequence the sufS gene in this resulting construct to rule out the possibility of selecting a sufS mutant that enabled this complementation? Along the same lines, could the authors comment on the expression levels of the Bacillus' enzymes when compared to the endogenous levels of PfSufS and PfMnmA?3) Although this reviewer favors the conclusions of this study on the proposed role of PfSufS and PfMnmA in 2-thiouridine tRNA modification and the genetic experiments supporting this role, the results described in this study do not provide a direct demonstration of their involvement or exclusive involvement in tRNA thiolation. That is, no analysis of tRNA modifications was included in this report. I'm certainly sensitive to the experimental challenges associated with these experiments and the complexity of analyzing any data resulting from samples of an organelle of an intracellular parasite. The purpose of this comment is not to invalidate the value of this contribution or to set an expectation for additional experiments, as the report is already describing several novel findings. But an ask is to revise the document to indicate that instead of "establishing" a role in tRNA thiolation, the results support this proposed role. Also, perhaps adding a comment in the discussion would add value to readers being able to appreciate the complexity of multiple pathways involving 2sU in the organism and host. Essential revisions:All three reviewers appreciated that this is an important and elegant study that substantially advances our understanding of the metabolic functions of Plasmodium apicoplast. The study as it stands provides compelling evidence that SufS plays a dual role in the FeS synthesis pathway and apicoplast maintenance. However, there are a few points that should be addressed in a revised version of the manuscript:1) The claim that SufS role in apicoplast maintenance is linked with tRNA modification is inferred from the genetic complementation experiments and the assumption that SufS plays a similar enzymatic role as the bacterial enzymes. However, this is not directly supported by data. In the absence of analysis of tRNA thiolation levels in the parasite (which would strengthen the study and make it complete), the authors should modify the text (and title) to clarify that the effect on tRNA remains a hypothesis that will require confirmation in follow-up work.We modified the text throughout the manuscript to address this issue. These changes and an explanation for why we didn’t measure tRNA thiolation levels are described in more detail in the responses to the reviewers below. A basic conceptual problem with measuring changes in tRNA thiolation levels in the SufS or MnmA KO parasites is the loss of the apicoplast genome and loss of all apicoplast tRNAs.2) A more detailed phylogenetic analysis and sequence comparisons not only of MnmA but also of SufS should be included (see comments from reviewers #1 and #2).We performed the suggested phylogenetic and sequence analyses and made some interesting observations that may impact how MnmA receives sulfur. Please, see our response to Reviewer #1, second part of point #1.3) The authors need to clarify the experimental conditions for the experiments shown in Figure 5F and 6F (see reviewer 1 point #2), if necessary through additional experiments.Further suggestions to improve the manuscript are listed below in the reviewers' recommendations for the authors.We clarified our experimental conditions. Please, see our detailed response to reviewer #1, point #2.Reviewer #1 (Recommendations for the authors):P. falciparum as YrvO in Bacillus (i.e. sulfur transfer to MnmA for tRNA thiolation). However this is not formally demonstrated, and the impact on apicoplast integrity could reflect other functions of the protein. Experiments directly showing the alteration of apicoplast tRNA thiolation would make the story even more complete.1) This work is elegant and robust. However, the claim that SufS (via MnmA) mediates tRNA modification remains a hypothesis but is not supported by data. The observation that BsYrvO and BsMnmA can complement the function of PfSufS and PfMnmA, respectively, strongly suggests that SufS plays a similar enzymatic role in We appreciate the reviewer’s enthusiasm for our findings. The reviewer agreed that our complementation experiments of PfSufS and PfMnmA activity with biochemically and genetically well-characterized bacterial orthologs strongly suggest dual function of PfSufS: both in FeS cluster formation and tRNA modification. As the reviewer points out, we did not measure tRNA modifications directly to show that they are affected by the loss of PfSufS and/or PfMnmA. We thought a lot about how to measure tRNA modifications, but concluded that there are significant technical limitations:P. falciparum express tRNALysUUU, tRNAGluUUC, and tRNAGlnUUG. In the apicoplast, MnmA should produce 2-thioU at residue 34 of these tRNAs. In the cytosol, an enzyme that does not have homology to MnmA (NCS2) is predicted to produce the same 2-thioU modification at the same residue of the three tRNAs expressed by the nuclear genome. Current methods of detecting tRNA base modifications involve hydrolyzing tRNA pools followed by LC-MS detection of modified bases. Thus, we would likely have to devise methods to separate apicoplast tRNAs from nuclear tRNAs prior to conducting an analysis of tRNA base modifications.i) Both the apicoplast and the nuclear genomes of P. falciparum tRNA base modifications (PMID: 30287681). In that study, the authors showed that total apicoplast RNA is a relatively minor population (rRNAs 0.5–2% of nuclear-encoded rRNAs). Due to the relatively poor contribution of the apicoplast RNAs, the authors speculated that most modifications from this organelle would have been below the detection limit of the LC-MS method they employed.Assuming the issues with detecting apicoplast tRNA modifications could be resolved, it would be difficult to link deletion of SufS or MnmA to specific changes in tRNA modifications. This is because deletion of either SufS or MnmA causes loss of the apicoplast genome and loss of all apicoplast tRNAs.ii) Low levels of apicoplast tRNAs would complicate efforts to study base modifications. Microarray analysis of apicoplast tRNAs shows that they are expressed at low levels compared to their nuclear counterparts (PMID: 32059044). Thus, we would probably also have to increase the parasite mass significantly to match the amount of tRNAs used in the previously described analysis of iii) Assuming the issues with detecting apicoplast tRNA modifications could be resolved, it would be difficult to link deletion of SufS or MnmA to specific changes in tRNA modifications. This is because deletion of either SufS or MnmA causes loss of the apicoplast genome and loss of all apicoplast tRNAs.iv) Another way to test the enzymatic role of PfMnmA in tRNA modification is expression and purification of a recombinant version of the protein and test in vitro activity on its substrate tRNAs. We set out to do this, however, we could not get the PfMnmA gene PCR amplified or synthesized due to its large size and extreme AT content despite multiple attempts. This prevented us from producing recombinant PfMnmA protein or conducting complementation experiments in bacterial systems.In light of these technical challenges, we consider the direct measurement of apicoplast tRNA modifications to be a separate project that will have technical challenges and require the development of new methods. Instead of tackling these challenges in this manuscript, we instead softened the claims made in the text by replacing words such as ‘demonstrating’ and ‘showing’ with statements about how the data support a role in tRNA thiolation. For example, the abstract now reads (line 42-44):“Complementation of MnmA and SufS activity with a bacterial MnmA and its cognate cysteine desulfurase, strongly suggests that the parasite SufS provides sulfur for both FeS biosynthesis and tRNA modification in the apicoplast.”E. coli…), which do not transfer sulfur to MnmA?Furthermore, the authors show comparisons between Pf and Bs MnmA, but it would be useful to also include information on the sequence/structural similarities between SufS and YrvO. In particular, what are the common features of SufS and YrvO that would not be observed in other bacterial cysteine desulfurases . In fact, our analysis shows that PfSufS and BsYrvO are members of two different classes of cysteine desulfurases. These results do not allow us to make any suggestions as to why these two would function in a similar manner. We added statements in the discussion considering our findings from this bioinformatic analysis (line 482-486):Pf SufS and Bs YrvO which would be indicative of their ability to transfer sulfur directly to their respective MnmA proteins. In fact, we found that these proteins represent two different classes of cysteine desulfurase . Further studies are necessary to understand what allows Pf SufS (and Bs YrvO) able to transfer sulfur directly.”“We could not identify any conserved motif(s) between P. falciparum numbering) region of PfMnmA and in other apicomplexan orthologs. Phylogenetic analysis of this domain, though, shows diversity among the MnmA orthologs in recent times and there is no clustering among the MnmA enzymes that receive sulfur through intermediate sulfur transferases. These results are now presented as Figure 3—figure supplement 3. Interestingly, homology modelling also predicted the possible presence of a SufE-like domain in the long N-terminal ends  of apicomplexan MnmA orthologs including P. falciparum. This new result is shown as Figure 3—figure supplement 8. The SufE-like domain in the N-terminus of PfMnmA might facilitate the transfer of sulfur from PfSufS and explain why SufE (or other protein intermediates) are not required for this process. We included these new insights in the Discussion section (line 473477).We also performed a detailed sequence and structural analysis of PfMnmA and its orthologs from prokaryotes (including cyanobacteria) and eukaryotes. Homology based structure prediction of MnmA orthologs revealed a classical MnmA-like domain in residues 4221084 (2) In the genetic complementation experiments in Figure 5F and 6F, the authors show that under non-permissive conditions (at day 8) there is disruption of the apicoplast. However, these experiments were apparently performed in the absence of mevalonate rescue, in conditions where a severe growth defect is observed. Therefore the effect on the apicoplast could be related to the parasite death. A more convincing approach would be to document the disruption of the apicoplast in a well-growing parasite in the presence of mevalonate. This would strengthen the conclusion that BsMnmA complements PfMnmA specific function in apicoplast maintenance.The genetic complementation experiments shown in in 5F and 6F were performed in the absence of mevalonate rescue. The question here is whether loss of MnmA activity leads to parasite death and subsequent apicoplast disruption, or whether apicoplasts are disrupted in a way that can be rescued with mevalonate. To address this, we conducted a time-course of apicoplast disruption in the presence of mevalonate (as suggested). These new results are presented as Figure 3 —figure supplement 5, 6, and 7 and show that significant apicoplast loss occurs on day 4 of the 8-day experiment while parasite growth remains robust due to the presence of mevalonate. Since this question is now addressed in Figure 3 —figure supplement 5, 6, and 7, we did not modify Figures 5F or 6F.3) In Figure 3F, how do the authors explain that knockdown has no effect on growth during the 4 first days, despite efficient protein depletion as shown in Figure S3A? Is it due to the inertia of the system for metabolite depletion?Pf MnmA. The slow kinetics of growth arrest shown in Figure 3F combined with the more rapid reduction in protein levels shown in current Figure 3-5A are not unusual, in our experience. We observed very similar results using the same knockdown method targeting the apicoplast ubiquitin-like protein (AUBL) (PMID: 32817449). There may be a ‘delayed death’ phenomenon at work since we see that the apicoplast organelles are intact on day 2 of the knockdown time course, but there is a significant increase in apicoplast disruption observed by day 4 .In Figure 3F, we used the TetR-DOZI inducible knockdown tool to control expression of endogenous 4) While the paper is very well written with a clear narrative, some aspects of the presentation of data or experiments could be improved:– In Figure 1D, it would be useful to show a normal apicoplast in the parental lineThe suggested images of the fluorescently labeled apicoplast in the PfMev parental line have already been published previously in the article “A mevalonate bypass system facilitates elucidation of plastid biology in malaria parasites”, PMID: 32059044. We would have included these images, but since they have already been published and can be referenced elsewhere, we chose not to include them.– In Fig3E, the apicoplast looks different  than in other parasites, is it representative of the normal apicoplast morphology?The data presented in Figure 3E are from an immunofluorescence labelling assay (IFA) where the cells were fixed and permeabilized, before labelling with specific antibodies. All the other microscopy images presented in this manuscript are from live cell fluorescence imaging. In general, 5-15% reduction in cell length have been observed when treated with various commonly used fixatives in IFAs (PMID: 33912815), which could explain the difference in appearance of the apicoplast in Figure 3E from other images presented in this manuscript.– Line 338-340: The rationale for the experiment in Figure 7 could be explained in a more explicit way. The experiment is designed to test if YrvO is capable of complementing PfSufS functions, especially for apicoplast maintenance.We have clarified the rationale for the experiment presented in Figure 7 in the revised manuscript (line 344-352):sufS with continuous supplementation of mevalonate to determine whether the Bs MnmA-YrvO protein can complement the essential activity of SufS. We tested this because not only have Bs MnmA and Bs YrvO been functionally characterized , but because Pf SufS and Bs YrvO share conserved catalytic residues required for cysteine desulfurase activity . We expected successful complementation of Pf SufS with Bs MnmA-YrvO to result in a parasite line with intact apicoplasts and a mevalonate-dependent phenotype , which would suggest that sulfur acquired by Bs YrvO is only transferred to MnmA but not to the components of the parasite SUF pathway.”“In these parasites, we attempted to delete Reviewer #2 (Recommendations for the authors):This is a very complete study, presenting an already considerable amount of data that adequately support the conclusions drawn by the authors. However, I feel that the tRNA modification part of the work  could have been explored further. The complementation assays with the bacterial BsMnmA strongly suggest that PfSufS would be able to directly transfer sulfur to the bacterial protein, however, tRNA analysis in the PfSufS and PfMnmA (and its complemented cell line) would provide compelling evidence that the key impact of depleting these proteins is indeed on thiolation of tRNAs. tRNAs can be purified and then identified and quantified by performing size exclusion chromatography and mass spectrometry analyses (see PubmedID 30287681 for a recent example in Plasmodium).The suggestion here is to measure tRNA thiolation in the knockout and complemented parasite lines to link SufS and/or MnmA to tRNA modification. We addressed this point as described above for point 1 of reviewer 1 by modifying the strength of the claims made in the paper. As described above, it would be difficult using current methods to measure apicoplast tRNA modifications versus nuclear tRNA modifications. The same thiolated base is predicted to be independently produced in different compartments of the cell. There is also a conceptual problem with analyzing tRNA modifications in SufS or MnmA deletion lines since these lines lose the apicoplast genome and do not contain any apicoplast tRNAs.In the same line of thoughts, several bacterial Mnma seem to show strong specificity to their partner desulfurase and in some bacterial lineages there is even indirect sulfur transfer to the tRNA via sulfur-carrier protein, so it is in fact quite unexpected to see that PfSufS could be a direct sulfur donor to a procaryotic MnmA, which in turn is potentially able to perform sulfur transfer directly on the parasite tRNA . Recent work has shown that some bacterial sulfur transferases may contain specific features linked to the ability to directly incorporate sulfur into tRNAs (see PubmedID 31801798). Given the cyanobacterial origin of the apicoplast, would it be possible that PfMnma is more closely related to procaryotic Mnma enzymes that are able to perform direct sulfur transfer? To complement the protein sequence alignments currently provided in Figure 3 supplements 1&2, it may be quite informative to include a small phylogenetic analysis coupled with a comparison of the sequence features of apicomplexan MnmA with procaryotic MnmA .We thank the reviewer for the suggestion to include a sequence and phylogenetic analysis to compare MnmA orthologs. We analyzed MnmA sequences across kingdoms (including prokaryotic species) and specifically within apicomplexan species as well as sequence alignments that focus individually on the N-terminal and C-terminal domains. We also included an analysis of cysteine desulfurase enzymes and were able to add several interesting insights to the manuscript. Please see the response above to a similar suggestion from reviewer 1.Finally, in complement to the data presented on Figure 7, depleting PfSufS in the bsmnmA+ (thus not expressing the YrvO desulfurase) cell line is likely to lead to both a loss of the apicoplast and a loss of parasite viability. This would be strengthening the idea that SufS acts by transferring sulfur to BsMnmA and would rule out an unrelated effect of MnmA alone.bsmnmA+ΔsufS parasite line to show that MnmA cannot compensate in some way for the loss of SufS. By ‘unrelated effect of MnmA alone’ we assume the reviewer is interested in whether MnmA+BsMnmA might be able to catalyze the liberation of sulfur from cysteine. If this was the case, we would have observed an intact apicoplast and mevalonate-independent growth phenotype in the ΔsufS line.The suggestion here is to generate a Reviewer #3 (Recommendations for the authors):1) The authors could elaborate some more on the result of the Mev-dependent phenotype of δ mnmA construct . This genetic construct presumably would contain an intact Fe-S biosynthetic pathway; however, loss of the apicoplast genome encoding sufB, ultimately leads to defects in Fe-S biogenesis, explaining the result.We thank the reviewer for this suggestion. We added a statement elaborating the effect of the loss of sufB in the MnmA knockout line (line 250-253):mnmA parasites were dependent on exogenous mevalonate supplementation for survival , which can be explained by the loss of sufB resulting in defective FeS synthesis and the inability to synthesize isoprenoids .”“Additionally, ∆2) The authors commented on the experimental challenge of generating a bsmnmA+ on a deltamnmA parasite line. Did the authors sequence the sufS gene in this resulting construct to rule out the possibility of selecting a sufS mutant that enabled this complementation? Along the same lines, could the authors comment on the expression levels of the Bacillus' enzymes when compared to the endogenous levels of PfSufS and PfMnmA?bsmnmA+ ∆mnmA line to investigate the possibility of selecting a SufS mutant. We have now sequenced the sufS gene sequence from two clonal lines of the bsmnmA+ ∆mnmA parasites. We also sequenced sufS gene from one of the bsmnmA-yrvO+ ∆mnmA lines and the PfMevattB parental line. We did not observe any mutation in the clonal lines of the bsmnmA+ ∆mnmA or in the bsmnmA-yrvO+ ∆mnmA parasites.The suggestion here was to sequence the We added this finding in the Discussion section (line 437-439).bsmnmA+ and bsmnmA-yrvO+ lines, the Bacillus genes were expressed using the promoter for calmodulin, which is expressed at a level greater than 90% of other P. falciparum genes (and much higher than mnmA or sufS). We intentionally tried to produce the Bacillus proteins at high levels to facilitate imaging of the mCherry fusion proteins. Live fluorescence is difficult to detect in poorly expressed proteins and our imaging results suggest that the Bacillus proteins are produced at a high level.In both the 3) Although this reviewer favors the conclusions of this study on the proposed role of PfSufS and PfMnmA in 2-thiouridine tRNA modification and the genetic experiments supporting this role, the results described in this study do not provide a direct demonstration of their involvement or exclusive involvement in tRNA thiolation. That is, no analysis of tRNA modifications was included in this report. I'm certainly sensitive to the experimental challenges associated with these experiments and the complexity of analyzing any data resulting from samples of an organelle of an intracellular parasite. The purpose of this comment is not to invalidate the value of this contribution or to set an expectation for additional experiments, as the report is already describing several novel findings. But an ask is to revise the document to indicate that instead of "establishing" a role in tRNA thiolation, the results support this proposed role. Also, perhaps adding a comment in the discussion would add value to readers being able to appreciate the complexity of multiple pathways involving 2sU in the organism and host.We appreciate the reviewer’s consideration regarding the technical challenges of analyzing apicoplast tRNAs given their low expression and the presence of the same tRNA modifications in the apicoplast and nucleus. We discussed these challenges in response to a similar comment made by reviewer 1 (point 1). We followed the reviewer’s suggestion and softened our claims by replacing words such as ‘demonstrating’ and ‘showing’ with statements about how the data support a role in tRNA thiolation. For example, the abstract now reads (line 42-44):“Complementation of MnmA and Sufis activity with a bacterial MnmA and its cognate cysteine desulfurase, strongly suggests that the parasite SufS provides sulfur for both FeS biosynthesis and tRNA modification in the apicoplast.”2U modifications in P. falciparum parasites (line 420-422). It is also true that host white blood cells also have the ability to form s2U tRNA modifications and that these cells are present in our culture system unless rigorous leukodepletion methods are used.As suggested, we added a sentence to the discussion to name the two enzymes that are predicted to catalyze sP. falciparum, NCS2 (PF3D7_1441000) is predicted to catalyze s2U modification of nuclear tRNAs while MnmA is predicted to make this modification in the apicoplast.”“In


MC1R) gene, resulting in a new hypothesis on the function of the encoded protein in regulating the cascade signals and leading to the production of different pigmentation. This result showed that local untapped breeds can be the reservoir of interesting genetic variants useful to better understanding underlying basic biological functions in mammals.Autochthonous pig breeds are important genetic resources, well adapted to local climatic conditions, environments, and traditional production systems, where they are associated with local and niche markets. The Greek Black Pig breed is the only local pig breed recognized in Greece. In this study, we started a population genetic characterization of this breed by analyzing a few gene markers associated with morphological and production traits and that usually differentiate wild boars from domestic breeds. The obtained results showed that, in the past, this breed experienced genetic admixture from two sources, wild boars and cosmopolitan breeds. On the one hand, this situation might raise some concerns for the genetic integrity of this animal genetic resource. On the other hand, this might contribute to within-population genetic variability reducing the problem of inbreeding of the small breed population. In this breed, we also identified a novel allele in the melanocortin 1 receptor  and compared these data with population genetic information available in other Mediterranean and Western Balkan pig breeds and wild boars. None of the investigated gene markers were fixed for one allele, suggesting that, in the past, this breed experienced introgression from wild boars and admixture from cosmopolitan pig breeds, enriching the breed genetic pool that should be further investigated to design appropriate conservation genetic strategies. We identified a new MC1R allele, containing two missense mutations already reported in two other independent alleles, but here present in the same haplotype. This allele might be useful to disclose biological information that can lead to better understanding the cascade transmission of signals to produce melanin pigments. This study demonstrated that autochthonous genetic resources can be an interesting reservoir of unexpected genetic variants.The Greek Black Pig (or Greek Pig) is the only recognized autochthonous pig breed raised in Greece, usually in extensive or semi-extensive production systems. According to its name, the characteristic breed coat color is solid black. In this study, with the aim to start a systematic genetic characterization of the Greek Black Pig breed, we investigated polymorphisms in major genes well known to affect exterior and production traits ( Sus scrofa, artificial directional selection in pigs has been the main driving force that substantially shaped the domestic genetic pools and differentiated them from the original European and Asian wild boar genetic background  [MC1R haplotype carries the same nucleotide of the D2E (MC1R*3) allele, that means it carries nucleotide C (MC1R pig sequence has been deposited in the European Nucleotide Archive (ENA) with accession number PRJEB59417. According to the two other nomenclatures of the allele series at this gene [MC1R*10 and 0302 . The reticulate position of this new MC1R sequence in the phylogenetic tree is due to the uncertainty of its origin between two equally plausible hypotheses: a de novo but recurrent mutation  that occurred in an D2E haplotype sequence; a recombination between an D2E allele and an e allele, which constituted a new haplotype carrying two mutations (p.N124D and p.A243T) of the two original alleles  allele, which is common in white breeds and populations such as Large White and Landrace. The KIT allele associated with the belted phenotype (g.8:43597545T) was detected, in heterozygous state, only in two other pigs. All these pigs had a typical solid black coat color. All remaining pigs had the wild-type genotypes in the investigated KIT gene markers. The markers were in the Hardy–Weinberg equilibrium.Only one pig carried the duplication of the NR6A1 and VRTN, were not fixed for the domestic (or mutated) alleles, with some differences in allele frequencies. In the NR6A1 gene, the mutated g.265347265T allele was the prominent allele (83.9%) in the Black Greek Pig breed. The wild-type C allele was present in 11 pigs. This polymorphic site was not in the Hardy–Weinberg equilibrium due to the lack of heterozygous animals . The analyzed polymorphism in VRTN (the SINE insertion or deletion) showed a balanced allele frequency with the mutated Q allele (with the insertion) that had a frequency of 44.9% and the wild-type allele  that had a frequency of 55.1%. Again, at this polymorphic site, the investigated population was not in the Hardy–Weinberg equilibrium (p = 0.04) due to an excess of heterozygous animals .The two investigated genes affecting vertebral number and teat number, IGF2 gene marker (frequency: 0.856). The G allele, considered the ancestral allele [A prevalence of the g.3072A allele was detected for the l allele ,41, was Allele frequencies for the same markers analyzed above were obtained for other 22 pig breeds and one wild boar population using whole-genome sequencing data from DNA pools . Table SIn both analyses, the Black Greek Pig breed was not closely positioned or clustered together with the wild boar population, even if wild-type alleles at all markers were also reported in this domesticated population.+E in the MC1R gene and of the wild-type allele of the NR6A1 gene in several pig breeds, including the Greek Black Pig breed. Gene flow might be also derived from other cosmopolitan breeds, again as also evidenced by the presence of several domestic alleles in the MC1R and KIT genes. This signature of reverse gene flow is not only evident in the Greek Black Pig breed but also in several other autochthonous breeds , that is associated with the dominant black coat color and that is expected to produce a constitutive activation of the receptor with a downstream signal transmission, mediated by the cAMP pathway, that leads to eumelanin production in the melanocytes [e allele at the Extension locus in pig [OCA2), encoding the homolog of the mouse p (pink-eyed dilution) gene, located on SSC15, might play a relevant role in affecting coat color in Duroc pigs [The most interesting results that we obtained was the identification of a novel  lineage ,20,22; M GenBank ,37,38,39e effect ,36. Thisanocytes ,48; p.A2s in pig . Accordis in pig . More deroc pigs .MC1R allele was originated. According to the obtained MC1R*10 nucleotide sequence, two equal probable hypotheses, as mentioned above, can be constructed. The first hypothesis assumes that a de novo and recurrent missense mutation (c.727G > A) causing the amino acid substitution p.A243T occurred in the MC1R*3 sequence, producing a new haplotype. This hypothesis might be supported by the fact that in another informative position (c.491C > T), allele MC1R*10 had the same nucleotide of allele MC1R*3. However, as the c.727G > A is downstream from the other two informative nucleotide substitutions that distinguish ED2 from e, it is not possible to completely exclude that MC1R*10 could have been produced by a rare recombination event involving both D2E and e right in the middle between the c.491C > T and the c.727G > A polymorphic sites to obtain the new haplotype that contained both missense mutations in the same allelic form. To lead these hypotheses in one or the other direction, other informative nucleotide sites should be obtained by sequencing downstream flanking regions and reconstruct a larger haplotype structure of the new MC1R*10 allele.It remains to infer the way in which this novel MC1R*10 allele in the breed population can also suggest that this novel allele probably emerged quite recently in the Greek Black Pig breed, through a new genetic event that could have good chances to be occurred in this breed. In this context, it is interesting to note that alleles D2E and e  allele, are the two most frequent alleles in the Greek Black Pig breed. Therefore, a recombination event between these two alleles  would be possible in this breed. It also seems more plausible that this allele is a new allele that emerged in this breed as no other studies, as far as we know, described this form in any other pig populations .The low frequency of the Q allele in the VRTN gene, which has been associated with a larger number of vertebrae and teats than the wild-type allele in many pig breeds  h,50 hQ ale allele , is the The investigation of DNA markers in untapped pig breeds can disclose the genetic history of these autochthonous animal genetic resources, usually well adapted to the local production systems. The quite high genetic variability detected in the five genes investigated in the Greek Black Pig breed indicated that admixture with both wild boar populations and other pig breeds occurred quite frequently (probably before the beginning of the conservation program), suggesting that it would be important to establish a well-defined plan able to maintain the genetic integrity of this pig breed. Other studies, including the genotyping of additional candidate gene markers and the analysis of thousands of SNPs covering the whole genome of the animals are needed to obtain a more detailed and improved picture of the two directions of gene flow (from wild boars and from cosmopolitan breeds) that might have shaped the current population genomic structure of the Greek Black Pig breed.MC1R allele in this breed, not previously reported in any other pig populations, indicates that local animal genetic resources can be the reservoir of interesting genetic variants that can be useful to understand basic biological mechanisms underlying relevant processes and phenotypic traits, with potential applications in other fields, including the use of unique gene markers for local breed product authentication.The identification of a novel


Long term survivors of childhood cancer have a high prevalence of chronic pain. Novel, multidisciplinary approaches to manage pain, are needed to allow for a reduction in the use of opioids for pain management. Physical activity is highly effective in managing chronic pain in children and adolescents, however, evidence about the combination of physical activity intervention and pain medications in chronic pain management in childhood cancer survivors is lacking. The aim of this study is to investigate the feasibility, acceptability and preliminary effects of a behavioral physical activity intervention integrated into standard post cancer directed treatment care to mitigate chronic pain in this unique population.This is a single site pilot randomized controlled trial of a 16-week physical activity intervention coupled with standard care. The primary aim is to assess the feasibility and acceptability of the physical activity intervention in childhood cancer survivors with chronic pain. Secondary aims include evaluating the differences in functional and psychosocial outcomes along with self-reported pain scores and cumulative dose of pain medications between the exercise group and standard cancer care group. The physical activity intervention is a home-based program structured to increase patients' physical activity behavior and to favor low intensity bodily movement using aerobic exercise and resistance training.This study will demonstrate that behavioral supportive measures like physical activity may be a novel means to improve cancer related chronic pain in young survivors of childhood cancer and decrease medication usage for pain along with improvement in functional and psychosocial outcomes. •Integrating a behavioral PA program into standard post-cancer directed care has a strong potential to mitigate chronic pain in CCS.•Pilot data from this study will provide essential information to guide PA recommendations for CCS with chronic pain.•This study will inform the development of clinical care guidelines for multidisciplinary management of chronic pain in CCS. MoreoveTreating chronic pain early on is important because if pain continues into adulthood ,16, it iMultidisciplinary approaches to manage chronic pain are needed to improve the quality of life of the CCS and to reduce the use of opioids which would also have the benefits of improving CCS' physical and psychosocial health . There iWe recently hypothesized that integration of PA into the management of patients' chronic pain could be an effective strategy to effectively mitigate pain while simultaneously reducing the use of pain medications . We beliGiven the evidence that exercise training has the potential to mitigate chronic pain through reframing pain sensations and sensitivity, we planned a randomized controlled two arm exercise intervention trial to demonstrate that integration of PA into standard cancer care will improve patient reported chronic pain, decrease the need for medications to manage chronic pain and improve functional and psychosocial outcomes in young survivors of childhood cancer.2The primary objective of this single-center, pilot randomized controlled trial is to determine the acceptability and feasibility of a 16-week behavioral PA intervention coupled with standard post-cancer directed treatment care in children and adolescent survivors of childhood cancer. The secondary objectives are to compare changes in chronic pain levels, cumulative dose of pain medications, physical function, and patient-reported outcomes within-group  and between the two groups.3ClinicalTrial.gov (NCT05562193).This manuscript adheres to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) Checklist . The tri3.1This is a single-site pilot randomized controlled trial where participants will be randomized in a 1:1 ratio to the 16-week behavioral PA intervention coupled with standard post-cancer directed treatment care, or to the standard post-cancer directed treatment care.3.2This study will be conducted in accordance with the Declaration of Helsinki. The study protocol was approved by the Penn State University Institutional Review Board (#00020762).3.3Potential participants will be identified through the Division of Pediatric Hematology and Oncology's Comprehensive Operational Database within the Penn State Health Children's Hospital at the Milton S. Hershey Medical Center, and through Penn State Cancer Institute located in Hershey, Pennsylvania in the United-States. The research coordinator will contact the patient's medical oncologist for medical clearance and approval to approach the patient's family  to present the study. If research staff are not able to contact the patient's medical oncologist to obtain their approval to approach their patients, potential participants will not be approached or enrolled.3.4A total of 20 participants will be enrolled following these inclusion and exclusion criteria.3.4.11)Male and female CCS diagnosed with any type of cancer;2)Completed all standard/planned cancer treatment and are stable at the time of enrollment;3)< 1-year post cancer therapy;4)Have chronic pain ;5)Between 10 and 17 years old at the time of enrollment;6)Who are and who are not non-ambulatory/wheelchair bound;7)Parent/legal guardian must be able to speak, read and understand the English language;8)Parent or legal guardian must be able to provide and understand informed consent;9)Participants must be able to provide and understand assent;10)Participants must be able to attend visits at Penn State Health Children's Hospital;11)Participant and parent/legal guardian must have access to a computer, smartphone or tablet.3.4.21)CCS who have not completed all standard/planned cancer treatment and/or not in complete remission at the time of enrollment;2)> 1-year post cancer therapy;3)< 10 years old and >17 years old at the time of enrollment;4)CCS who have evidence in their medical record of an absolute contraindication to complete any of the physical assessments. This exclusion criterion is at the oncologist's discretion and can be applicable any time during the study. Research staff will be informed of the absolute contraindication when they contact the medical oncologists via secure email or secure messaging through the electronic medical record for approval to approach their patients for the study and for medical clearance.5)CCS who have history of refractory or recurrent cancer;6)Participants or their parent/legal guardian who are unable to speak, read, and understand the English language;7)CCS who are unable to access and complete online questionnaires.3.5The informed consent document will be reviewed in-person with the potential participants and their parents or legal guardians. All the potential questions related to the study will be answered. All eligible and interested participants will be consented for study participation by an approved study team member. To ensure that children and adolescents (between 10 and 17 years old at the time of enrollment) give assent without parental influence, they will be asked to give a fair explanation of the study without the help of their parents. They will be asked personally if they agree to participate before giving their assent. Written informed consent will be obtained from parents or legal guardians and assent will be obtained from the participants prior to the initiation of any study-related activities.Participants and their parents or legal guardians will be informed that their participation in the research is voluntary, that their specific information will be de-identified, and that their decision to participate in this research study will not affect their care. Specific language in the consent document will allow to clarify that the relationship with their medical oncologist will not be altered if they choose not to consent to the research or choose to withdraw later. If enrolled participants request withdrawal from the study, we will ask for their withdrawal reasons and no further contact will be made. The participants and their parents or legal guardians will be informed that their data that was already collected can be retained and/or not included in the analysis according to their decision. The request for previously collected data to be removed from the analysis must be provided to the study team in writing.3.6A computerized random number generator will be performed to randomly assign each participant to the behavioral PA intervention coupled with standard post-cancer directed treatment care group (n = 10) or the standard post-cancer directed treatment care group (n = 10) in the order they are enrolled . A staff member not associated with the enrollment of the participants will create an envelope for each of the participants to be enrolled (n = 20). The envelopes will be sealed. Thus, staff members involved in the recruitment will not have any influence on the allocation of the patients.3.7Participants who are randomized to the intervention group will participate in a 16-week behavioral PA intervention coupled with standard post-cancer directed treatment care. The intervention will be based on the Children's Oncology Group Guidelines for Diet and PA recommendations , the IntThe behavioral PA intervention will be structured to increase patients' PA behavior and to favor low intensity bodily movement  or intensity <5 on a scale of 0–10), starting with a session duration of <15-min, three days per week. The session duration and frequency will increase over time. Participants will receive a set of resistance bands and a Fitbit (Inspire 3) at the beginning of the intervention, and they will be asked to wear their Fitbit every day (day and night) for the duration of the study. To note, data generated from the Fitbits will not be collected since the purpose of giving a Fitbit to participants is only for them to keep track of their physical activities.The behavioral PA intervention will also include support calls and text messages from the research staff, as follows: weekly support calls (weeks 1–4), biweekly support calls (weeks 6 and 8) and texts (weeks 5 and 7), monthly support calls (weeks 12 and 16) and texts . The behavioral PA intervention will be individualized according to patients' health status, results from the physical function assessment, and the most recent week's achieved PA. The intervention will be modified and adapted during weekly support calls or text messages, including frequency, intensity, time and type to maximize participants' success. The behavioral PA intervention will be conducted by an exercise physiologist and an exercise expert with specialty training in pediatric oncology.3.8Participants who are randomized to the intervention or control group will continue to receive standard post-cancer directed treatment care at the time of enrollment and during the study.Of note, participants who are randomized to the control group will only receive standard post-cancer directed treatment care. At the end of the study, however, these participants will be offered an exercise consult along with physical activity recommendations, a set of resistance bands, and a Fitbit (Inspire 3).3.9All participants (n = 20) enrolled to participate in this study will receive baseline assessments the same day they provided assent, and their parents or legal guardians provided written informed consent. Moreover, they will receive follow-up assessments on weeks 4, 8, 12 and 16, as described in 3.10All participants will self-report their date of birth and gender. They will also describe their racial background, their ethnicity and the highest grade of school they completed at the time of enrollment.3.11All participants will self-report their age at cancer diagnosis, the duration of their cancer treatment, the time since their treatment ended, their cancer diagnosis, and the treatment regimen they received. If necessary, this information may be extracted from medical records.3.12All participants will self-report their pain levels using the PedsQL pediatric pain questionnaire which is a symptom-specific instrument for children (8–12 years of age), and adolescents (13–18 years of age) to assess pain in pediatric patients with chronic health conditions . The par3.13All participants will self-report their pain medication usage using a questionnaire developed by the study team. This questionnaire will record usage of prescribed and over-the-counter medications and will gather information about the use of non-prescription pain medications . Additional questions related to smoking behavior and cannabis/cannabinoids use  will als3.14•Chart review to gather information on prescription pain medication usage through medication reconciliation data. Information will be collected about number of pain medications prescribed, total dosage and length of time prescribed. This review will be conducted by the principal investigator who is a pediatric oncologist.•The PDMP (Prescription Drug Monitoring Program) will be accessed to gather information on all filled prescriptions for controlled substances within the state of Pennsylvania. This review will be conducted by the principal investigator who is a pediatric oncologist.•Morphine equivalent dose (MED) will be calculated for each patient after looking at the total daily amount of each opioid the patient is taking. Using the standard conversion factors developed by the CDC, the Morphine milligram equivalent (MME) equates the many different opioids into a standard value that is based on morphine and its potency providing an easily interpretable metric to represent opioid use . This caFor all participants, three other strategies will be used to collect data related to pain medications, as follows.3.15All participants will answer psychosocial questions using the PROMIS pediatric questionnaires , includi3.16All participants will self-report their PA behavior using the original version of the Godin Leisure Time Exercise Questionnaire (GLTEQ) to assess participants' weekly frequencies of strenuous, moderate and light activities for at least 15 min during a typical 7-day period (a week) . They wi3.17Maximum voluntary isometric handgrip strength of the forearm muscles will be measured, two times, using a calibrated Jamar hydraulic hand dynamometer. This assessment is a valid and reliable measure that has been reported to be easy for children and adolescents to understand and achieve .predVO2peak) will be used [Aerobic capacity will be measured using a 6-min walk test (6 MW T) . Partici be used .Physical functioning will be measured using two different tests. During the 30-s chair stand test, participants will be instructed to complete as many full sit-to-stands as possible within 30-s. During the “Timed Up and Go” test, participants will be instructed to rise from a chair, walk 3 m (10 feet) around an obstacle, and then walk back to the chair and sit down. These tests will be performed twice. Participants' perceived exertion  will be3.18Sample size calculations were not required for this single-site, pilot randomized controlled trial. The sample size of 20 is justified due to the limited availability of participants with the specific eligibility criteria being investigated.Acceptability will be assessed and expressed as a percentage as follows: (number of patients enrolled × 100/number of patients approached).Feasibility will be assessed by the number of participants randomized to the behavioral PA intervention coupled with standard post-cancer directed treatment care who complete more than 50% of the intervention calculated and expressed as a percentage, as follows: (number of interventions completed × 100/number of interventions planned). To note, the planned interventions are the support calls and text messages, as follows: weekly support calls (weeks 1–4), biweekly support calls (weeks 6 and 8) and texts (weeks 5 and 7), monthly support calls (weeks 12 and 16) and texts .d effect sizes [The effects of the behavioral PA activity intervention coupled with standard post-cancer directed treatment care on secondary outcomes  will be explored using Repeated Measures Analysis of Variance (RM-ANOVA). RM-ANOVA  will be ct sizes  will be Drop-out and missing data will be accounted for and reported. If the reason is an adverse event during the intervention, it will be reported as well. In case of dropouts or missing data, an intention-to-treat analysis will be performed.3.19To ensure high quality data collection, written standard operating procedures will be created by the principal investigator (SD) and co-investigators (MC and KS) and disseminated to the study team members. When possible, validated questionnaires and measures were chosen. All data will be managed and stored in Research Electronic Data Capture (REDCap).3.20Safety data including adverse events or serious adverse events will be collected through the Pediatric Module of the Patient-Reported Outcomes version of the Common Terminology Criteria for Adverse Events (Ped-PRO-CTCAE). The Ped-PRO-CTCAE will be used to monitor adverse events in both groups at baseline, 4, 8, 12 and 16-week follow-ups. The NIH NCI Ped-PRO-CTCAE form Builder to build a study-specific custom form related to the behavioral PA intervention to provide a more specific scope of the safety of the intervention. The form has been developed in collaboration with and reviewed by pediatric oncologists. The recall period for the Ped-PRO-CTCAE is the past 7 days. The Ped-PRO-CTCAE responses are scored from 0 to 4. Adverse events reported during the study procedure that are different from those assessed at baseline will be reported to the responsible IRB.The study will be monitored by the Data and Safety Monitoring Committee (DSMC) and will require a review every 6 months and an audit yearly.3.21The results generated from this study will be analyzed, reported and disseminated through peer-reviewed publications, presentations at conferences focused on the supportive care of patients in oncology, exercise-related meetings and academic conferences.3.22Findings from this study will document the acceptability and feasibility of a 16-week PA intervention coupled with standard post-cancer directed treatment care in children and adolescent survivors of childhood cancer, with chronic pain. Although our target sample size is small, pilot data from this study has the strong opportunity to demonstrate that the integrating a behavioral PA intervention into standard post-cancer directed treatment care will contribute to decreasing patient-reported chronic pain, decrease the need for pain medications, and improve functional and psychosocial outcomes in CCS.Assuming this study is successful, our results will provide a compelling rationale to further examine the integration of behavioral PA interventions to mitigate chronic pain level from both a functional and quality of life perspective. Moreover, exploratory mechanism outcomes will have the potential to be explored, such as pain sensation, body sensation, and pain tolerance in CCS with chronic pain. The central hypothesis would be that exercise training may have the effect of decreasing pain by reframing the pain sensation and improving body sensations, along with increasing pain tolerance.The literature is scarce regarding the potential of integrating PA into standard post-cancer directed care in CCS. It is possible that barriers such as fear of movement, which would limit the potential of promoting PA behavior changes. Future research in this area is greatly needed.4There is limited evidence on how PA can impact patient reported chronic pain, need for pain medications, physical function and psychosocial outcomes in young survivors of childhood cancer. Given the great morbidity associated with the late effects of cancer therapy, the proposed study will provide essential information to guide PA recommendations for CCS with chronic pain and could inform the development of clinical care guidelines and policy making decisions for the multidisciplinary management of chronic pain in CCS.Children and adolescents diagnosed with cancer are rare overall and the design of exercise oncology clinical trials in this population is practically challenging. A strength of our study is the inclusion of CCS diagnosed with any cancer type, though we won't have enough statistical power to perform subgroup analysis, as its a pilot study. Our pilot randomized controlled trial will help us gather data to progress toward a fully powered efficacy trial where specific cancer subtypes could be studied, assuming that the results from this current study justify doing so. Our long-term goal is to identify CCS with chronic pain that could benefit the most from our behavioral PA intervention. At this stage, we can only hypothesize that integration of a behavioral PA intervention will be an effective strategy to mitigate pain while simultaneously reducing the use of pain medications.In recognition of the urgent need to improve the management of chronic pain, the NIH recently issued a call for projects and announced increased funding for pain research and its “Helping to End Addiction Long-term (HEAL)" initiative to speed scientific solutions to the national opioid public health crisis. Our study is in line with the research funding plan from the NIH and places us at a unique vantage point to build an outstanding and successful research project that answers the NIH call and improves supportive care for CCS.SD wrote and obtained the grant funding. MC, SD and KS conceptualized and designed the protocol. KS provided input into the development of the protocol. SD and MC drafted the manuscript and KS reviewed the manuscript. All authors have reviewed and approved the final version of the manuscript.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.