Wnt/ß-catenin signalling is crucial for maintaining the balance between cell proliferation and differentiation, both during tissue morphogenesis and in tissue maintenance throughout postnatal life. Whereas the signalling activities of the core Wnt/ß-catenin pathway components are understood in great detail, far less is known about the precise role and regulation of the many different modulators of Wnt/ß-catenin signalling that have been identified to date. Here we describe TMEM98, a putative transmembrane protein of unknown function, as an interaction partner and regulator of the GSK3-binding protein FRAT2. We show that TMEM98 reduces FRAT2 protein levels and, accordingly, inhibits the FRAT2-mediated induction of ß-catenin/TCF signalling. We also characterize the intracellular trafficking of TMEM98 in more detail and show that it is recycled between the plasma membrane and the Golgi. Together, our findings not only reveal a new layer of regulation for Wnt/ß-catenin signalling, but also a new biological activity for TMEM98. Wnt/ß-catenin signalling is crucial for embryonic development and tissue homeostasis in all multicellular animals. In mammals, it is first required for induction of the primitive streak at the onset of gastrulation . It contThe dynamic expression of 19 different WNT proteins results in a complex signalling landscape . SecreteUnder physiological conditions, activity of the Wnt/ß-catenin pathway is tightly controlled. A so-called ‘destruction complex’, containing AXIN1, APC, CSNK1 and GSK3, continuously binds and phosphorylates free CTNNB1. Because phosphorylated CTNNB1 is rapidly degraded by the proteasome, this ensures that low levels of cytoplasmic and nuclear CTNNB1 are maintained in the absence of a WNT signal. When WNT proteins engage FZD/LRP at the cell surface, the destruction complex is inactivated, resulting in an increase in the nucleocytoplasmic levels of CTNNB1 and the concomitant induction of CTNNB1/TCF transcriptional activity –10.FRAT/GBP proteins are potent activators of CTNNB1/TCF signalling independent from WNT/FZD activity due to their capacity to bind GSK3 –13. FRATXenopus FRAT homologue, GBP, is critically required for dorsoventral axis formation as part of the maternal Wnt pathway Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: NoReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The FRAT family of proteins have been shown to be activators of Wnt/β-catenin signalling. They act by binding to GSK3 thereby interfering with the assembly of the complex that phosphorylates free CTNNB1 and targets it for degradation. However, FRAT proteins are not required for Wnt signalling in mammals and so they are thought to be modulators. In this paper the authors carry out a yeast 2-hybrid experiment in order to detect proteins that interact with FRAT2 and identify the transmembrane protein TMEM98 as a potential binding partner. They go on to confirm the interaction between the two proteins by co-immunoprecipitation. They then investigate the sub-cellular localisation and topology of TMEM98 and find that it is recycled between the plasma membrane and the Golgi. They also examine the effect of TMEM98 on FRAT activity and protein levels.Whilst I find the yeast 2-hybrid, interaction data between FRAT2 and TMEM98, the topology data and some of the localisation data to be convincing I do not find some of the other data compelling enough to fully support the conclusions drawn by the authors as outline below.1) It does seem strange that GSK3 was not found in the yeast 2-hybrid experiment, this is worthy of discussion.2) In Westerns TMEM98-FLAG appears to be a double band . It appears to be a single band in other Westerns, for example Supplementary Fig 5. Also there are no size markers shown for any of the Westerns. Can the authors comment on this?3) In Fig 2A there is clear plasma membrane staining for the tagged TMEM98. Fig 2C shows localisation of TMEM98 to the outer periphery following release from Brefeldin A block. The arrowheads purport to indicate TMEM98 at the periphery 180 minutes after release. I do not find this particularly convincing, it is very hard to see on the printed picture and when enlarged on the screen I can see just a faint blue edge. This is nothing like the pattern of staining seen in Fig 2A. What does an untransfected cell look like? Is there no fluorescent signal?4) Fig 2D. For the TMEM98ΔN-AcGFP transfection again all the cells look blue, is this low or no signal?5) Fig2E. For the TM1-AcGFP transfection arrowheads are supposed to point to signal at distal protrusions of the ER. Again. very low signal and difficult to see. The authors describe the protein AcGFP as being cytosolic. However the intensity of the heat map suggests to me that the principal place the protein is found is in the nucleus not the cytoplasm.6) In the TOPFLASH reporter assays shown in Fig 3 is the effect of TMEM98 on FRAT-induced reporter activity statistically significant? Although we should not be slaves to p values it would be informative to know what they were as there is considerable spread and overlap in the data points. The authors state that TMEM98 reduced the level of CTNNB1/TCF signalling in a dose-dependent manner  but for the highest amount of TMEM98 the median level of signalling is higher than for all the lower amounts of TMEM98 so this statement is not justified.7) The authors conclude that there is a dose-dependent reduction in both FRAT1 and FRAT2 levels caused by increasing amounts of TMEM98 . By eye it is not convincing to me, the protein levels need to be quantitated to conclude this. The authors do some quantitation shown in Fig 4B and C but the amount of TMEM98-sGFP2 does not look to me as though it increases with increased amount of transfected plasmid. In Fig 4D the level of myc-FRAT1 appears much lower when transfected alone that when it was transfected along with TMEM98-FLAG (compare lanes 1 and 4). Also, there does not appear to be a difference between the signal for FRAT proteins in the presence or absence of full-length TMEM98 in Supplementary Fig 5A. In Supplementary Fig 7A increasing amounts of TMEM98-FLAG are transfected along with a constant amount of myc-FRAT2. However in lane 4 there appears to be less TMEM98-FLAG than in lane 3. In lane 4 the signal for myc-FRAT2 is lower than the adjacent lanes where the amount of TMEM98-FLAG detected is higher. Overall there is a lot of variability among the different experiments. The authors themselves say this on page 7 of the manuscript.8) Statistics should be done to test the significance of the TOPFLASH reporter assays shown in Fig 4E and F. There is a great deal of overlap in the pink box (just TMEM98 RNAi) and the blue and green boxes .9) In Supplementary Fig 7B and C the amount of TMEM98-FLAG is kept constant and the amounts of myc-FRAT1 and myc-FRAT2 are increased. The authors conclude that increased myc-FRAT2 increased TMEM98-FLAG levels but to me the signal from lane 1  looks extremely similar to the signal in 3 out of 4 of the lanes where increasing amounts of myc-FRAT2 were transfected. It is difficult to reach a convincing conclusion as to the effect of these two proteins on each other’s stability.10) What do the yellow and blue arrowheads indicate in Fig. 6?11) The reference list is duplicated.Reviewer #2: In this manuscript, van der Wal described that TMEM98 transmembrane protein interacts with FRAT2 and regulates its activity. The findings are interesting as they reveal a new layer of regulation of Wnt/b-catenin signaling. However, there are several major concerns that need to be addressed prior to publication.1. Figure 1：The binding between TMEM98 and FRAT2 appears to be very weak, as judged by the ratio between the FRAT2 band to the background bands which are faint in aFLAG but extremely strong in a-myc staining. This should be explained and discussed.2. In consideration of the false positive signal in yeast-two-hybrid assay, the interaction between TMEM98-ΔN and FRAT2 is not shown and should be repeated by co-immunoprecipitation in the presence of the proteasome inhibitor MG132, including Fig S5. It is necessary to show the binding between TMEM98 and FRAT2-ΔN as well.3. Figure 2: The data in Fig 2C showed that TMEM98-GFP signals were found to be closely associated with the nucleus in the absence of treatment, suggesting its presence in ER as well. In the presence of Brefeldin A, the signals were restricted to the Golgi, does Brefeldin A disturb the export from distal Golgi compartments to the cell surface , and does Brefeldin A inhibit protein transport from the Golgi to the ER? If so, these data should suggest that TMEM98 is transported from Golgi to the ER? The extracellular location of TMEM98 C-terminus in Fig 2A was not well verified . Other methods should be performed to substantiate the subcellular localization of TMEM98, such as using the technique defined in . And, the TMEM98ΔN-AcGFP was invisible  in Fig 2D.4. Figure 2H: it is mentioned in methods that cells were treated with trypsin in FACS analysis. However, trypsin will remove the extracellular C-terminus of TMEM98-Flag. Did this affect the results?5. Figure 3: since FRAT1 is a more potent activator of CTNNB1/TCF signaling than FRAT2 showed in Fig 3B, is it better to test the inhibition of FRAT1 activities in the presence of TMEM98 in Fig 3A? Meanwhile, the conclusion is not convincing that TMEM98 consistently reduced the level of CTNNB1/TCF signaling in a dose-dependent manner according to Fig 3A (maybe due to the non-linear increase in TMEM98 protein levels). Did the level of phosphorylated β-catenin proteins change when transfected with TMEM98 and FRAT1/2?6. As TMEM98-AcGFP protein does not inhibit FRAT2 activity to the same extent as TMEM98-FLAG and TMEM98-mCherry, it may not be suitable for subcellular tracing TMEM98 proteins in Fig 2 and Fig 6.7. If the model in Fig 7B is correct, we would expect that the region between two transmembrane domains would interact with FRAT. Is this true?References:Lorenz H,Hailey DW and Lippincott-Schwartz J. Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization. Nat Methods, 2006, 3(3): 205-210.Miller SG,Carnell L and Moore HH. Post-Golgi membrane traffic: brefeldin A inhibits export from distal Golgi compartments to the cell surface but not recycling. 1992, 118(2): 267-283.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  10 Nov 2019Response to specific editor comments:As indicated in your decision letter (PONE-D-19-18523), we have paid particular attention to the following:- For the presentation and interpretation of the imaging data in Figure 2, we have changed the LUT for some of the figure panels to provide better contrast. We have also modified some of the main text in our manuscript to prevent overinterpretation of our data.- For Figures 3-5 we have performed statistical testing as requested. We have also included new Western blots with quantification, since both reviewers commented on the variability of the data.Please note, that the overall interpretation of our data has not changed. Additional details and background are provided in the attached rebuttal letter. As for compliance with PLOS ONE journal requirements, we have ensured the following:1. All of the files in our resubmission meet PLOS ONE style requirements. 2. The sources of all cell lines have been indicated in the methods section.https://osf.io/ef74w/ (DOI provided in the methods section). 3. All original uncropped and unadjusted images underlying the blot and gel results have been posted at a public data repository at 4. All instances of data not shown have been removed from the manuscript.5. We have removed funding-related text from the Acknowledgements section. The funding statement as originally provided is still correct.Response to specific reviewer comments :We thank both Reviewer #1 and Reviewer #2 for their time and effort. Based on their constructive feedback, we have made the required changes to our manuscript. We are confident that we have addressed all of their concerns and that our manuscript has improved as a result.Reviewer #1 The FRAT family of proteins have been shown to be activators of Wnt/β-catenin signalling. They act by binding to GSK3 thereby interfering with the assembly of the complex that phosphorylates free CTNNB1 and targets it for degradation. However, FRAT proteins are not required for Wnt signalling in mammals and so they are thought to be modulators. In this paper the authors carry out a yeast 2-hybrid experiment in order to detect proteins that interact with FRAT2 and identify the transmembrane protein TMEM98 as a potential binding partner. They go on to confirm the interaction between the two proteins by co-immunoprecipitation. They then investigate the sub-cellular localisation and topology of TMEM98 and find that it is recycled between the plasma membrane and the Golgi. They also examine the effect of TMEM98 on FRAT activity and protein levels. Whilst I find the yeast 2-hybrid, interaction data between FRAT2 and TMEM98, the topology data and some of the localisation data to be convincing I do not find some of the other data compelling enough to fully support the conclusions drawn by the authors as outline below.We thank Reviewer #1 for their critical reading and evaluation of our manuscript. We are happy that they find the yeast-two-hybrid data, FRAT2 and TMEM98 interaction data, TMEM98 topology data and most of the localization data convincing, since these data are the foundation for the core message of our manuscript. We also appreciate the reviewer’s detailed comments and questions regarding some of the other data, and we want to take the opportunity to address all of their points in more detail below. 1) It does seem strange that GSK3 was not found in the yeast 2-hybrid experiment, this is worthy of discussion.Like the reviewer, we were initially surprised that we did not pick up GSK3 in our yeast-two-hybrid screen with full-length FRAT2. We reasoned that perhaps the FRAT2 N-terminus was masking the GSK3-interacting domain under the conditions used for the screen. This was one of the main reasons for repeating the yeast-two-hybrid screen with the N-terminal deletion mutant (FRAT2�N). However, as the reviewer rightly notes, we did not identify GSK3 in the FRAT2�N screen either. The simplest explanation is that for whatever reason the correct GSK3 clone/functional GSK3 fragment was not present in the yeast-two-hybrid cDNA library used for our Y2H screens. Another reason could be that the interaction between FRAT2 and GSK3 is relatively weak and/or transient. We don’t have any actual data to support either one of these scenarios. However, the reviewer might be interested to know, that the lab of Dr. Anton Berns (where this work was initiated) also never picked up GSK3 in any of the FRAT1 yeast-two-hybrid screens that were performed in the mid 1990s (this work remains unpublished).Since we do not have a clear explanation as to why GSK3 did not show up as an interactor in our yeast-two-hybrid screen , we chose not to spend too much space in the manuscript speculating about the potential reasons. 2) In Westerns TMEM98-FLAG appears to be a double band . It appears to be a single band in other Westerns, for example Supplementary Fig 5. Also there are no size markers shown for any of the Westerns. Can the authors comment on this?https://osf.io/ef74w/ (as per PLOS ONE guidelines). We apologize for the absence of the protein size markers. This was an omission on our part, which has now been corrected in the revised version of the manuscript. In addition, all of the original, uncropped blots have now also been made available via As for why TMEM98-FLAG appears as a double band on some of our blots but not on others: We hypothesize that the two bands represent different post-translationally modified species of TMEM98, but we have not investigated this further as this was beyond the scope of the current study. We suspect that the double bands are not visible on all of our blots because not all experiments were performed under the exact same conditions. For instance, some Western blots contain protein lysates made with RIPA lysis buffer that were run on a 4-12% NuPage pre-cast gel, whereas others depict protein lysates made with Promega’s passive lysis buffer  that were run on home-made 12% SDS-page gels. These differences are detailed in the materials and methods section . 3) In Fig 2A there is clear plasma membrane staining for the tagged TMEM98. Fig 2C shows localisation of TMEM98 to the outer periphery following release from Brefeldin A block. The arrowheads purport to indicate TMEM98 at the periphery 180 minutes after release. I do not find this particularly convincing, it is very hard to see on the printed picture and when enlarged on the screen I can see just a faint blue edge. This is nothing like the pattern of staining seen in Fig 2A. What does an untransfected cell look like? Is there no fluorescent signal?We agree with the reviewer that plasma membrane staining is more clear in some experiments than in others. We can offer multiple explanations for this: First, the experiments were performed in different cell lines (e.g. HeLa versus HEK293A) and with different fixations . We observed the clearest plasma membrane staining in living, unfixed HeLa cells . Second, the most prominent plasma membrane signal is not always detected in the same plane (or Z-position) as the ER or the Golgi signal . Third, the reviewer is correct in noting that the plasma membrane signal is relatively faint compared to other subcellular structures (including the Golgi), particularly in fixed cells . In these images, we set our PMT/gain so as not to oversaturate the most intense signal (Golgi/ER), causing the plasma membrane signal to be relatively low.In our revised manuscript, we have changed the LUT for Figures 2C-E to improve the contrast/visualization. We think that as a result, the plasma membrane signal (indicated by arrowheads) is now better visible and we hope the reviewer agrees. 4) Fig 2D. For the TMEM98ΔN-AcGFP transfection again all the cells look blue, is this low or no signal?A similar point was raised by Reviewer #2. We include the image below for the reviewers, which also contains the negative control sample for the experiment depicted in Figure 2D. In the image below, we have enhanced the contrast and brightness such that the untransfected control cells (3rd panel) are just barely visible. Thus, the signal for TMEM98�N  is indeed low signal. When the contrast and brightness of the TMEM98�N transfected cells are adjusted further , the untransfected cells in the well also become visible. In our revision, we have indicated the position of some of these cells with an asterisk in Figure 2D . In addition, we have changed the LUT for Figures 2C-E for better contrast/visualization. Thus, we hope that the reviewer agrees that the cells transfected with TMEM98�N are indeed expressing low levels of the deletion mutant, which is in agreement with our Western blots result presented in Figure 1.5) Fig2E. For the TM1-AcGFP transfection arrowheads are supposed to point to signal at distal protrusions of the ER. Again. very low signal and difficult to see. Again, we agree with the reviewer that the signal is subtle. In our revised manuscript, we have changed the LUT for Figures 2C-E to improve the contrast/visualization. Because we do not want to over interpret our data, we have removed the specific statement that signal reflects distal protrusions of the ER from the legend of Figure 2 . We have also removed the arrowheads from panel 2E accordingly.The authors describe the protein AcGFP as being cytosolic. However the intensity of the heat map suggests to me that the principal place the protein is found is in the nucleus not the cytoplasm.We agree with the reviewer that in the image depicted, the signal is more intense in the nucleus. We have reworded the figure legend to reflect that AcGFP (when not fused to TMEM98) is a free floating fluorescent protein . https://www.ncbi.nlm.nih.gov/pubmed/17586454).We want to point out that it is not uncommon for free floating fluorescent proteins to be distributed across the nucleus and the cytoplasm as a result of diffusion . This may well result in the signal appearing to be more intense in the nucleus than in the cytoplasm. For a reference, see Seibel et al. 2007 (6) In the TOPFLASH reporter assays shown in Fig 3 is the effect of TMEM98 on FRAT-induced reporter activity statistically significant? Although we should not be slaves to p values it would be informative to know what they were as there is considerable spread and overlap in the data points. We have performed statistical testing for Figures 3-5 as requested and have updated the figures, figure legends and materials and methods sections accordingly. As can be seen in Figure 3, the inhibitory effect of TMEM98 on FRAT induced reporter activity is statistically significant. The authors state that TMEM98 reduced the level of CTNNB1/TCF signalling in a dose-dependent manner  but for the highest amount of TMEM98 the median level of signalling is higher than for all the lower amounts of TMEM98 so this statement is not justified.We thank the reviewer for pointing this out. A similar point was raised by Reviewer #2. Given that we only had two data points for this particular condition (i.e. the highest amount of Tmem98), this precludes meaningful statistical testing. Therefore, we have removed this condition from Figure 3A. We have modified the text so as not to overinterpret our data and have removed all instances of “dose-dependent” inhibition. Page 13, lines 263-264 now reads: “Co-transfection experiments showed that TMEM98 reduced the level of CTNNB1/TCF signalling induced by FRAT2 .” See also point 7 below.7) The authors conclude that there is a dose-dependent reduction in both FRAT1 and FRAT2 levels caused by increasing amounts of TMEM98 . By eye it is not convincing to me, the protein levels need to be quantitated to conclude this. The authors do some quantitation shown in Fig 4B and C but the amount of TMEM98-sGFP2 does not look to me as though it increases with increased amount of transfected plasmid. In Fig 4D the level of myc-FRAT1 appears much lower when transfected alone that when it was transfected along with TMEM98-FLAG (compare lanes 1 and 4). Also, there does not appear to be a difference between the signal for FRAT proteins in the presence or absence of full-length TMEM98 in Supplementary Fig 5A. In Supplementary Fig 7A increasing amounts of TMEM98-FLAG are transfected along with a constant amount of myc-FRAT2. However in lane 4 there appears to be less TMEM98-FLAG than in lane 3. In lane 4 the signal for myc-FRAT2 is lower than the adjacent lanes where the amount of TMEM98-FLAG detected is higher. Overall there is a lot of variability among the different experiments. The authors themselves say this on page 7 of the manuscript.We appreciate the critical eye of this reviewer. First, the reviewer is correct in noting that “the amount of TMEM98-sGFP2 does not look to me as though it increases with increased amount of transfected plasmid”. This is indeed the case when TMEM98 is co-transfected with FRAT2 . This observation is the reason for proposing the model in new Supplementary Figure 7E . Given that we don’t have definitive proof for this model, we have moved the model from the main figures to the supplementary figures. Accordingly, we moved this section from the results to the discussion, which now reads : “Because TMEM98 protein levels did not increase linearly in the presence of FRAT2, as opposed to when TMEM98 was transfected alone , our findings are consistent with a model in which TMEM98 and FRAT2 form a negative feedback loop . As such, TMEM98 and FRAT2 may constitute a dynamic regulatory switch with the capacity to fine-tune CTNNB1/TCF signalling activity.”Second, the reviewer is also correct in noting that “Overall there is a lot of variability among the different experiments. The authors themselves say this on page 7 of the manuscript.”We initially suspected that these were pipetting errors, but after careful optimization of our experiments we suspect that this is actual, biological variation. We reported this variability to be transparent, but we now realize that it may have distracted from the main message of our manuscript, which is that TMEM98 binds and inhibits FRAT2. In our revised manuscript, we have modified Figure 4 and the corresponding main text to i) properly illustrate the inherent spread in our data, ii) allow proper quantification of the results and iii) perform statistical testing:- We have removed the data in which we compare the effects of TMEM98 overexpression on FRAT1 and FRAT2 protein levels, so as not to confuse the reader or dilute our main message (which focuses on FRAT2). The text has been modified accordingly. The discussion  now reads: “TMEM98 also binds and inhibits FRAT1, but whether TMEM98 affects the biological activity of both homologues to the same extent remains to be determined.”- We have included additional Western blots  to quantify the effects of TMEM98 overexpression on FRAT2 protein levels . The corresponding text  now reads: “Quantitative Western blot analysis revealed a variable, but consistent reduction in FRAT2 protein levels in the presence of full length TMEM98 .” - Some of the Western blots that were used in the first version of our manuscript  may have been overexposed , thus preventing accurate/meaningful quantification. We have replaced these Western blots with new, non-saturated Western blots from new experiments using fluorescent antibodies and LICOR detection . Although the corresponding results section , has been rewritten to focus on the TMEM98 mediated reduction of FRAT protein levels we want to stress that none of the changes we have made changes our original message or interpretation of the data.We believe that as a result of these changes the flow of our story has improved and more speculative (e.g. the negative feedback loop model) and potentially confusing aspects (e.g. inherent variability) have been moved to the discussion and supplementary data. 8) Statistics should be done to test the significance of the TOPFLASH reporter assays shown in Fig 4E and F. There is a great deal of overlap in the pink box (just TMEM98 RNAi) and the blue and green boxes .We have performed statistical testing for Figures 3-5 as requested and have updated the figures, figure legends and materials and methods accordingly. As can be seen in Figure 4E-F, only the increase in FRAT1 induced TOPFLASH reporter activity is statistically significant. We suspect that this is due to the fact that FRAT1 is a more potent activator of CTNNB1/TCF signaling than FRAT2 to begin with , which allows for a larger dynamic range.9) In Supplementary Fig 7B and C the amount of TMEM98-FLAG is kept constant and the amounts of myc-FRAT1 and myc-FRAT2 are increased. The authors conclude that increased myc-FRAT2 increased TMEM98-FLAG levels but to me the signal from lane 1  looks extremely similar to the signal in 3 out of 4 of the lanes where increasing amounts of myc-FRAT2 were transfected. It is difficult to reach a convincing conclusion as to the effect of these two proteins on each other’s stability.Once again, we thank the reviewer for their careful consideration of our data and we agree that it is difficult to reach a convincing conclusion as to the effect of TMEM98 and FRAT2 on each other’s stability. As detailed in our response to point 7) above, we have moved this more speculative aspect to the discussion and we have rewritten the results section to improve the flow and not distract from our main message. 10) What do the yellow and blue arrowheads indicate in Fig. 6?We apologize for not including this in the figure legend.Yellow arrowheads indicate co-localization, whereas blue arrowheads indicate examples without co-localization. This has been corrected in the figure legend . 11) The reference list is duplicated.We apologize for this error. This has been fixed in the revised manuscript.Reviewer #2In this manuscript, van der Wal described that TMEM98 transmembrane protein interacts with FRAT2 and regulates its activity. The findings are interesting as they reveal a new layer of regulation of Wnt/b-catenin signaling. However, there are several major concerns that need to be addressed prior to publication.We thank Reviewer #2 for their comments and constructive feedback. We are confident that we have addressed all of their concerns in the revised version of our manuscript. Below, we will address all of their points in more detail.1. Figure 1：The binding between TMEM98 and FRAT2 appears to be very weak, as judged by the ratio between the FRAT2 band to the background bands which are faint in aFLAG but extremely strong in a-myc staining. This should be explained and discussed.The reviewer is correct in noting that not all of the FRAT2 protein pool is bound by TMEM98. We agree that this can be explained by low affinity binding. However, we also offer another explanation: If our model, depicted in new Figure 6A , is correct, then only a small portion of the FRAT2 protein pool can come into contact with TMEM98. Thus, we think our immunoprecipitation data lend further support for a model in which the only interaction between FRAT2 and TMEM98 can take place inside MVBs. This has now been included in the discussion .”). We thank reviewer 2 for pointing out this fact.2. In consideration of the false positive signal in yeast-two-hybrid assay, the interaction between TMEM98-ΔN and FRAT2 is not shown and should be repeated by co-immunoprecipitation in the presence of the proteasome inhibitor MG132, including Fig S5. It is necessary to show the binding between TMEM98 and FRAT2-ΔN as well.We have not been able to detect an interaction between TMEM98�N and FRAT2 or between TMEM98 and FRAT2�N in co-immunoprecipitation experiments. We suspect that this is at least in part due to the fact that both deletion mutants (TMEM98�N and FRAT2�N) are mislocalized. As an example, we provide Figure 2 for the reviewer below, which illustrates the tendency of FRAT2�N to accumulate in the nucleus . A precise dissection of the interaction between TMEM98 and FRAT2 would thus require an entirely different experimental approach, and we argue that this is beyond the scope of the current manuscript.However, we want to point out that we provide multiple lines of evidence that support a physical and functional interaction between TMEM98 and FRAT2, including co-immunoprecipitation of the full length proteins , the fact that TMEM98�N is stabilized when FRAT2 is co-expressed , and the effect of both TMEM98 overexpression and knockdown on FRAT1 and FRAT2 protein levels and activity .3. Figure 2: The data in Fig 2C showed that TMEM98-GFP signals were found to be closely associated with the nucleus in the absence of treatment, suggesting its presence in ER as well. In the presence of Brefeldin A, the signals were restricted to the Golgi, does Brefeldin A disturb the export from distal Golgi compartments to the cell surface , and does Brefeldin A inhibit protein transport from the Golgi to the ER? If so, these data should suggest that TMEM98 is transported from Golgi to the ER? We suspect that the reviewer has perhaps misinterpreted Figure 2C and we apologize for any confusion we may have caused. As the legend indicates, the “0 minutes” timepoint in Figure 2C refers to the chase time after release from a 4-hour treatment with Brefeldin A . Thus, the “0 minutes” timepoint does not reflect the absence of treatment, but the result of 4 hours of treatment with Brefeldin A. The reviewer is correct in noting that at this point, most of the TMEM98 signal is indeed restricted to the ER. This is in agreement with previous reports demonstrating a role for TMEM98 in this compartment . After a longer chase (60 minutes and longer after release from the Brefeldin A treatment) TMEM98 again traffics to the Golgi and the cell surface (see arrowheads in the 180 minute chase panel). The extracellular location of TMEM98 C-terminus in Fig 2A was not well verified . Other methods should be performed to substantiate the subcellular localization of TMEM98, such as using the technique defined in . We thank the reviewer for suggesting that we perform a fluorescence protease protection (FPP) assay. We have considered this option to determine the orientation of TMEM98 in the plasma membrane. However, it would require us to also tag TMEM98 on the N-terminus and this is not possible due the presence of the N-terminal signal peptide and membrane anchor, which results in improper targeting and localization . This is one of the reasons why we chose to perform the FACS experiment depicted in Figure 2 instead. To our opinion, this experiment provides sufficient evidence that the C-terminus is located on the outside of the cell, confirming the experiments of Fu et al. . And, the TMEM98ΔN-AcGFP was invisible in Fig 2D.A similar point was raised by Reviewer #1. We include the image below for the reviewers, which also contains the negative control sample for the experiment depicted in Figure 2D. In the image below, we have enhanced the contrast and brightness such that the untransfected control cells (3rd panel) are just barely visible. Thus, the signal for TMEM98�N  is indeed low signal. When the contrast and brightness of the TMEM98�N transfected cells are adjusted further , the untransfected cells in the well also become visible. In our revision, we have indicated the position of some of these cells with an asterisk in Figure 2D . In addition, we have changed the LUT for Figures 2C-E for better contrast/visualization. Thus, we hope that the reviewer agrees that the cells transfected with TMEM98�N are indeed expressing low levels of the deletion mutant, which is in agreement with our Western blots result presented in Figure 1.4. Figure 2H: it is mentioned in methods that cells were treated with trypsin in FACS analysis. However, trypsin will remove the extracellular C-terminus of TMEM98-Flag. Did this affect the results?We thank the reviewer for pointing this out. We cannot exclude that indeed some signal may have been lost due to cleavage by trypsin. In fact, we believe that this might explain why less than 100% of the cell population shows up as positive in the extracellular staining. We have included a remark to this effect in the materials and methods section . However, we stress that this does not affect our main conclusion, which is that the C-terminus of TMEM98 is on the outside of the cell.5. Figure 3: since FRAT1 is a more potent activator of CTNNB1/TCF signaling than FRAT2 showed in Fig 3B, is it better to test the inhibition of FRAT1 activities in the presence of TMEM98 in Fig 3A? We have performed our most elaborate set of experiments with FRAT2, given that this is how we initially identified TMEM98. We do find that FRAT1 also binds to TMEM98  and indeed, knockdown of TMEM98 increases FRAT1 mediated CTNNB1/TCF signaling . Thus, we propose that TMEM98 biologically interacts with both FRAT homologues. However, it is beyond the scope of the current study to investigate how similar and/or different the activities of TMEM98 towards FRAT1 and FRAT2 are. We have removed the data in which we compare the effects of TMEM98 overexpression on FRAT1 and FRAT2 protein levels, so as not to confuse the reader or dilute our main message (which focuses on FRAT2). The text has been modified accordingly. This is also mentioned in the discussion : “TMEM98 also binds and inhibits FRAT1, but whether TMEM98 affects the biological activity of both homologues to the same extent remains to be determined.”Meanwhile, the conclusion is not convincing that TMEM98 consistently reduced the level of CTNNB1/TCF signaling in a dose-dependent manner according to Fig 3A (maybe due to the non-linear increase in TMEM98 protein levels). We thank the reviewer for pointing this out. A similar point was raised by Reviewer #1. Given that we only had two data points for this particular condition (i.e. the highest amount of Tmem98), this precludes meaningful statistical testing. Therefore, we have removed this condition from Figure 3A. We have modified the text so as not to overinterpret our data and have removed all instances of “dose-dependent” inhibition. Page 13, lines 263-264 now reads: “Co-transfection experiments showed that TMEM98 reduced the level of CTNNB1/TCF signalling induced by FRAT2 .”In our revised manuscript, we have modified Figure 4 and the corresponding main text to i) properly illustrate the inherent spread in our data, ii) allow proper quantification of the results and iii) perform statistical testing.We have included additional Western blots  to quantify the effects of TMEM98 overexpression on FRAT2 protein levels . The corresponding text  now reads: “Quantitative Western blot analysis revealed a variable, but consistent reduction in FRAT2 protein levels in the presence of full length TMEM98 .” Some of the Western blots that were used in the first version of our manuscript  may have been overexposed , thus preventing accurate/meaningful quantification. We have replaced these Western blots with new, non-saturated Western blots from new experiments using fluorescent antibodies and LICOR detection . Although the corresponding results section , has been rewritten to focus on the TMEM98 mediated reduction of FRAT protein levels we want to stress that none of the changes we have made changes our original message or interpretation of the data.Did the level of phosphorylated β-catenin proteins change when transfected with TMEM98 and FRAT1/2?Since TMEM98 reduces FRAT2 protein levels, it is expected that the levels of non-phosphorylated or active beta-catenin indeed change accordingly. However, this is likely to be a secondary effect, due to the change if FRAT2 levels and therefore we have not investigated this further.6. As TMEM98-AcGFP protein does not inhibit FRAT2 activity to the same extent as TMEM98-FLAG and TMEM98-mCherry, it may not be suitable for subcellular tracing TMEM98 proteins in Fig 2 and Fig 6.Cranfill et al. reported the tendency of AcGFP to dimerize , which is why we re-cloned our existing constructs into sGFP2 and mTq2 fusion proteins for our co-localization studies as a pre-caution. However, there is no reason to assume that an AcGFP fusion protein would result in major differences in trafficking at low levels of protein expression. In support of this, the plasma membrane and Golgi localization of TMEM98-AcGFP are confirmed by our results with TMEM98-sGFP2 , as are other subcellular localization patterns in Figure 5 , which were confirmed with co-localization of TMEM98-sGFP2 and/or TMEM98-mTq2 with Rab-fusion proteins . 7. If the model in Fig 7B is correct, we would expect that the region between two transmembrane domains would interact with FRAT. Is this true?The reviewer is correct that this is indeed our hypothesis. However, as we also point out, this would be hard to reconcile with some of the topology findings by Fu et al. (2015). At this point, both of our models are speculative. Future studies would be needed to determine the exact FRAT/TMEM98 binding interface. We have rewritten the discussion around Figure 6  to stress this point .AttachmentResponse_to_Reviewers.pdfSubmitted filename: Click here for additional data file. 12 Dec 2019PONE-D-19-18523R1TMEM98 is a negative regulator of FRAT mediated Wnt/β-catenin signallingPLOS ONEDear dr. van Amerongen,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Both reviewers agree that the manuscript is considerably improved. However there remain some concerns that need to be addressed. Please ensure that all the statistics are complete and all figure legends are included in the manuscript.  Also, issues related to markers for immunoblots and the signal strength of plasma membrane labeling also need to be addressed.https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.We would appreciate receiving your revised manuscript by Jan 26 2020 11:59PM. 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This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.We look forward to receiving your revised manuscript.Kind regards,Michael KovalAcademic EditorPLOS ONE[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: (No Response)Reviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The paper is much improved. I just have a few minor comments to make.Size markers are still not shown for all the Westerns, for example Fig 1B.The authors say “In our revised manuscript, we have changed the LUT for Figures 2C-E to improve the contrast/visualization. We think that as a result, the plasma membrane signal (indicated by arrowheads) is now better visible and we hope the reviewer agrees.” To me the signal at the cell periphery is still quite faint.In figure 3B please show the P value for the luciferase activity of myc-FRAT2 with and without TMEM98-FLAG.Please show the P value for the luciferase activity of myc-FRAT2 with and without TMEM98 RNAi #3 in Fig 4E. I am not convinced Fig 4F adds anything. Is it just a way of showing the data in Fig 4E in a different way?Apologies but I could not find the figure legends for the supplementary figures in the revised submission.Reviewer #2: The reviewers have adequately addressed my questions and concerns, and explained why some experiments can not be done at this stage.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  16 Dec 2019Please see our official cover letter for a direct response to the points highlighted by the editor. Our response to the reviewers is also attached as a separate rebuttal, and pasted below:We thank both reviewers for assessing the revised version of our manuscript. Below we want to take the opportunity to address the remaining comments of Reviewer #1.Reviewer #1: The paper is much improved. I just have a few minor comments to make.Size markers are still not shown for all the Westerns, for example Fig 1B.As shown in Figure 1, we have used the heavy chain (~50 kDa) and light chain (~25 kDa) bands from the antibodies used for the immunoprecipitation experiments as size markers . Given that we had 12 samples to run for these pull downs on a 12-slot gel, this precluded the inclusion of a protein size marker. Therefore, we gave preference to running the lysates and the pull downs on a single gel over the inclusion of a separate marker. As shown in the original data deposited at DOI 10.17605/OSF.IO/EF74W, these Western blots are clean and the heavy and light chain can be used reliably to get an impression of protein size distribution on the blot.Please note that the panel on the left comes from the same gel as the panel on the right . These markers thus refer to both panels (direct lysates and pull downs). We have double-checked all other Western blots in the main figures and supplementary figures, and all of them now have size markers. We apologize for not catching all of these omissions in our previous resubmission. Please note that the sole exception is the experiment depicted in Figure 4C/D, where we unfortunately forgot to copy the size markers onto the film after developing the Western blot (ECL detection). Here, we ask the reviewer to please also assess all of the original data deposited at DOI 10.17605/OSF.IO/EF74W, which shows that myc-Frat1/2 and Tmem98-FLAG reliably run at the expected sizes in all experiments (i.e. between 25 kDa and 35 kDa). The authors say “In our revised manuscript, we have changed the LUT for Figures 2C-E to improve the contrast/visualization. We think that as a result, the plasma membrane signal (indicated by arrowheads) is now better visible and we hope the reviewer agrees.” To me the signal at the cell periphery is still quite faint.We agree with this reviewer that the signal in the plasma membrane is “still quite faint” for the images depicted in Figure 2C-E. However, we want to emphasize that our message is not that TMEM98 is predominantly located in the plasma membrane, but that it is present in the plasma membrane at all. As described on pages 8-9, TMEM98 is both predicted to be secreted as well as to be a transmembrane protein. Our experiments  support that at least part of the TMEM98 pool resides in the plasma membrane, which we believe is an important confirmation of the resident location of TMEM98. Figure 2C supports this conclusion, since it shows that after being trapped in the ER following Brefeldin A treatment, part of the TMEM98 pool again reaches the plasma membrane 3 hours after release . To show the reviewer additional examples, we provide the images below. These also show TMEM98 returning to the plasma membrane 2 hours (top left) and 3 hours (following 3 images) after release from Brefeldin A treatment. Please note that the image depicted in 2C is also shown in the images below (bottom right). 2 hours after release. Note plasma membrane signal at the cell-cell border. 3 hours after release. Note the subtle outline of the plasma membrane. 3 hours after release. Note the subtle outline of the plasma membrane. 3 hours after release (same as image 2C). Note the subtle outline of the plasma membrane and the visibility of the distal cell membrane protrusions.The grey scale enhances the contrast, but the reviewer is right: the signal in the plasma membrane is subtle. As we already indicated in our previous rebuttal: We observed the clearest plasma membrane staining in living, unfixed HeLa cells . Please note that Figure 2C was made using PFA fixed cells, which in our hands reduced the intensity of the plasma membrane signal. The plasma membrane signal is also faint compared to other subcellular structures , particularly in fixed cells . In these images, we set our PMT/gain so as not to oversaturate the most intense signal (Golgi/ER), causing the plasma membrane signal to be relatively low. Please note that all of these subtleties are indicated in the Legend for Figure 2.Taken all of this together, we stand by our conclusion as stated in the manuscript on pages 8-9: “A TMEM98-GFP fusion protein indeed localizes to the plasma membrane , in addition to showing more prominent localization to the Golgi and intracellular vesicles .”In figure 3B please show the P value for the luciferase activity of myc-FRAT2 with and without TMEM98-FLAG.We analyzed our data with Prism, using a one-way ANOVA and Tukey’s multiple comparisons test (comparing every condition to every other condition). In this analysis, neither the difference between vector and myc-Frat2 alone (first condition vs. fifth condition), nor the difference between myc-Frat2 alone and myc-Frat2 + TMEM98-FLAG is statistically significant . One explanation is the large intrinsic difference in the ability of Frat1 and Frat2 to induce TOPFLASH luciferase reporter activity, with Frat2 being a less potent activator of CTNNB1/TCF signaling . We chose to show only significant comparisons in the figure (as stated in the figure legend), which is why this P-value is not shown/depicted. The figures would get really cluttered if all non-significant comparisons are also included. We have modified the last sentence of the figure legend (page 15), which now states “Only statistically significant differences  are indicated with asterisks“. Please show the P value for the luciferase activity of myc-FRAT2 with and without TMEM98 RNAi #3 in Fig 4E. As above: When analyzed using a one-way ANOVA and Tukey’s multiple comparisons test (comparing every condition to every other condition), neither the difference between vector and myc-Frat2 alone (first condition vs. fifth condition), nor the difference between myc-Frat2 alone and myc-Frat2 + RNAi #3 is statistically significant . We chose to show only significant comparisons in the figure (as stated in the figure legend), which is why this P-value is not shown/depicted. The figures would get really cluttered if all non-significant comparisons are also included. We have now modified the figure to include all significant comparisons  and we have changed the last sentence of the figure legend (page 16), which now states “Only statistically significant differences  are indicated with asterisks“. I am not convinced Fig 4F adds anything. Is it just a way of showing the data in Fig 4E in a different way?The reviewer is correct, this is a different representation of the same data, as stated in the figure legend on page 16. Apologies but I could not find the figure legends for the supplementary figures in the revised submission.Please note that the supplementary figure legends should have been included as a separate file (Supplementary Files.docx / Supplementary Files.pdf) with our last submission, since it was our impression that they should not be included with the main manuscript as per PLOS ONE manuscript preparation guidelines. This time they have again been uploaded as a separate file, again named Supplementary Files.docx / Supplementary Files.pdfReviewer #2: The reviewers have adequately addressed my questions and concerns, and explained why some experiments can not be done at this stage.We thank Reviewer #2 for their time and effort.AttachmentResponse_To_Reviewers.pdfSubmitted filename: Click here for additional data file. 19 Dec 2019TMEM98 is a negative regulator of FRAT-mediated WNT/β-catenin signalingPONE-D-19-18523R2Dear Dr. van Amerongen,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Michael KovalAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 9 Jan 2020PONE-D-19-18523R2 TMEM98 is a negative regulator of FRAT mediated Wnt/β-catenin signalling Dear Dr. van Amerongen:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Michael Koval Academic EditorPLOS ONE


Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG), starting from known compounds with activity against a related parasite PKG orthologue, is reported. Examination of key sub-structural elements led to new compounds with good levels of inhibitory activity against the recombinant kinase and in vitro activity against the parasite. Key examples were shown to possess encouraging in vitro ADME properties, and computational analysis provided valuable insight into the origins of the observed activity profiles.Development of a class of bicyclic inhibitors of the  Plasmodium, with most deaths caused by the P. falciparum species. Despite being largely preventable and treatable, it is currently responsible for almost 0.5 million deaths per year, with young children and pregnant women in sub-Saharan Africa particularly at risk.Malaria is one of the most prevalent infectious diseases of the developing world whose causative agent in humans is the protozoan parasite P. falciparum kinome play important roles in multiple stages of the parasite lifecycle.P. falciparum cGMP-dependent protein kinase (PfPKG) is attracting considerable interest as a promising antimalarial drug target. Chemical inhibitors of this enzyme have demonstrated prevention of merozoite egressPfPKG remains an appealing drug target for developing new anti-malarial small molecules.There is a growing body of evidence to suggest that members of the 1b]pyridines as potent and selective inhibitors of PfPKG.2in vitro blood stage anti-parasite activity, good selectivity against human kinases and significant in vivo target-driven efficacy.2 in ways that would address these important physicochemical property considerations, whilst maintaining cell potency and lipophilic ligand efficiency.We have recently reported studies on thiazoles2 would aid the achievement of these objectives. The pyrimidine and its 2-substituent offered opportunities to influence potency and to probe the hinge binding motif against recent crystallographic datain vitro profiles as described above.We considered that three important initial areas of focus on the structure of 5 was obtained from 4-thiomethyl-6-methylpyrimidine 34, transformation of the thiomethyl motif and introduction of the diaminomethyl side chain  assays.78 was also nearly 40-fold less biochemically active, and other heterocycles such as 9 showed no improvement.10. Introduction of an aryl piperazine motif in 11 resulted in a further drop in potency, in stark contrast to our previous observations on similarly extended aminopyrimidines in the thiazole series.2 was likely to provide an optimal interaction with the hinge region of the enzyme.These structural alterations significantly influenced levels of enzyme inhibition . The reg2, 8 and 11 into a recently published PfPKG crystal structure (PDB:5DYK)2 and 8 to enable charge interactions with two acidic residues at the solvent interface (E625 and D682). Classical bidentate hydrogen bond formation to hinge residue V621 contributes significantly to the high affinity of 2 and explains the lower affinity of 7 and 8. However, pyridine 8 requires a larger rotation out of plane from the bicyclic core , in order to avoid steric clash between hydrogen atoms. Introduction of the larger phenyl piperazine in 11 appeared to allow an edge-face aromatic interaction with Y822 and a relatively long-range charge interaction with E625 at the pocket mouth. Formation of these interactions had been proposed as a route to obtaining a boost in potency in the thiazole series.N,N-dimethylamine group and E625. It also appears that re-introduction of a bidentate hinge-binding motif in aminopyridine11 is not sufficient to regain affinity. Though this favours a more coplanar arrangement of the bicyclic core and hinge-binding heterocycle, steric clash between the two rings and the increase in strain energy largely negates any affinity gain. This is reflected in an intermediate calculated dihedral angle (26.2°) for 11. Hence the current data suggested retaining the amino pyrimidine motif as a more appropriate choice of hinge binding motif, and one which would best allow for effective substituent growth towards the mouth of the binding pocket in future analogues.Docking of key examples in vitro metabolism. In addition, larger aminopyrimidine substituents had previously been shown to provide significant additional in vitro potency,12 or 13 could be assembled using similar chemistry to that described above. The sulfide 12 could be oxidised and easily displaced with more reactive amines, as shown in 13 proved to be more suitable, allowing rapid and efficient preparation of the desired analogues.We turned our attention to the benzylic tertiary amine group attached to the bicyclic core, and hypothesised that re-positioning it to become part of the aminopyrimidine substituent would be beneficial for metabolic stability whilst maintaining potency. In doing so, it was thought that the deletion of both a tertiary amine and a labile benzylic methylene group would remove two likely sites for primary 14 showed a promising level of in vitro potency  cell-based assay were obtained. While metabolic stability for 16 remained reasonably good, other aspects of physicochemistry were likely to be driven by poorer solubility. Adjusting basicity (as in piperidine 17), or combining with a change in vector (e.g. pyrazole 18) provided only isolated improvements in ADME properties (eg. solubility for 18).16–18 was unexpected given their measured logD values, for which there was no convincing explanation. Taken together, these data suggested that this type of aryl linked analogue was in many cases able to provide very good levels of potency, but that a well-balanced overall in vitro profile was not easy to locate in compounds lacking the basic substituent on the bicyclic core.We were encouraged to find that early examples such as the simple alkyl amine  potency . Conform19 and 20 could be synthesised from the regioisomer of 3 and an appropriate aminopyrimidine in the same way as described for 5, with separation of the regioisomeric bicyclic intermediates. Benzimidazole 23 could be prepared by oxidation and displacement of intermediate 2122 and reductive amination  regioisomer. Transformation of the desired regioisomeric product using known chemistry provided the pyrazolopyridine 29.The imidazopyridazine  ammonia . Then diin vitro activity and ADME profile. The imidazopyrimidine examples 19 and 20 could not in either case match the biochemical activity, anti-parasite activity or lipophilic efficiency of 2, though both possessed broadly similar ADME profiles , but appeared to be significantly weakened in the case of benzimidazole 23 (19 and 20, higher desolvation penalties due to increased polarity could explain the lowering of affinity for those molecules. The imidazopyridazine 26 showed an electronegative field isosurface very similar to 2 and 29, but two additional factors may contribute more significantly to its lower affinity. Firstly, the electrostatic repulsion between the nitrogen atoms on the core and pyrimidine ring appears likely to deplanarise that part of the molecule, weakening key interactions in doing so. Secondly, the pendent amine employed may result in less optimal positioning of the charged group, despite increased polarity and flexibility.Computational calculations again suggested a plausible framework by which to understand these data. Each bicyclic system bears a conserved nitrogen atom in the 5-membered ring, but this does not appear to engage the side chain of residue K570 in a bonding interaction in any case. A similar observation was made for a crystal structure of the azole 23 . AlthougPfPKG inhibitors, based upon a previously described chemical starting point. By focusing on key sub-structural motifs, we have both confirmed and enhanced important SAR and physicochemical trends, resulting in key examples such as 16 and 29. These two compounds showed good levels of biochemical and in vitro anti-malarial activity; 29 in particular possessed equivalent mLogD, improved passive permeability and retained a good level of lipophilic ligand efficiency. Docking and computational analyses provided additional insight into the origins of the observed structure activity relationships. Studies to refine, expand and apply this new knowledge to the preparation of further improved compounds will be reported in due course.This report has described our initial efforts to develop a series of bicyclic


The possible oncologic benefit of radical lymphadenectomy has been blurred by a number of reasons. In most of the trials the strategies under comparison were made similar after protocol violations. Imperfect design of the trials could not exclude the influence of cofounding factors. Inappropriate endpoints could not detect evidently the difference between the two surgical strategies. On the other hand radical lymphadenectomy was characterized by increased morbidity and mortality. This was mostly caused by the addition of pancreatico-splenectomy in all D2-dissections, even when not indicated. A careful analysis of the available evidence indicates that D2-lymphadenectomy performed by adequately trained surgeons without resection of the pancreas and/or spleen, unless otherwise indicated, decreases Gastric Cancer Related Deaths and increases Disease Specific Survival. This evidence is not compelling but cannot be ignored. D2-lymphadendctomy is nowadays considered to be the standard of care for resectable gastric cancer.The role of extended lymphadenectomy in the surgical treatment of gastric cancer has been debated for many years. So far six prospective randomized trials and a number of meta-analyses comparing D The benefit of resection of regional lymph nodes in gastric cancer been debated for many years and still remains under question. Τhe failure of limited surgery to control disease loco-regionally was reported as early as the 1950s by Gordon McNeer. In his autopsy series he found more than 20% cancer recurrence in the non-resected perigastric nodes or the gastric bed  and didn1 lymphadenectomy (formerly known as R1-resection), removal of the second tier D2 lymphadenectomy (formerly known as R2 resection), removal of the third a D3 lymphadenectomy. In the 1960’s the Japanese Society suggested that removal of the appropriate number of tiers would increase the chance of negative “lymphadenectomy margins”. Furthermore, under the influence of the work of Kanai, Kajitani and Aikou  however the outcome of multivariate analysis was not reported   . Neverth2 column most of them suffer from heterogeneity. Also the duration of follow-up differs among the included studies. Mocellin et al  . .2-lymph2-dissection leads to increased morbidity which on the other hand can be decreased significantly by avoiding resection of the spleen and the pancreas unless properly indicated.Taken all these together we may conclude that D2-dissection plus pancreaticosplenectomy presented the poorest survival. It was speculated that the possible benefits of extended lymphadenectomy were thus blurred. In the Dutch trial splenectomy or pancreaticosplenectomy decreased mean Overall Survival after 15 years in both the D1 and D2 dissection group . After excluding both non-compliant and contaminated operations the difference became 10%  but still remained not significant  was higher than the survival of the non-contaminated D1-group (those who had a proper D1-dissection); and more importantly it was higher than the survival of the remaining 192 patients who underwent what the authors perceived as “D2 dissection” (A strict adherence to the study protocol is of paramount importance in any clinical trial. Quality control in the comparison of two lymphadenectomy strategies should ensure the complete removal of the lymph node stations as predetermined by the protocol, and only these. Removing more or less stations constitutes protocol violation. If fewer stations are removed then compliance to the protocol is defective, if more stations are resected then contamination ensues. In particular for lymphadenectomy in gastric cancer, contamination has been defined as the resection of more than two lymph node stations that should not have been removed i.e., over-resection . On the nificant . Howeversection” .1- or D2- lymph node dissection and being able to discriminate between these two operations demands appropriate training. This improves operative time, lymph node yield, morbidity and mortality. Parkih et al in UK reported that the curve reaches a plateau after 18—24 months or 15–25 procedures (2 dissections due to their participation in the Italian multicenter phase II study (2-arm in the first 10 months of the study i.e., the training period and some more months. The Japanese instructor operated 34 patients and the supervising surgeons 33 (2 dissections (The role of training and of the learning curve also cannot be overemphasized. Conducting a proper Docedures , Lee et ocedures  while Wuocedures . Furtherocedures . Such anII study . ConsequII study  or 6 monII study  one JapaII study  or 11 (1II study  regionalII study . 67 patigeons 33 . In othesections . The trisections  are fulf2 arm were accidentally older in the South African trial (Beyond any doubt the comparison of two different surgical strategies against a known lethal disease such as gastric cancer should have survival as a chief endpoint. However Overall Survival includes also all those patients who died from causes unrelated to gastric cancer. Although theoretically these patients will be equally distributed in the arms of a well conducted randomized comparison, this is not always the case. For example, the patients of the Dan trial , and thean trial .1 arm and a 12% improvement with D2 dissection (1-dissection was found to be around 30% (Any trial should have the power to prove the expected difference in the treatment outcome. One can speculate that this was not probably the case in the trials of Dent et al  and Robessection . These eound 30% . To provound 30% , which b2- lymphadenectomy. If morbidity and mortality are kept to a minimum, then probably D2 lymphadenectomy is advantageous, in particular for patients with locally advanced tumors. However it seems that this advantage is easily wiped out by unrequired resection of the pancreas and/or spleen and by inadequately trained surgeons. It has to be noted however that the existing studies are neither recent nor flawless and the conclusions drawn may not pertain to today’s modern medicine. Since the closure of the last trial on the row, some 12 years ago, much progress has been made in the diagnosis, staging, peri-, and post-operative care of patients with gastric cancer. Perhaps in the near future as medicine advances, the necessity and extend of lymphadenectomy will have to be redefined. The new framework of surgical oncology should be individualized and take into consideration “novel” parameters like neoadjuvant treatment, biological therapies and, more importantly, the molecular characteristics of each tumor. Until new solid evidence appears, with new, contemporary, well designed and properly conducted randomized trials, implementation of D2-lymphadenectomy into the daily practice of well-trained surgeons seems justified. For this reason, the vast majority of the scientific societies worldwide issued guidelines with D2- lymphadenectomy as the standard treatment for resectable gastric cancer (From the studies conducted so far there is plenty of evidence on the equivalence, and there is limited evidence on the superiority of Dc cancer  providedIK and AM both participated on the conception and design of the manuscript; data acquisition, interpretation and analysis; drafting, revising and final approval of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest


In each node, patient samples were analysed via class imbalance and hybrid feature selection techniques to build the prediction model. The outcome from analysis of each node was a set of genes that could differentiate each Gleason group from the remaining groups. To validate the proposed method, the set of identified genes were used to classify a second dataset of 499 prostate cancer patients collected from cBioportal. (3) Results: The overall accuracy of applying this novel method to the first dataset was 93.3%; the method was further validated to have 87% accuracy using the second dataset. This method also identified genes that were not previously reported as potential biomarkers for specific Gleason groups. In particular, PIAS3 was identified as a potential biomarker for Gleason score 4 + 3 = 7, and UBE2V2 for Gleason score 6. (4) Insight: Previous reports show that the genes predicted by this newly proposed method strongly correlate with prostate cancer development and progression. Furthermore, pathway analysis shows that both PIAS3 and UBE2V2 share similar protein interaction pathways, the JAK/STAT signaling process.(1) Background:One of the most common cancers that affect North American men and men worldwide is prostate cancer. The Gleason score is a pathological grading system to examine the potential aggressiveness of the disease in the prostate tissue. Advancements in computing and next-generation sequencing technology now allow us to study the genomic profiles of patients in association with their different Gleason scores more accurately and effectively. (2) Methods: In this study, we used a novel machine learning method to analyse gene expression of prostate tumours with different Gleason scores, and identify potential genetic biomarkers for each Gleason group. We obtained a publicly-available RNA-Seq dataset of a cohort of 104 prostate cancer patients from the National Center for Biotechnology Information’s (NCBI) Gene Expression Omnibus (GEO) repository, and categorised patients based on their Gleason scores to create a hierarchy of disease progression. A hierarchical model with standard classifiers in different Gleason groups, also known as  Cancer is among the main causes of death worldwide. Among males, prostate cancer is the cancer type with the highest incidence; 1.276 million new cases were diagnosed in 2019 [In the 1950s, Pierre Denoix devised a system that categorises solid tumours into different stages . The claRecent prostate cancer research has greatly focused on identifying gene expression patterns that correlate with disease progression, and can be used as predictive tools for patient treatment and outcome. Moreover, advances in next-generation sequencing (NGS) technology have made genomic data analysis widely available. The output of NGS sequencers requires preprocessing algorithms to do things such as align the reads to a reference human genome and assemble them into transcripts. Many genomic tools that align the RNA-Seq reads to the human genome have been proposed, especially BLAST is one of the first tools developed to align reads . TopHat2In addition, machine learning applications in genomic analysis have become a solid approach to analysing RNA-Seq data for studying a multitude of diseases. Alkhateeb et al. proposed a supervised method to discover biomarkers that can predict the likelihood that a prostate cancer tumour will progress to the next stage . ArvanitThe focus of this study was to identify genes that can be used to differentiate specific Gleason groups. This work is an extension of our previously proposed prediction model, which was based on analysing the RNA-Seq data from patients with different Gleason scores . The metThe first dataset used in this study is a collection of 104 samples and their TPM values. Stated as a classification problem, this study designates five classes obtained from joint Gleason groups. The distribution of each group is shown in The model also identified six gene transcripts that are differentially expressed in the five different Gleason scores. Of these, the corresponding genes shown in Naïve Bayes outperformed the other classifiers, as it distinguished the first Gleason score node from the rest with 94% accuracy, the second node with a higher accuracy of 98%, and the last two Gleason score nodes with 100% accuracy, as shown in To further validate the model, we applied the method on a second publicly-available dataset  obtainedUBE2V2, whose gene’s transcript was selected in the third node of our hierarchical model, has been shown to protect cells by mediating DNA repair functions [Many of the genes that encode the differentially expressed transcripts identified in this study have been previously shown to play various roles in cancer. Some have been shown to promote cancer progression, while other play a protective role. For example, unctions . In famiunctions . In our unctions .GPR137 expression and EPB41L1 is associated with tumours of Gleason scores 3 + 4 = 7 and 8, respectively. Earlier studies show that proteins encoded by EPB41L1 are associated with the proper organisation of the cell cytoskeleton, and that EPB41L1 plays an important role in the negative regulation of cell metastasis, migration, and invasion. Expression of EPB41L1 has been observed to be lower in prostate cancer compared to normal cells. Although it remains unclear, disruption of normal EPB41L1 expression may play an important role in disorganised cell and tissue structures associated with higher grade prostate cancer [EPB41L1 plays an important role in recurrence and has been associated with highly metastatic lung and breast cancer [EPB41L1 was also shown to be differentially expressed in gastric cancer [GPR137 expression has been shown to be upregulated in prostate cancer tissues compared with paracancerous tissues. Moreover, knockdown of GPR137 resulted in decreased cell proliferation and colony formation in PC-3 and DU145 prostate cancer cell lines, and was associated with cell cycle arrest at G0/G1 phase. GPR137 suppression also decreases the migration and invasive abilities of PC-3 cells, suggesting that GPR137 plays a role in prostate cancer progression and metastasis [Our study also revealed that the differential expression of e cancer , and thut cancer . EPB41L1c cancer . On the tastasis .PIAS3 and Rest Corepressor 3 (Rcor3) were both associated with tumours of Gleason score 4 + 3 = 7. While very little is known about the role of Rest Corepressor 3 (Rcor3) in prostate cancer, it has been shown to act as an antagonist of cell differentiation [PIAS3 expression has been observed in a variety of human cancers, including lung, breast, prostate, colorectal, and brain [PIAS3 is expressed in prostate cancer cells, and its expression is induced in response to androgens [PIAS has been shown to enhance the transcriptional activity of androgen receptors (AR) in prostate cancer cells, other studies have revealed that ectopic overexpression of PIAS3 suppresses AR-mediated gene activation induced by dihydrotestosterone (DHT) [PIAS3 acts as a negative regulator of AR transcriptional activity and signaling through direct protein–protein interaction. Recent findings have also revealed that AR is also differentially correlated with Gleason score patterns in both primary and metastatic prostate cancer, where it is upregulated in Gleason group 4 and downregulated in Gleason pattern 5.Differential expression of ntiation , a charand brain . PIAS3 indrogens ,26. Althne (DHT) . PIAS3 aPIAS3 is a member of the mammalian PIAS family consisting of four members: PIAS1, PIAS2, PIAS3, and PIAS4 [PIAS3 protein directly binds to several transcription factors and either blocks or enhances their activity. PIAS3 is also specific inhibitor of signal transducer and activator of transcription 3 (STAT3), a transcription factor and member of the Janus kinase (JAK)/STAT signaling pathway [PIAS3 blocks the transactivation and binding of STAT3 to specific DNA elements via protein–protein interactions, thereby inhibiting STAT3-mediated gene activation. https://www.proteomicsdb.org/proteomicsdb/#human/proteinDetails/86810/interactions) based on experimental and epidemiological evidence. The Figure shows that both PIAS3 and UBE2V2 share the same protein interaction network.nd PIAS4 . PIAS3 p pathway ,29. This pathway ,31. As aPIAS3 is also the only member of the PIAS family that has been shown to directly interact with Stat5a/b and repress Stat5-mediated transcription [PIAS3 against Stat5a/b-driven gene transcription and disease progression in breast cancer, the predominant Stat5a/b protein that binds to DNA has been shown to be N-terminally truncated in human prostate cancer cells and clinical prostate cancers [PIAS3. Hence, the truncated form of Stat5 in prostate cancer cells evades PIAS3-mediated transcriptional inhibition, thereby increasing prostate cancer growth and progression. Thus, the proteolytic cleavage of the N-terminus of Stat5a/b may be a mechanism by which Stat5 evades the transcriptional repression by PIAS3 in prostate cancer cells. This further indicates the complexity of intracellular protein interactions and its role in disease progression.cription . Stat5a/cription , associacription , and a pcription . Transcrcription ,37. Morecription ,38. In v cancers . FurtherOur study applied a novel machine learning model to identify differentially expressed, prostate cancer stage-specific transcripts. Although the application of this model to other related datasets is required to further valid our findings, the use of this model in conjunction with in vitro and in vivo biological studies will aid in elucidating the intricate molecular relationships between the identified transcripts. Moreover, this will provide more insight into predicted prognostic outcomes and the development of effective therapeutic strategies against prostate cancer progression.The primary dataset used in this study was retrieved from the National Center for Biotechnology Information (NCBI) and is referenced with Gene Expression Omnibus (GEO) number GSE54460 . This RNThis dataset was generated by using the Illumina HiSeq 2000 NGS on paired-end sequences of length 51 bp each. The pre-processing pipeline model starts by obtaining the RNA-Seq samples and pre-processing it using SRAtools , as depiNGS technology allows us to read the patient’s genome and generate a significant amount of raw data in a snapshot. However, the underlying process yields artefacts, and pre-processing must be done before the downstream analysis. These artefacts include duplication and bias reads , among oSome classes have a markedly lower number of samples than the others, which may cause some classifiers to become biased towards the majority class. To solve this problem, multiple resampling methods were deployed and tested to identify the specific method that would yield the best solution for a particular dataset. After applying multiple oversampling and under-sampling methods, the best option was found to be the synthetic minority oversampling technique (SMOTE)  for overNCL works by removing any sample whose class is different from the class of at least two of its three nearest neighbours. SMOTE, instead, introduces a new way of creating new samples, by utilising the feature vector that connects each sample and introduces a new synthetic sample along the line that connects the two underlying samples. The exact location of the new sample on the line itself is calculated by measuring the Euclidean distance between the two samples and multiplying that value by a random number between 0 and 1. X concerning class vector A is defined as follows:As the output of the pre-processing step, the method retrieved 41,971 transcripts along with their corresponding quantifications measured by TPM. Such a large number of transcripts leads to a complex classification model, mostly due to the curse of dimensionality . Thus, fA and A given X.Here, S is the set of features, h is the class, in our case, the five Gleason groups.After filtering the transcripts based on their IG scores, a wrapper-based feature selection algorithm that uses minimum redundancy maximum relevance (mRMR) is used to narrow down the most relevant, least redundant transcripts to a few per group; mRMR has the capability of incorporating any classifier to select features (transcripts) that minimise the redundancy while increasing the correlation to the class vector . The wraThe problem dealt with is multi-class classification, which was solved using the one-versus-rest approach. There are five different classes, which correspond to the five distinct Gleason groups. To apply a one-versus-rest approach, we created five different datasets from the actual data. For each dataset, we set one of the classes to form the positive class, while the rest of the classes were combined to form the negative class. The classification pipeline resembles a binary tree structure, where each internal node is a binary classification problem see . StartinWe used the Scitkit-learn  library Identifying novel biomarkers that are clinically associated with specific Gleason groups in prostate cancer is vital for the diagnosis and treatment of the disease. Utilising NGS data and machine learning techniques, a supervised learning method was proposed to find group-specific sets of transcripts with significant different levels of quantification values. The transcripts, along with the corresponding genes, identified by the proposed machine learning method, were found in the literature to play crucial roles in cancer pathogenesis; key transcripts were strongly correlated to prostate cancer. To validate the model, we also tested it on a gene expression dataset, showing that the resulting genes are related to prostate cancer progression.The work presented in this paper opens the way for future directions of research. One of these is to apply and adjust the same method to other cancer types. Another possible avenue would be to consider analysing samples from patients who have progressed through more than one Gleason group. This method aims to eliminate confounding factors between patients, potentially leading to a clearer analysis of differential gene expression between different grades of prostate cancer. In addition, a multi-omics model based on different types of genomics data for this problem could be investigated, which may provide a comprehensive analysis of the progression, diagnosis, and treatment of the disease.