Saccharomyces cerevisiae, including the oxidative stress response. We also identify and characterize the first known Ser57 ubiquitin kinases in yeast and human cells, and we report that two Ser57 ubiquitin kinases regulate the oxidative stress response in yeast. These studies implicate ubiquitin phosphorylation at the Ser57 position as an important modifier of ubiquitin function, particularly in response to proteotoxic stress.Ubiquitination regulates many different cellular processes, including protein quality control, membrane trafficking, and stress responses. The diversity of ubiquitin functions in the cell is partly due to its ability to form chains with distinct linkages that can alter the fate of substrate proteins in unique ways. The complexity of the ubiquitin code is further enhanced by post-translational modifications on ubiquitin itself, the biological functions of which are not well understood. Here, we present genetic and biochemical evidence that serine 57 (Ser57) phosphorylation of ubiquitin functions in stress responses in  Ubiquitin is a post-translational modifier that regulates diverse cellular processes in eukaryotic cells. The broad utility of ubiquitin as a regulatory modification is due to the high degree of complexity associated with ubiquitin polymers, which are added to substrate proteins by the activity of ubiquitin conjugation machinery (E1-E2-E3 cascades) and removed from substrates by deubiquitylases (DUBs). Ubiquitin can be conjugated recursively at any of seven internal lysines or the N-terminus to generate polymers with distinct topological features . ComplexMany proteotoxic stresses activate ubiquitin networks to promote protein quality control and protect the cell from damage associated with systemic protein misfolding. Oxidative stress is highly damaging to the cell, triggering deployment and re-distribution of existing ubiquitin pools and induction of ubiquitin biosynthesis to promote survival by activating a repertoire of ubiquitin-mediated responses . During Although ubiquitin networks play a critical role in the eukaryotic cellular response to proteotoxic stress, precisely how these networks are tuned to enhance protein quality control and other protective functions remain unclear. Given that Ser65 phosphorylation of ubiquitin regulates the clearance of damaged mitochondria , we hypoTo probe potential biological functions of Ser57 ubiquitin phosphorylation in yeast, we generated yeast strains expressing exclusively wildtype, Ser57Ala  or Ser57Asp  ubiquitin. It is important to emphasize that such complete ubiquitin replacement may exaggerate effects associated with physiological ubiquitin phosphorylation, which occurs at very low stoichiometry  and prob2O2), cells expressing S57A ubiquitin were deficient in this response and only arrested growth in response to more severe oxidative stress (>2 mM H2O2) (2O2 (1.0 mM) resulted in an increased abundance of total ubiquitin conjugates  . The faimM H2O2) . Since omM H2O2)  we testenjugates  as well njugates , consistnjugates . Comparenjugates . By contnjugates . Since snjugates .  did not confer canavanine or thialysine resistance  ubiquitin  ubiquitin  of ubiquitin in response to stress. However, the data also indicate that Sks1 and Vhs1 are not required for the production of Ser57 phosphorylated ubiquitin during normal growth or oxidative stress . This mabiquitin . In this2O2). An important limitation of this genetic analysis is the built-in assumption that ubiquitin variants (phosphomimetic or phosphorylation resistant) behave as expected and do not alter other biochemical properties of ubiquitin in a cellular context. Additionally, such phenotypes associated with ubiquitin variants are likely to over-estimate the physiological effects of ubiquitin phosphorylation. In the case of phospho-mimetic (S57D) ubiquitin, this is because physiological ubiquitin phosphorylation is sub-stoichiometric. In the case of phosphorylation resistant (S57A) ubiquitin, this may be due to redundancy of kinases. Such redundancy seems likely since we identified multiple Ser57 ubiquitin kinases  and the deletion of two kinases did not decrease the production of Ser57 phosphorylated ubiquitin  while replacement with phosphorylation resistant ubiquitin phenocopies kinase loss of function  by counterselection on URA-dropout SDM plates containing DL-2-aminoadipic acid as previously described (RPS31 and RPL40B) and its derivatives. Cells were cultured at 26°C in yeast-peptone-dextrose (YPD) or synthetic dextrose minimal medium  at 200 rpm agitation. SILAC media were supplemented with light lysine (12C614N2 L-Lys) and arginine (12C614N4 L-Arg), or heavy isotopes of lysine (13C615N2 L-Lys) and arginine (13C615N4 L-Arg). For spot plate dilution assay, cells were grown for 18–24 hr, normalized to OD600 of 1.0, and sequentially diluted at 1:10 dilution onto SDM agar plates containing amino acid dropout mixture in the absence or presence of 2.5 mM NaH2AsO4, 200 mM hydroxyurea, 2.0 µg/mL canavanine, and 6.0 µg/mL thialysine. For H2O2 sensitivity assay, yeast cells grown to the mid-log phase (OD600 of 0.7) were diluted to OD600 of 0.025 with 1.5 mM H2O2 in SDM and terminal OD600 of cells was measured after 1–3 days of incubation. For growth sensitivity assay of liquid cultures, cells were grown for 18 hr at 26°C, normalized to OD600 of 5.0, and subcultured into fresh media with a starting OD600 of 0.1. OD600 of cells was recorded at different time points until cells reach the stationary phase. For bacterial cultures, the yeast kinase and ubiquitin were heterologously co-expressed in E. coli Rosetta 2 (DE3) by 1 mM IPTG induction at 26°C for 16 hr.All escribed . Such SUYeast proteins were precipitated by adding ice-cold 10% trichloroacetate in TE buffer , washed with 100% acetone, and lyophilized by vacuum centrifugation. Desiccated protein was resolubilized in urea sample buffer.E. coli C41(DE3) cells cultured in LB Media. Cells were induced at an OD600 of 0.6 with 1 mM IPTG and allowed to express for 4 hr at 37°C. Cells were pelleted by centrifugation at 6000 × g for 25 min and flash-frozen with liquid nitrogen. Before purification, the cells were thawed on ice, resuspended in 5 mL of lysis buffer  per 1 g of cells, and sonicated . Cell lysates were cleared by centrifugation  and filtered through a 0.45 μM filter. For purification, lysates were applied to Ni-NTA resin  that had been equilibrated with lysis buffer containing 20 mM imidazole. The protein was eluted with lysis buffer containing 400 mM imidazole. Protein was buffer exchanged to remove imidazole  and the purification tag was cleaved. The protein was loaded on a HiPrep Q FF Anion Exchange Column  and eluted by buffer with 300 mM NaCl. Recombinant ubiquitin variants were purified using the Ni-NTA affinity protocol described above, followed by size exclusion chromatography using a HiLoad Superdex 75 pg column . The protein was eluted in a buffer containing . Ubiquitin-containing fractions were pooled and concentrated by centrifugation to 1 mM.N-terminally tagged Vhs1 or Sks1 were produced in E. coli lysates) or urea sample buffer (for TCA-precipitated yeast proteins) containing 10% β-mercaptoethanol were resolved in 12–14% Bis-Tris PAGE gel by electrophoresis. Separated proteins were transferred onto PVDF membrane  and immunoblotting was performed using the following primary antibodies: anti-ubiquitin , anti-K48 , anti-K63 , and anti-G6PDH . Anti-mouse (IRDye 680RD-Goat anti-mouse) or anti-rabbit (IRDye 800CW-Goat anti-rabbit) secondary antibodies were purchased from LI-COR. Blots were visualized using Odyssey CLx (LI-COR Biosciences) and signal intensity was quantified using Image Studio Lite (LI-COR Biosciences).Proteins in Laemmli buffer (for 3VO4). Lysate was clarified by centrifugation at 21,000 × g for 10 min at 4°C and supernatant was transferred into a new tube and diluted with three-fold volume of ice-cold TBS . 3XFLAG-ubiquitin in 12 mL diluted lysate was enriched by incubation with 50 µL of EZview anti-FLAG M2 resin slurry (Sigma) for 2 hr at 4°C with rotation. The resin was washed three times with cold TBS and incubated with 90 µL elution buffer  at 98°C for 5 min. The collected eluate was reduced with 10 mM DTT, alkylated with 20 mM iodoacetamide, and precipitated with 300 µL PPT solution . Light and heavy protein pellets were dissolved with Urea-Tris solution . Heavy and light samples were combined, diluted four-fold with water, and digested with 1 µg MS-grade trypsin  by overnight incubation at 37°C. Phosphopeptides were enriched by immobilized metal affinity chromatography (IMAC) using Fe(III)-nitrilotriacetic acid resin as previously described (SILAC-based mass spectrometry for quantitation and mapping of ubiquitin phosphorylation sites was performed as previously described . Brieflyescribed  and dissKinase activity assays were performed in a reaction mixture containing 50 mM Tris (pH 7.4), 150 mM NaCl, 10 mM MgCl2, 0.1 mM ATP, 1 mM DTT, 0.5 µM ubiquitin, and 50 nM kinase. Reactions on linkage-specific ubiquitin polymers were carried out at 30°C for 30 min and quenched by adding an equal volume of Laemmli buffer with 10% β-mercaptoethanol and heating at 98°C for 10 min. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This work identifies candidate kinases for Ubiquitin in yeast, as well as their potential role in stress response poses foundations for further work in that undeniably interesting field.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Benoît Kornmann as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Reviewing Editor and David Ron as the Senior Editor.Thank you for submitting your article "Identification of ubiquitin Ser57 kinases regulating the oxidative stress response in yeast" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at E. coli, which homes in two kinases, Vhs1 and Sks1.This manuscript sets out to identify a role for ubiquitin phosphorylation and to identify the kinases necessary for it. The same group has previously shown that serine 57 phosphorylation can be detected in yeast cells. Here they generate strains expressing only phosphomimetic or phosphonull mutants and asses their phenotype in terms of Ubiquitin linkage alone and effect on cell physiology. Among other phenotypes, they find that a strain expressing a non phosphorylable ubiquitin likely fails to mount a response to low doses of H2O2, leading to a slightly increased sensitivity to this chemical. THey also find that treatment with H2O2 slightly increases the amount of phosphorylated Ubiquitin. They then go on to identify the kinases responsible for this phosphorylation using a screen in They delete both kinase and show that this, to a large extent phenocopies the expression of a non-phosphorylable Ubiquitin, and that expression of a phosphomimetic rescued some of the phenotypes of the kinase deleted strain. They also show that overexpressing one of the kinase increases the amount of phosphorylation on ubiquitin.E. coli.Finally, they perform a similar screen using human kinases and human ubiquitin and identify a family of kinases that have the ability to phosphorylate ubiquitin in All three reviewers found the work of interest. Yet, because pSer57-Ubiquitin is so rare, they expressed concerns that the observed phosphorylation of Ubiquitin could be an epiphenomenon of little incidence to cell function.First, the phenotypes of the alanine and aspartate mutants may be due to general effects on Ubiquitin rather than true phospho-Null and -mimetics effects. This concern is minimized by showing that the deletion and overexpression of the kinases phenocopy the ubiquitin mutants. Indeed, analysis of the ubiquitin mutant is only valid in the light of this phenocopy. Yet, because of its importance, this point can and should be pushed further. For instance, while the asp mutant is sensitive to hydroxyurea, the ala mutant behaves as a wildtype. This is at odds with the fact that the KO of each kinase individually increase HU resistance. In this case at least, the effect of deleting the kinase does not appear to involve a decrease in the level of ser57 phosphorylation. How can this be reconciled? Also, while you show that expressing the 57Asp bypasses the need for the kinase in the H2O2 sensitivity assay, is it also the case for the HU and tunicamicin resistance bestowed by the deletion of the kinases? Please find in the specific points a list of experiments required to better pinpoint the phenocopy that is so essential for the relevance of this study. Also, overexpression Vhs1 causes a slight canavanine resistance, reminiscent of canavanine resistance confered by s57d expression. Vhs1 overexpression should therefore not confer canavanine resistance if expressed in a s57a background. This is important to strengthen the phenocopy.Second, while you show that both kinases can phosphorylate Ubiquitin in bacteria and in vitro, and that the overexpression of one of them increases the phosphorylation levels, you do not show how deletion of the kinase affect phosphorylation. This can and should be done, in particular to show if the observed increase in phosphorylation upon oxidative stress is mediated by these kinases.Third, given the low abundance of pS57 ubiquitin, it is hard to conceive that this modification has an important effect on global chain linkage, unless this rare modification is applied to an equally rare set of substrates . This should be better emphasized throughout the manuscript so as not to mislead readers into believing that a substantial fraction of ubiquitin is subjected to phosphorylation.Fourth, in many cases, the experiments are not described to a sufficient amount of detail. For instance, vectors used herein are not described anywhere, nor is the way that all copies of ubiquitin have been replaced with mutant forms. The Supplementary file 2 is absent, so is Supplementary file 1. A much better method section is required to ensure the reproducibility of the experiments. Better descriptions of numbers pertaining to quantitative analysis, statistical test employed an p-value threshold, description of error bars  are also needed in figures and legends.Essential revisions:1) In Figure 1A and Figure 3—figure supplement 1, the authors test the effect of ubiquitin phospho-mutants and absence of kinases, respectively, on the ability of yeast cells to recover from acute heat stress. Firstly, it is puzzling though the experimental conditions are the same (39ᵒC for 18 hours and shifted back to 26ᵒC for recovery) in both cases, the wild-type strain is as good as dead in Figure 1A while it grows fine in Figure 3—figure supplement 1. Importantly, to validate the resistance phenotype of the S57D mutant, the authors should rather over-express the kinases and see that cells grow better in this condition compared to the wild-type and much better compared to the S57A mutant.2) In Figure 1F, the authors employ anti-K48 ubiquitin and anti-K63 ubiquitin antibodies to show the specificity varies between S57A and the S57D mutant. The concern here is whether the serine mutants affect the binding of the antibodies. For instance, the epitope recognized by the anti-K63 ubiquitin antibody could involve the serine 57, however, when mutated to aspartate, the antibody can lose its ability to bind K63-linked ubiquitin. Is there a way to rule this out?3) The authors show that S57D increase K48 but decrease K63-linkages whereas S57A decrease K48 but increase K63-linkages upon H2O2 treatment . What about overexpression or deletion of Vhs1 and Sks1? Does absence of the kinases impact the mutual abundance of ubiquitin K48 and K63 linkages in vivo? Gly-Gly peptides analysis of the data in the experiment from Figure 2G might answer this.4) Deletion of the kinases increase resistance to tunicamycin. However, expression of S57A does not. To strengthen the case of the phenocopy, it is important to check if kinases have ubiquitin-independet effects and how much of the phenocopy is actually wrought by independent mechanisms.5) In general, the growth assays on tunicamycin, hydroxyurea or canavanin in Figure 1—figure supplement 1, Figure 3—figure supplement 2, Figure 3—figure supplement 3 and Figure3—figure supplement 4 should rather be moved to the main figures.6) In Figure 4, human MARK kinases are found to trigger phosphorylation on UbS57 in vitro. It would be insightful to validate this finding in vivo and check whether phosphorylation of UbS57 also regulate the oxidative stress response in mammalian cells. I understand however that this might be take much longer to do than the timeframe which is allocated for revision. In this context, the authors may consider avoiding finishing the paper with these preliminary mammalian data and move them elsewhere in the manuscript. For instance, splitting data from Figure 2 in Figure 2 and Figure 3 and moving Figure 4C in the new Figure 2  would save some space to end the paper with the current Figure 3 and its supplements.Reviewer #1:In this manuscript, Hepowit et al., unravel the yeast and the human kinases that phosphorylate ubiquitin at serine 57 and shed some light on the importance of this modification in conditions of stress. The authors back their claims with well designed and straightforward experiments. Phosphorylation of ubiquitin plays important roles, the extent of which is not yet fully understood. The findings in this paper are intriguing, important and pave the way to further research.Essential revisions:1) In Figure 1A and Figure 3—figure supplement 1, the authors test the effect of ubiquitin phospho-mutants and absence of kinases, respectively, on the ability of yeast cells to recover from acute heat stress. Firstly, it is puzzling though the experimental conditions are the same (39ᵒC for 18 hours and shifted back to 26ᵒC for recovery) in both cases, the wild-type strain is as good as dead in Figure 1A while it grows fine in Figure 3—figure supplement 1. Importantly, to validate the resistance phenotype of the S57D mutant, the authors should rather over-express the kinases and see that cells grow better in this condition compared to the wild-type and much better compared to the S57A mutant.2) In Figure 1F, the authors employ anti-K48 ubiquitin and anti-K63 ubiquitin antibodies to show the specificity varies between S57A and the S57D mutant. The concern here is whether the serine mutants affect the binding of the antibodies. For instance, the epitope recognized by the anti-K63 ubiquitin antibody could involve the serine 57, however, when mutated to aspartate, the antibody can lose its ability to bind K63 ubiquitin. Is there a way to rule this out?Reviewer #3:eLife. Besides aspects that are clearly out of scope for this short report (identification of ligases or substrates), I must admit that my suggestions for improvement of this study are only cosmetic.Modifying the modifier is a principle that took all its meaning few years ago when Ubiquitin was found to be phosphorylated on Serine 65 to regulate mitophagy in mammalian cells through a crosstalk between the ubiquitin ligase Parkin and the mitochondrial kinase PINK1. Since then, MacGurn and colleagues have shown that Ubiquitin also undergoes phosphorylation in yeast on Serine 57 but kinases involved in this phosphorylation remain to be identified. In this short report, the same lab now finds that phosphorylation of UbSer57 is required for heat shock and oxidative stress responses in yeast and identifies two kinases of the Snf1-related family that mediate this phosphorylation. The experimental design of this study is very solid, the manuscript is very well written and the identification of kinases promoting ubiquitin phosphorylation in yeast is of high enough significance to warrant rapid publication in Essential revisions:- The authors show that S57D increase K48 but decrease K63-linkages whereas S57A decrease K48 but increase K63-linkages upon H2O2 treatment . What about overexpression or deletion of Vhs1 and Sks1? Does absence of the kinases impact the mutual abundance of ubiquitin K48 and K63 linkages in vivo? Any Gly-Gly peptides detected in the experiment from Figure 2G?- Deletion of the kinases mimic the growth effects of S57A on tunicamycin and hydroxyurea. It would be nice to check whether UbS57D can bypass the absence of the kinases in these conditions even if this has been evaluated for the responses to oxidative stress in Figure 3.- In general, the growth assays on tunicamycin, hydroxyurea or canavanin in Figure 1—figure supplement 1, Figure 3—figure supplement 2, Figure 3—figure supplement 3 and Figure3—figure supplement 4 should rather be moved to the main figures.- In Figure 4, human MARK kinases are found to trigger phosphorylation on UbS57 in vitro. It would be insightful to validate this finding in vivo and check whether phosphorylation of UbS57 also regulate the oxidative stress response in mammalian cells. I understand however that this might be take much longer to do than the timeframe which is allocated for revision. In this context, the authors may consider avoiding finishing the paper with these preliminary mammalian data and move them elsewhere in the manuscript. For instance, splitting data from Figure 2 in Figure 2 and Figure 3 and moving Figure 4C in the new Figure 2  would save some space to end the paper with the current Figure 3 and its supplements.Reviewer #4:This manuscript by Hepowitt et al., focusses on the mechanism and phenotypic consequences of ubiquitin post-translational modification (PTM). Specifically, one of the first identified modifications – phosphorylation of Ser57. This loosely follows on from previous work conducted in the MacGurn lab in which they identified ubiquitin Ser57 phosphorylation (Ser57P) as a regulator of endocytic trafficking and ubiquitin turnover .The current manuscript begins by presenting a number of varied phenotypes associated with yeast solely expressing Ser57Asp (a phosphomimetic) and Ser57Ala (non phosphorylatable) ubiquitin mutants (presumably from an endogenous ubiquitin loci). Yeast expressing Ser57Asp have a conspicuous heat tolerance phenotype  and an increased sensitivity to hydroxyurea relative to wildtype yeast . Curiously, the opposite trend is not observed with Ser57Ala ubiquitin. Conversely, Ser57Asp and Ser57Ala ubiquitin mutants do possess contrasting phenotypes, with respect to wildtype yeast, in terms of tunicamycin sensitivity  .The authors then move towards the principal thesis of their manuscript; that modification of Ser57 is important for the proper regulation of the yeast oxidative stress response. In support of this tenet Hepowitt et al. present the somewhat contradictory observations that Ser57Ala ubiquitin permits more growth in the presence of H2O2  but reduced viability in response to a shorter H2O2 stress . Ser57Asp mutation does not perturb the yeast in these assays, relative to wildtype strains. The authors suggest that these differences in response to oxidative stress may play out at the level of different polyubiquitin linkage types favoured by the different amino acids at residue 57 (as a proxy for different phosphorylation statuses). This is supported by the observation that Ser57Asp yeast have significantly elevated levels of poly-K48-linked ubiquitin and decreased levels of poly-K63-linked in response to H2O2 exposure and also seemingly under basal/0 mM H2O2 conditions, relative to wildtype . The reciprocal phenotypes were also observed for Ser57Ala yeast. This observation, in turn, stimulates the authors to speculate that oxidative stress may induce the phosphorylation of Ser57 which subsequently favours poly-K48-linked ubiquitination and thus proteasomal degradation of the increased burden of oxidised/damaged protein. Accordingly, reduced carbonylated proteins are observed in Ser57Asp strains . However, given the contrast between Ser57Asp and wildtype cells in terms of ubiquitin linkage both {plus minus} H2O2  the above hypothesis is conceptually difficult to marry with the observation of seeming parity between wildtype and Ser57Asp expressing yeast in terms of oxidative stress growth response and post-stress viability  and with the observation that wildtype cells both increase K48 and K63-linked polyubiquitin in response to oxidative stress .E. coli co-expression system and when SksI is overexpressed in yeast . Crucially, however, the authors do not go on to show that endogenous VhsI and/or SksI directly influence the modification of endogenous ubiquitin at Ser57 in yeast and, by extension, they also do not show that these kinases regulate Ser57P in an oxidative stress dependent manner. That is to say, in order to substantiate their claim that these kinases regulate yeast oxidative stress response by phosphorylating Ser57 a similar experiment to Figure 1H, comparing the relative abundance of Ser57P peptides in wildtype and Δsks1Δvhs1 cells {plus minus} H2O2, should have been undertaken.An important experiment, and the first time the authors address the phosphorylation of Ser57 directly in this manuscript, is presented in Figure 1F. Based on this SILAC experiment it appears that oxidative stress (H2O2) can induce up to around a 1.7-fold increase in the amount of Ser57P ubiquitin. The authors then go on to demonstrate quite nicely that two SnfI-related Ser/Thr kinases VhsI and SksI can phosphorylate ubiquitin Ser57 in vitro, in an Instead, the authors compare kinase knockout phenotypes to the previous Ser51Asp and Ser51Ala phenotypes. Unfortunately, even these are performed in a rather haphazard fashion. For example, although Ser57Asp was shown to produce a robust heat tolerance phenotype and also a tunicamycin sensitivity , in Figure S3C wildtype cells were only compared in this regard to Δsks1Δvhs1 and not also to SksI/VhsI over-expression  to be responsible for Ser57P dephosphorylation and surprisingly not mentioned or utilised in this paper). With respect to assaying the role of these kinases (and by extension the function of Ser57P) in an oxidative stress response the authors only examine the growth response to H2O2 . The presence of both kinases appears necessary for growth arrest, aligning with the lack of growth arrest previously observed in the Ser57Ala context. In order to more definitively demonstrate that this phenotype is due to an inability to phosphorylate ubiquitin's Ser57 the analysis in Figure 3 should have been extended. That is to say, the phenotypic rescue of Δsks1Δvhs1 by overexpression of SksI+VhsI should have been tested in different ubiquitin backgrounds. If the rescue is dependent on SksI+VhsI mediated phosphorylation of Ser57 then the growth Δsks1Δvhs1 phenotype should not be rescuable in the background of Ser57Ala mutant ubiquitin.In summary, the authors do not adequately demonstrate that endogenous SksI and VhsI kinases are responsible for phosphorylation of endogenous ubiquitin at Ser57 either in the absence or presence of an oxidative stressor. As such, the claim that these kinases and by extension ubiquitin phosphorylation at Ser57 is important in the yeast oxidative stress response is poorly supported. The authors imply, largely based on replacement of the entire ubiquitin pool with either Ser57Ala or Ser57Asp mutants, that in response to oxidative stress SksI/VhsI phosphorylates Ser57 . This, in turn, results in an increased propensity for poly-K48-linkages and thus increased proteasomal clearance of oxidised or damaged proteins and by some mechanism an oxidative stress induced growth retardation . However, what is conspicuous by its absence is a lack of discussion regarding the stoichiometry of this modification. It was previously estimated by this group, using mass spectrometry, that in yeast lacking the Ser57 phosphatases (Ppz1 and Ppz2), which possessed a ca. 3-fold elevation in Ser57P modification, that the amount of ubiquitin modified at Ser57 was still less than 0.05% of the entire ubiquitin population . In this manuscript the authors find that oxidative stress causes less than a 2-fold upregulation in Ser57P levels, this would therefore represent less than 0.03% of the ubiquitin population being phosphorylated at Ser57 even after H2O2 application. The inferences that can therefore be drawn from replacing all of the endogenous ubiquitin with Ser57Ala and Ser57Asp  are extremely limited. The mechanism by which such a minor fraction of the ubiquitin pool could affect the oxidative stress response is a conundrum which is not at all addressed by Hepowit et al.eLife. Your article has been reviewed by three peer reviewers, including Benoît Kornmann as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Reviewing Editor and David Ron as the Senior Editor.Thank you for submitting your article "Identification of ubiquitin Ser57 kinases regulating the oxidative stress response in yeast" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.You will be glad to learn that, despite some disagreement among reviewers, the consensus was that your story might deserve publication.While all three reviewers acknowledge the potential importance of your work, they also acknowledge that you have not formally proven that VHS1 and SKS1 are kinases that phosphorylate ubiquitin in vivo and that this is important for stress response. What you have shown is that these two kinases are sufficient for phosphorylation in bacteria, but that they are not necessary for phosphorylation in vivo.Your interpretation that Vhs1 and Sks1 are necessary for the phosphorylation of a defined subset of ubiquitin molecules (as is the case for PINK1) is plausible, but far from being the only interpretation.Indeed, many of your experiments that assess fitness of strains with ubiquitin variants and kinase KO or overexpression hinge on the prerequisite that phosphomimic or phosphonull mutants behave as expected and do not affect any other aspect of ubiquitin biology, a point that you cannot ascertain.Moreover the phosphomimic and phosphonull mutants are bound to have effects beyond what can be attributed to Vhs1 and Sks1, the mimics because they affect 100% of ubiquitins, while phospho-ubiquitins at maximum represent a small fraction, and the null mutants because they will affect non-Vhs1/Sks1-mediated phosphorylation, which is the majority of phosphorylation. Moreover, simply missing a hydroxyl group on the surface of ubiquitin might alter its properties, irrespective of phosphorylation.So an equally plausible explanation is that ubiquitin biology is altered by your mutations in way which happens to phenocopy the kinases KO and overexpression, respectively, at least at the level of general fitness and stress survival.The disagreement between the reviewers hinged upon allowing you the benefit of the doubt, or considering that the doubt was too great to constitute a publishable story. The consensus reached was that your paper deserves publication, provided that you make the following amendments to it:1) The new proteomics results, as disappointing as you might find them, are an important part of the story and should constitute part of a main figure and not be buried in a Supplementary file. They should be the basis of an important discussion point (see below).2) Instead of a result and Discussion section, the paper should have a stand-alone Discussion section, where these concerns should be thoroughly put forward.3) In this discussion you should explicitly state that you are asking readers to accept the proposition that the phenotype in question arises from a small Sks1/Vhs1-dependent pool of Ub_pS57, which is undetectable on the background levels of Ub_pS57, which are themselves a very small fraction of Ub.All reviewers sincerely hope that your views will be vindicated in due course, and that you will enjoy the satisfaction of knowing that you persevered for good reason in face of considerable skepticism, but in the meantime, it is important for all interested parties, to lay bare the limitations of your current study.The individual reviews are appended for your perusal.Reviewer #1:The revised manuscript submitted here by Hepowit et al. addresses many of the concerns raised by the reviewers. In the first round of review and the manuscript presented here is therefore much improved. Still, the new data are not necessarily aligned with the authors’ original model. Some clarifications are therefore required.1) Importantly, the model is that two kinases phosphorylate ubiquitin under stress. When directly tested in strains deficient for these two kinases, it appears that the kinases are not necessary for stress-induced phosphorylation of ubiquitin. This is an important result that deserves better than being mentioned and kept for a Supplementary file 3. Moreover, it is unclear what a SILAC ratio of 1 actually means since in all other figures, SILAC ratios are presented as log2. Here it appears to be on a linear scale (with 1 meaning 'no change'). This should be clarified and the data from Supplementary file 1 presented in a more visible fashion in the main figure. The fact that neither Sks1 nor Vph1 are required for stress induced phosphorylation of ubiquitin is too important to the comprehension of the implications of this paper.2) It was shown in the first version of the paper that individual mutation of the kinases led to HU resistance . This was at odd with the finding that the expression of a non-phosphorylatable ubiquitin did not cause the same phenotype, indicating that both kinases exert their function on HU resistance through ubiquitin-independent mechanisms. These data have now disappeared from the manuscript. Although the interpretation of these results is not straightforward, it is something that readers will want to see, nonetheless.Reviewer #2:Taken together, the authors have made a good job in revising the manuscript and in taking reviewers comments into consideration. It is clearly disappointing that the combined absence of Vhs1 and Sks1 fails to induce detectable decrease in phosphorylation of UbS57. Yet, this can be explained by a possible specificity of Vhs1 and Sks1 toward limited ubiquitinated substrates and by the fact that other kinases, distinct form Vhs1 and Sks1, have the capacity to phosphorylate UbS57. While the authors emphasize these possibilities, they also provide several data that support this interpretation. As shown in the initial version of the manuscript, Gin4 and Kcc4 are two other kinases that can trigger UbS57 phosphorylation. Most importantly, new data from the revised version now support a possible specificity of Vhs1-mediated phosphorylation of UbS57 in protein misfolding stress  and of Sks1-mediated phosphorylation of UbS57 in DNA damage stress . Notably, Figures 3E and 3F also demonstrate that the overexpression of Sks1 and of Vhs1 both stimulate the phosphorylation of endogenous Ubiquitin at S57.In the eyes of this reviewer, the revised manuscript is really improved and the new data appropriately support the claims of the authors.Reviewer #3:The claim for discovery here is that phosphorylation of Ub on serine 57 affects stress responsiveness of yeast.The key evidence in support of this conclusion is the concordance in phenotype between a replacement allele that renders Ser57 unphosphorylateable (S57A) and inactivating mutations of two kinases demonstrated in a heterologous system to be capable of phosphorylating Ub_S57 How credible is this interpretation?The stoichiometry of S57 phosphorylation is exceedingly low, which is to say that in stressed cells most of the Ub remains unphosphorylated. To account for the phenotypic consequences of kinase inactivation and/or Ub_S57A one would have to conjure a scenario whereby phosphorylation affects a small pool of Ub that is critical to the stress adaptation. Given the potential precedent set by PINK1, this was a speculation we as reviewers were willing to sanction when we invited a revised version of the paper without insisting that evidence for the existence of such a pool be provided.In the revised version of the manuscript we now learn that the kinases in question, Sks1 and Vhs1 are not essential for the stress-induced Ub phosphorylation – other kinases can compensate for this biochemical event, but not, apparently, for its phenotypic consequences. Thus, to uphold the paper's key conclusion we must now be willing to take an additional leap of faith, namely that the already small pool of Ub_pS57 is itself comprised of two sub-pools, one that is Sks1 and Vhs1-dependent – and important to the phenotype arising from the Ub_S57A mutation (and/or the inactivation of the kinases) – and a second pool that is irrelevant to this stress response.As reviewers we need to ask ourselves if we are willing to endorse the publication of a paper that makes the aforementioned claim, supports it with the data provided here and then qualifies the conclusion with these key caveats relating to the unproven existence of a critical small pool of Sks1 and Vhs1-dependent Ub_pS57. All three reviewers found the work of interest. Yet, because pSer57-Ubiquitin is so rare, they expressed concerns that the observed phosphorylation of Ubiquitin could be an epiphenomenon of little incidence to cell function.We are extremely grateful for the reviewers’ interest in the manuscript, and we agree with the concern that ubiquitin phosphorylation could be an epiphenomenon. However, we think these concerns are mitigated by the following:- Several new lines of genetic evidence have been added to the revised manuscript which expand upon the genetic relationship between the Ser57 ubiquitin kinases that we identified and the phosphorylation of ubiquitin at the Ser57 position. The details of these new experiments are outlined in the following point-by-point response to reviewer comments.- We think the data strongly indicate that Ser57 phosphorylation of ubiquitin plays a physiological role in cell stress responses. Biochemical data indicates that Ser57 phosphorylated ubiquitin is produced in response to conditions of oxidative stress, and genetic data indicates Ser57 phosphorylated ubiquitin is a requirement for the oxidative stress response. Our data also implicates a functional role for Ser57 phosphorylation in other proteotoxic stress responses.- Although we cannot exclude the possibility that ubiquitin phosphorylation is an epiphenomenon, the stress tolerance phenotypes are real and therefore may have synthetic value. For example, mechanisms that promote cellular tolerance to proteotoxic stresses may ultimately have therapeutic value for diseases associated with protein misfolding. Additionally, yeast variants that exhibit thermal tolerance may have industrial applications. Therefore, the results reported in this study have potential impact beyond the understanding of cellular responses.Overall, we believe the new experiments and revisions incorporated have resulted in a stronger manuscript that will be of significant interest to many labs in different fields – and we hope the editors and reviewers agree.First, the phenotypes of the alanine and aspartate mutants may be due to general effects on Ubiquitin rather than true phospho-Null and -mimetics effects. This concern is minimized by showing that the deletion and overexpression of the kinases phenocopy the ubiquitin mutants. Indeed, analysis of the ubiquitin mutant is only valid in the light of this phenocopy. Yet, because of its importance, this point can and should be pushed further. For instance, while the asp mutant is sensitive to hydroxyurea, the ala mutant behaves as a wildtype. This is at odds with the fact that the KO of each kinase individually increase HU resistance. In this case at least, the effect of deleting the kinase does not appear to involve a decrease in the level of ser57 phosphorylation. How can this be reconciled? Also, while you show that expressing the 57Asp bypasses the need for the kinase in the H2O2 sensitivity assay, is it also the case for the HU and tunicamicin resistance bestowed by the deletion of the kinases? Please find in the specific points a list of experiments required to better pinpoint the phenocopy that is so essential for the relevance of this study. Also, overexpression Vhs1 causes a slight canavanine resistance, reminiscent of canavanine resistance confered by s57d expression. Vhs1 overexpression should therefore not confer canavanine resistance if expressed in a s57a background. This is important to strengthen the phenocopy.These are excellent points raised by reviewers. In the revised manuscript, we have expanded our genetic analysis, which has revealed additional genetic interactions between the Ser57 ubiquitin kinases and the Ser57 position of ubiquitin. Specifically, we have included the following new experiments in Figure 4:- Overexpression of VHS1 phenocopies the canavanine resistance phenotype observed with expression of S57D ubiquitin. This phenotype requires catalytic activity of Vhs1, and it is suppressed by expression of S57A ubiquitin.- Overexpression of VHS1 phenocopies the thialysine resistance phenotype observed with expression of S57D ubiquitin. This phenotype requires catalytic activity of Vhs1, and it is suppressed by expression of S57A ubiquitin.- Overexpression of SKS1 phenocopies the hydroxyurea sensitivity phenotype observed with expression of S57D ubiquitin. This phenotype is suppressed by expression of S57A ubiquitin.All of these findings indicate that overexpression of SKS1 and VHS1 drive production of Ser57 phosphorylated ubiquitin , which is required for the observed phenotypes. Combined with our original genetic analysis of these kinases in the context of oxidative stress, we think these results strengthen the main conclusions of the paper.Δsks1Δvhs1 double mutant cells is subtle and will require additional genetic analysis in follow-up studies. However, we do not believe this exclusion changes the main conclusions or significance of the manuscript.) and following a 30 minute exposure to H2O2 (Experiment #2) and in neither case did we observe significant change in the amount of Ser57 phospho-ubiquitin. This result indicates that absence of Sks1 and Vhs1 does not affect global levels of Ser57 phosphorylated ubiquitin following acute exposure to H2O2. However, it does not exclude the possibility that these kinases may participate in the response to prolonged oxidative stress, and it does not account for localized production of phosphorylated ubiquitin. Redundancy with other kinases  may also be a factor – although redundancy is not consistent with the genetic requirement of SKS1 and VHS1 for growth arrest during oxidative stress. We have tried to objectively interpret the data (Results section) and we hope reviewers and editors will appreciate our effort despite the outcome.This experiment was the major bottleneck to our resubmission, since it required us to generate new strains and due to significant delays at our proteomics core facility. We performed the requested experiment, and the data are now presented in Figure 4, Supplementary file 3. In this SILAC experiment, we compared wildtype and Third, given the low abundance of pS57 ubiquitin, it is hard to conceive that this modification has an important effect on global chain linkage, unless this rare modification is applied to an equally rare set of substrates . This should be better emphasized throughout the manuscript so as not to mislead readers into believing that a substantial fraction of ubiquitin is subjected to phosphorylation.We agree that in physiological circumstances the production of phospho-ubiquitin is highly localized – as is the case for Pink1-mediated production of Ser65 phosphorylated ubiquitin. Indeed, this interpretation is consistent with many of our observations presented throughout the manuscript, and this is also the reason why genetics has been a powerful tool for accessing the biology of Ser57 phosphorylated ubiquitin. To emphasize this point, we have made the following changes and additions to the text:- Results section: By way of introducing the yeast ubiquitin replacement strains used in this study (S57A and S57D) we emphasize that this strategy inherently over-estimates the stoichiometry of ubiquitin phosphorylation, and thus the phenotypes likely exaggerate effects that are likely localized and transient in a physiological context.- Results section: In discussing the impact S57A and S57D have on linkage types used in conjugation, we re-emphasize the point that complete ubiquitin replacement in the strains used exaggerates the effects expected at physiological phosphorylation levels. We also link this to discussion of our result that Ser57 phosphorylation is induced by oxidative stress, but the stoichiometry is still sufficiently low that the impact is likely highly localized.- Results section: In this concluding paragraph, we emphasize the low stoichiometry of ubiquitin phosphorylation in a physiological context. We also compare Ser57 kinases to Pink1, speculating that activity of these kinases on localized pools of ubiquitin likely drives the biology that is underscored by the genetics.We think these changes capture the spirit of the reviewer/editorial critique. In doing so, we have more clearly articulated the limitations associated with the data we present, and we believe this strengthens the manuscript as a whole.Fourth, in many cases, the experiments are not described to a sufficient amount of detail. For instance, vectors used herein are not described anywhere, nor is the way that all copies of ubiquitin have been replaced with mutant forms. The Supplementary file 2 is absent, so is Supplementary file 1. A much better method section is required to ensure the reproducibility of the experiments. Better descriptions of numbers pertaining to quantitative analysis, statistical test employed an p-value threshold, description of error bars  are also needed in figures and legends.We apologize for these oversights, which have been addressed in the revised manuscript. Here is a list of specific changes and additions to address this concern:- All strains and plasmids used in this study are now described in a Key Resources Table.- We have also provided in the Materials and methods section a better description of our ubiquitin replacement yeast strains and how those were generated.- In Figure Legends, we now provide a thorough description of statistical tests, error bars, significance thresholds, and numbers of biological replicates for all relevant experiments depicted in main and supplemental figures.- We have included source data files corresponding to each figure, which includes all numerical data and statistical analysis that was used to generate all graphs presented in this study.We have expanded the experimental details provided in our Materials and methods section to ensure reproducibility of the experiments.Essential revisions:1) In Figure 1A and Figure 3—figure supplement 1, the authors test the effect of ubiquitin phospho-mutants and absence of kinases, respectively, on the ability of yeast cells to recover from acute heat stress. Firstly, it is puzzling though the experimental conditions are the same (39ᵒC for 18 hours and shifted back to 26ᵒC for recovery) in both cases, the wild-type strain is as good as dead in Figure 1A while it grows fine in Figure 3—figure supplement 1.This disparity is due to differences in strain background between these two experiments. For cells expressing only WT, S57A, or S57D ubiquitin, we have used the SUB280 background, which was designed to express ubiquitin from a single source and is therefore used in experiments for phenotypic analysis of strains expressing S57A and S57D ubiquitin.  was performed in the SEY6210 strain background, which has greater thermal tolerance than SUB280. In the revised manuscript, we have added text in the Materials and methods to clarify why SUB280 and SEY6210 background were used in different experiments.Importantly, to validate the resistance phenotype of the S57D mutant, the authors should rather over-express the kinases and see that cells grow better in this condition compared to the wild-type and much better compared to the S57A mutant.These are good suggestions raised by reviewers. Our analysis did not uncover any thermal tolerance phenotypes associated with the deletion or overexpression of Ser57 ubiquitin kinases identified in this study . However, this does not exclude a role for Ser57 phosphorylation in the heat stress response, and we hypothesize there may be additional (and as yet unidentified) Ser57 ubiquitin kinases that function specifically in the heat stress response.We have also included new data revealing that yeast cells expressing S57A ubiquitin exhibit thermal sensitivity . This provides additional evidence of a role for Ser57 phosphorylated ubiquitin in promoting thermal tolerance, which we think strengthens the revised manuscript.2) In Figure 1F, the authors employ anti-K48 ubiquitin and anti-K63 ubiquitin antibodies to show the specificity varies between S57A and the S57D mutant. The concern here is whether the serine mutants affect the binding of the antibodies. For instance, the epitope recognized by the anti-K63 ubiquitin antibody could involve the serine 57, however, when mutated to aspartate, the antibody can lose its ability to bind K63-linked ubiquitin. Is there a way to rule this out?This is an excellent point raised by reviewers. Signal from blotting with the anti-K63 antibody is typically very weak, but we actually detect a baseline signal for K63 linkage detection that declines with oxidative stress . We have also now quantified this over triplicate experiments . These data suggest that S57D ubiquitin is recognized by the anti-K63 antibody but the signal decreases with oxidative stress.To validate the findings of Figure 2C-E using methods that do not rely on linkage-specific antibodies, we turned to SILAC-MS combined with enrichment of ubiquitin-remnant (di-Gly) peptides comparing cells expressing wildtype (heavy-labelled) and S57D (light-labelled) ubiquitin. In this analysis, we resolved linkage-specific peptides corresponding to K6-, K33- and K48-linked polymers . Consistent with our blotting analysis, SILAC quantification revealed a 39% increase in K48-linked polymers associated with S57D ubiquitin. Importantly, K63-linked polymers are a blind spot of this analysis, since the Ser57Asp mutation occurs on the same peptide that harbors K63.  Nevertheless, these results are consistent with immunoblotting results presented in Figure 2, and they provide additional quantitative analysis of other linkage-types not amenable to immunoblot analysis . We believe the addition of this new experimental data addresses the reviewers concerns and strengthens the manuscript.3) The authors show that S57D increase K48 but decrease K63-linkages whereas S57A decrease K48 but increase K63-linkages upon H2O2 treatment . What about overexpression or deletion of Vhs1 and Sks1? Does absence of the kinases impact the mutual abundance of ubiquitin K48 and K63 linkages in vivo? Gly-Gly peptides analysis of the data in the experiment from Figure 2G might answer this.This is an excellent idea – and we went back over the data for Figures 3E-F . Although some linkage-specific diGly peptides were resolved in this analysis, there are no consistent trends for any peptides other than the Ser57 phosphopeptide in these experiments. This finding suggests that production of Ser57 phospho-ubiquitin, at least at the levels attained in these experiments, is not sufficient to substantially affect global ubiquitin linkage patterns. The source data for these experiments is now provided in Figure 3—source data 1.4) Deletion of the kinases increase resistance to tunicamycin. However, expression of S57A does not. To strengthen the case of the phenocopy, it is important to check if kinases have ubiquitin-independet effects and how much of the phenocopy is actually wrought by independent mechanisms.Actually, the initial submission included data revealing that kinase deletion and S57A expression both resulted in slight resistance to tunicamycin, but because this phenotype was subtle in both cases we decided to remove the tunicamycin results from this study in favor of stronger phenotypes. Instead, we have included new data for canavanine, thialysine, and hydroxyurea phenotypes that link the kinases to ubiquitin-dependent effects. We think this strengthens the revised manuscript.5) In general, the growth assays on tunicamycin, hydroxyurea or canavanin in Figure 1—figure supplement 1, Figure 3—figure supplement 2, Figure 3—figure supplement 3 and Figure3—figure supplement 4 should rather be moved to the main figures.This is an excellent suggestion – and we have moved more phenotypic data to the main figures  in the revised manuscript.6) In Figure 4, human MARK kinases are found to trigger phosphorylation on UbS57 in vitro. It would be insightful to validate this finding in vivo and check whether phosphorylation of UbS57 also regulate the oxidative stress response in mammalian cells. I understand however that this might be take much longer to do than the timeframe which is allocated for revision. In this context, the authors may consider avoiding finishing the paper with these preliminary mammalian data and move them elsewhere in the manuscript. For instance, splitting data from Figure 2 in Figure 2 and Figure 3 and moving Figure 4C in the new Figure 2  would save some space to end the paper with the current Figure 3 and its supplements.This is an excellent suggestion, and we have re-structured the revised manuscript accordingly.Reviewer #4:This manuscript by Hepowitt et al., focusses on the mechanism and phenotypic consequences of ubiquitin post-translational modification (PTM). Specifically, one of the first identified modifications – phosphorylation of Ser57. This loosely follows on from previous work conducted in the MacGurn lab in which they identified ubiquitin Ser57 phosphorylation (Ser57P) as a regulator of endocytic trafficking and ubiquitin turnover .The current manuscript begins by presenting a number of varied phenotypes associated with yeast solely expressing Ser57Asp (a phosphomimetic) and Ser57Ala (non phosphorylatable) ubiquitin mutants (presumably from an endogenous ubiquitin loci). Yeast expressing Ser57Asp have a conspicuous heat tolerance phenotype  and an increased sensitivity to hydroxyurea relative to wildtype yeast . Curiously, the opposite trend is not observed with Ser57Ala ubiquitin. Conversely, Ser57Asp and Ser57Ala ubiquitin mutants do possess contrasting phenotypes, with respect to wildtype yeast, in terms of tunicamycin sensitivity  .Δsks1Δvhs1 double mutant cells is subtle and will require additional genetic analysis in follow-up studies. However, we do not believe this exclusion changes the main conclusions or significance of the manuscript.These are excellent observations. We note that we have included additional data in the revised manuscript that reveal a heat sensitivity phenotype of yeast expression S57A ubiquitin . We have also decided to remove our analysis of growth in the presence of tunicamycin. The tunicamycin resistance phenotype of The authors then move towards the principal thesis of their manuscript; that modification of Ser57 is important for the proper regulation of the yeast oxidative stress response. […] However, given the contrast between Ser57Asp and wildtype cells in terms of ubiquitin linkage both {plus minus} H2O2  the above hypothesis is conceptually difficult to marry with the observation of seeming parity between wildtype and Ser57Asp expressing yeast in terms of oxidative stress growth response and post-stress viability  and with the observation that wildtype cells both increase K48 and K63-linked polyubiquitin in response to oxidative stress .In consideration of these excellent points raised by reviewer #4, we have more rigorously addressed and quantified the linkage-type affects associated with expression of S57A and S57D ubiquitin in yeast . While the phenotypic experiments  do not reveal any difference between yeast cells expressing wildtype and those expression S57D ubiquitin variants, it is notable that no loss of viability is detected in wildtype cells  so we cannot conclude phenotypic parity between wildtype and S57D expressing yeast. Furthermore, while there does appear to be an increase in K63-linked polyubiquitin in response to oxidative stress, quantification of our results does not reveal this to be statistically significant. This may reflect a limitation of the sensitivity (or specificity) of the anti-K63 antibody and so we refrain from making strong conclusions on this point in the manuscript. In contrast, we believe the most significant observations in these experiments pertain to abundance of K48-linked polyubiquitin, which is appears much elevated in the presence of S57D and at the expense of K63-linked polymers. Additionally, we include new SILAC-MS analysis of polyubiquitin linkage abundance in these strains, and we have revised the text to include a more nuanced discussion of these observations – which includes consideration of the limitations associated with using ubiquitin replacement yeast strains, where ubiquitin is exclusively S57A or S57D. We hope reviewer #4 will agree that these revisions have helped to strengthen the manuscript, and that despite these limitations the results are still significant and worthy of publication.Instead, the authors compare kinase knockout phenotypes to the previous Ser51Asp and Ser51Ala phenotypes. Unfortunately, even these are performed in a rather haphazard fashion. For example, although Ser57Asp was shown to produce a robust heat tolerance phenotype and also a tunicamycin sensitivity , in Figure S3C wildtype cells were only compared in this regard to Δsks1Δvhs1 and not also to SksI/VhsI over-expression  to be responsible for Ser57P dephosphorylation and surprisingly not mentioned or utilised in this paper).These are excellent points raised by reviewer #4. While our data do implicate Ser57 phosphorylation of ubiquitin in thermal tolerance, our analysis did not uncover a kinase responsible for this particular phenotype. We suspect that there may be additional, as yet unidentified ubiquitin kinases which may confer thermal tolerance.Δsks1Δvhs1 double mutant cells is subtle and will require additional genetic analysis in follow-up studies. However, we do not believe this exclusion changes the main conclusions or significance of the manuscript.Please note that we have also decided to remove our analysis of growth in the presence of tunicamycin. The tunicamycin resistance phenotype of Examining a role for Ppz phosphatases as antagonists of these kinases is also an excellent idea. While we have some genetic data to support this relationship, we think this will require a deeper genetic and biochemical analysis that is outside the scope of the current manuscript.1) The new proteomics results, as disappointing as you might find them, are an important part of the story and should constitute part of a main figure and not be buried in a Supplementary file. They should be the basis of an important discussion point (see below).eLife editorial staff that tables cannot be incorporated into figures. As such, we decided to move the data from Supplementary file 3 into Table 1, which is now part of the main article. To link this to Figure 4, we now reference Table 1 in the Figure 4 legend (and vice versa). Additionally, to highlight this further, we have added the chromatography data that is the basis of quantification, which is now displayed in Figure 4H. We agree with reviewers that these revisions help to emphasize this important result.We were advised by At the request of reviewer 1, we have also log transformed the data in Table 1, to make it more consistent with our treatment of other similar data throughout the manuscript. This is described in the revised legend for Table 1.2) Instead of a result and Discussion section, the paper should have a stand-alone Discussion section, where these concerns should be thoroughly put forward.We agree, and we have implemented this suggestion in the revised manuscript.3) In this discussion you should explicitly state that you are asking readers to accept the proposition that the phenotype in question arises from a small Sks1/Vhs1-dependent pool of Ub_pS57, which is undetectable on the background levels of Ub_pS57, which are themselves a very small fraction of Ub.We have now expanded our discussion and included a more thorough explanation of our proposed interpretation, as well as the limitations associated with it. We have also included additional discussion of the limitations associated with genetic analysis of ubiquitin replacement yeast strains. These revisions are all found in the new Discussion section of the manuscript.


The mechanism of how ice crystal form has been extensively studied by many researchers but remains an open question. Molecular dynamics (MD) simulations are a useful tool for investigating the molecular-scale mechanism of crystal formation. However, the timescale of phenomena that can be analyzed by MD simulations is typically restricted to microseconds or less, which is far too short to explore ice crystal formation that occurs in real systems. In this study, a metadynamics (MTD) method was adopted to overcome this timescale limitation of MD simulations. An MD simulation combined with the MTD method, in which two discrete oxygen–oxygen radial distribution functions represented by Gaussian window functions were used as collective variables, successfully reproduced the formation of several different ice crystals when the Gaussian window functions were set at appropriate oxygen–oxygen distances: cubic ice, stacking disordered ice consisting of cubic ice and hexagonal ice, high-pressure ice VII, layered ice with an ice VII structure, and layered ice with an unknown structure. The free-energy landscape generated by the MTD method suggests that the formation of each ice crystal occurred via high-density water with a similar structure to the formed ice crystal. The present method can be used not only to study the mechanism of crystal formation but also to search for new crystals in real systems. Ice polymorphs play essential roles in the Earth’s environment, the life cycles of organisms living in cold environments, our daily life, and industry. Therefore, numerous studies have investigated ice polymorphs to determine the phase diagram, identify suitable formation conditions, and so forth4.Ice crystals have a variety of polymorphs depending on the temperature and pressureh), the formation mechanism is still unclear. One of the reasons for this is that appropriate methods for determining the formation mechanisms of ice polymorphs are lacking. To resolve this issue, it is necessary to analyze the molecular-scale processes of the structural changes that occur during the formation of ice polymorphs. Computer simulations, such as molecular dynamics (MD), are often useful for this purpose5. However, the formation of each ice polymorph occurs via nucleation, which is an activation process that occurs on a much longer timescale than the typical microsecond-order run time of an MD simulation. Thus, the timescale of MD simulations is far too short to study the molecular-scale processes of the structural changes that occur in the formation of ice polymorphs.However, the formation mechanisms of most ice polymorphs remain elusive. Even for ordinary hexagonal ice I  for a system of interest. This free-energy landscape quantitatively represents the relative thermodynamic stabilities of many different states of the system. Therefore, it is possible to search for a kinetically favorable pathway for the transition between different states, on the assumption that this transition occurs according to a pathway on the free-energy landscape. Thus, application of the MTD method to supercooled water is anticipated to yield valuable information for elucidating the formation mechanisms of ice polymorphs.Metadynamics (MTD) is an enhanced sampling method that increases the probability of reaching high free-energy states by adding a Gaussian bias potential to the Hamiltonian of a stateU, as CVs26. Although the formation of ice I occurred, the formation of other ice polymorphs did not. In principle, the number of states that are reproduced in an MTD simulation increases with an increasing number of CVs. However, the number of CVs should be set as small as possible to conserve computational time and facilitate the determination of pathways for the formation of each polymorph7. Moreover, it is preferable to use experimentally measurable CVs to enable experimental verification of the formation mechanism. Recently, Niu et al. applied the MTD method to examine the formation mechanism of a silica crystal using X-ray diffraction peak intensities, which can be measured experimentally, as CVs31.Quigley and Rodger adopted the MTD method in an MD simulation of supercooled water using the Steinhardt order parameters and the potential energy, 40 represented by Gaussian window functions with different intervals of integration. RDFs are measurable experimentally. The obtained results demonstrate that by selecting an appropriate set of intervals of integration, the formation of different ice polymorphs, including an unknown ice structure, can occur in a single MTD simulation. Kinetically favorable pathways for the formation of each observed ice polymorph, which were predicted by the free-energy landscape, are also presented.In this study, MD simulations in which the MTD method was implemented (MTD simulations) were performed for low-density water (LDW) using only two CVs, which were two discrete oxygen–oxygen radial distribution functions (RDFs)40 were selected as CVs, with each represented by a Gaussian window function, w(r), as a function of the oxygen–oxygen distance, r, so that CVs were differentiable with r40. Each discrete RDF represented the number of r in a given range. Four different sets of CV pairs with different intervals of integration for w(r) were examined  was set in the short-r region for each CV of sets 1 and 2 and in the long-r region for each CV of sets 3 and 4. Previous studies indicate that the use of U as a CV is convenient for reproducing crystallization in MTD simulations27. Thus, a CV set of both the RDFs and U was also examined (set 5).Two discrete RDFs implemented in PLUMED 1.3Interestingly, during the MTD simulations for sets 1–4, not only spatially uniform structures but also spatially nonuniform structures in which high-density areas and low-density (or vacuum) areas coexisted appeared in the system. Notably, the structure in the high-density areas can be regarded as having been formed at high pressure. Thus, in addition to the formation of ice I, the formation of high-pressure ice polymorphs can also be expected in a single MTD simulation with these CV sets.c) also occurred for sets 3 and 4. In principle, the probability of the formation of different ice polymorphs in a single MTD simulation increases if w(r) is set in the long-r region for each CV because the difference in the CV value between ice polymorphs increases upon increasing the r region at which w(r) is set , and high-density ice polymorphs at 200 K and 1 atm in the r region selected for sets 3 and 4 displayed distinct differences , layered ice VII (ice VII layers), and layered ice with an unknown structure (unknown ice layers)  and the distribution function of the angle, θ, formed by the three nearest-neighbor oxygen atoms (P) at a1 and a2, suggest that the HDW structures at these points were different; the HDW at a1 (HDW1) had a disordered structure, whereas that at a2 (HDW2) had an ordered structure resembling the structure of ice VII , where the first minimum of g appears for most of the other ice polymorphs   was not sufficiently small. Similarly, the accuracy of the gradients of the free-energy landscape near the local minima were also not enough high to judge whether the gradient along a pathway for the formation of Ic (and Isd) directly from LDW was gentle. The MTD simulation with the well-tempered MTD method45 is needed to determine the number of local minima, their relative depths, and the gradient of the free-energy landscape near the local minima precisely.The free-energy landscape shown in Fig. c, and Isd. The frequent formation of these high-energy ice structures in spite of their low thermodynamic stabilities suggests that their formation was kinetically favorable compared to the formation of other structures in the corresponding CV regions.It is obvious that the thermodynamic stabilities of ice VII, ice VII layers, and unknown ice layers, which appeared only at high free-energy regions, were much lower than those of LDW, IIn the present simulations, the formation of other ice polymorphs did not occur. A possible reason for it is that the formation of water with the same CV values as other ice polymorphs prevented the formation of them, which can be speculated from the result that the CV values for ice IV and VI were close to the values for HDW Fig. . Systema47, suggesting that the formation of ice VII is kinetically more favorable than the formation of ice VI or ice VIII. The structure of water at high pressure is known to be closer to that of ice VII than to that of ice VI48, which explains the kinetically favorable formation of ice VII in the stable region for ice VI. These findings are qualitatively consistent with the present result that ice VII appeared from HDW2, which had a similar structure to ice VII, although the simulation temperature of 200 K was considerably lower than the experimental temperature at which the formation of ice VII has been observed in the stable region of ice VI or ice VIII (room temperature or higher).In real systems, ice VII appears not only in the region where it is thermodynamically stable on the phase diagram but also in the region where ice VI or ice VIII is thermodynamically stablec and Isd appeared but Ih did not. This result suggests that the formation of Ic was kinetically more favorable than that of Ih, which is consistent with the experimental finding of Takahashi and Kobayashi that, at temperatures much lower than the melting point of Ih, the formation of Ic is kinetically more favorable than the formation of Ih49. It should be further noted that the appearance of Ic and Isd rather than Ih was also reported in an MTD simulation study by Quigley and Rodger26, an MD simulation study by Moore and Molinero50, and an MD simulation study by Malkin et al51.In the present MTD simulation, I2O) as the aforementioned system (216 H2O), using almost the same CVs as for set 4. The formation of both Ic and ice VII occurred even in the MTD simulations of the large system  with different intervals of integration as CVs, successfully reproduced the formation of various ice structures when the interval of integration for the w(r) of each CV was set appropriately: Ic, Isd, ice VII, ice VII layers, and unknown ice layers. To our knowledge, the present study is the first to report the formation of both Ic (or Isd) and high-pressure ice polymorphs in a single MTD simulation.MTD simulations of supercooled water using two discrete RDFs, represented by c, and Isd corresponded to local minimum structures, whereas the formation of the high-energy structures of ice VII, ice VII layers, and unknown ice layers occurred because they were kinetically favorable. The present results indicate that the formation of ice VII from LDW occurred via HDW2, which has an ordered structure similar to that of ice VII. The present results also suggest that the formation of Ic and Isd was kinetically more favorable than that of Ih. These findings of the present MTD simulation are consistent with earlier MTD, MD, and experimental studies51.The free-energy landscape generated via the present MTD method indicated that LDW, Ic (and Isd) and ice VII and also point toward the possibility of discovering a new ice structure. The MTD simulations with the present CVs generated not only spatially uniform structures but also nonuniform structures in the system, so it is anticipated that the present method can be used to analyze not only the thermodynamic stability and formation mechanism of bulk phases but also those of phase separation processes, such as separation into high- and low-density water phases or amorphous phases53. The MTD method using appropriately selected CVs therefore represents a new strategy for studying crystal growth physics, phase transition physics, materials science, and materials technology.In conclusion, MTD simulations using CVs of discrete RDFs show great potential for elucidating the formation mechanisms of ice polymorphs in real systems. The present MTD simulations have provided valuable information on the formation mechanisms of Ih consisting of 3 × 3 × 3 unit cells, each of which had an antiferroelectric structure containing eight water molecules54, was heated by an MD simulation at 800 K for 1 ns. Subsequently, the system was cooled by an MD simulation at 200 K and 1 atm for 20 ns. The final structure obtained after the cooling MD simulation was used as the initial structure of the system. The dimensions of the system were 1.367 × 2.277 × 2.137 nm3. The constant-pressure algorithm was not used with the present MTD method because it might cause large changes in the volume of the system55, which would create a substantially large number of different states including vapor states.The simulations were performed for a constant-volume rectangular parallelepiped system consisting of 216 water molecules. The initial structure of the system was obtained as follows. Initially, the system in which all water molecules were arranged into the lattice sites of bulk I2O56, which was proposed for the simulation of ice crystal growth from water near the real melting point57, was used to estimate the intermolecular interactions. The long-range Coulomb interaction was calculated using the Ewald method with a convergence parameter of 5.0333 nm−1, 7 × 11 × 11 reciprocal lattice vectors, and a real-space cut-off distance of 0.65 nm. The short-range Lennard-Jones interaction was cut off at an intermolecular distance of 0.65 nm.A modified version of the six-site model of H58. The MD simulations were performed using DL_POLY 2.2059 in which PLUMED 1.340 was implemented to permit combination with the MTD method. For the analysis of P, the oxygen atoms in a selected oxygen atom pair was judged to be nearest neighbors to each other if the interatomic distance was shorter than 0.3 nm.The computations were performed using the leapfrog algorithm with a time step of 2 fs. The temperature was maintained at 200 K using the Nosé–Hoover thermostat with a coupling constant of 0.1 psSupplementary Information.


Childhood wasting is among the most prevalent forms of undernutrition globally. The Southeast Asia region is home to many wasted children, but wasting is not recognized as a public health problem and its epidemiology is yet to be fully examined. This analysis aimed to determine the burden of wasting, its predictors, and the level of wasting and stunting concurrence. Datasets from Demographic and Health Surveys and Multiple Indicator Cluster Surveys in six countries in the region were analyzed. The pooled weighted prevalence for wasting and concurrent wasting and stunting among children 0–59 months in the six countries was 8.9%, 95% CI (8.0–9.9) and 1.6%, 95% CI (1.5–1.8), respectively. This prevalence is approximately 12-fold higher than the 0.7% prevalence of high-income countries; and translated into an absolute number of 1,088,747 children affected by wasting and 272,563 concurrent wasting and stunting. Wasting prevalence was 50 percent higher in the 0–23-month age group. Predictors for wasting included source of drinking water, wealth index, urban residence, child’s age and history of illness and mother’s body mass index. In conclusion, our analysis showed that wasting is a serious public health problem in the region that should be addressed urgently using both preventive and curative approaches. Undernutrition in the first 1000 days post-conception has both short- and long-term detrimental consequences for the health and nutrition status of children, and adversely affects the economic productivity of nations ,3,4,5, pThe standardization of the SAM management, by the World Health Organization (WHO) and the adoption of the community-based management of acute malnutrition (CMAM) have contributed to the tremendous reduction in SAM case fatality ,24,25,26This article is based on a review of nationally representative surveys of selected countries in the Southeast Asia region to determine wasting burden, determinants and the overlap with stunting. It aims to contribute to the existing and growing body of evidence needed to support the prioritization of treatment of wasting in addition to the ongoing effort to prevent both wasting and stunting.The present study is a secondary data analysis of the latest Demographic and Health Survey (DHS) and Multiple Indicator Cluster Surveys (MICS) or National Food and Nutrition Survey (NFNS) (when DHS or MICS data is unavailable) from Cambodia, Lao PDR, Myanmar, Thailand, Timor-Leste and Vietnam.Data were from nationally representative cross-sectional surveys conducted between 2013 and 2017 in six Southeast Asian countries that had publicly available data. All the countries in the region were eligible, but nationally representative survey data were not publicly available all countries. These datasets used included the Cambodia Demographic and Health Survey (DHS) 2014, Lao PDR Multiple Indicator Cluster Surveys (MICS) 2017, Myanmar DHS 2015, Thailand MICS 2015/6, Timor-Leste National Food and Nutrition Survey (NFNS) 2013 and Vietnam MICS 2011.All the surveys used multistage cluster sampling. Detailed sampling plans are available from the final survey reports. All the datasets included sampling weights used for the calculation of nationally representative statistics. For this regional analysis, data from each country were imported into Stata14.1 (Stata Corp) and merged for analysis.Children under five years of age were included in the analysis if they had a plausible value for WHZ .The primary outcomes for our analyses were wasting, stunting, and concurrence of wasting and stunting. Wasting was diagnosed when WHZ of WHO child growth standards was <−2 SD, and severe wasting when WHZ was <−3 SD and <−2. Stunting was diagnosed when length/height-for-age z-score (HAZ) was <−2 SD and severe stunting when HAZ was <−3 SD. Wasting and stunting concurrence was diagnosed when both WHZ and HAZ are <−2 SD. Entries with missing values of the child age, sex and any anthropometric measurement were excluded in the analysis. A two-stage approach to anthropometric data cleaning was applied. First we applied the biological plausibility criteria where values were set to missing if weight > 50 kg or if height > 200 cm. Second, we applied the WHO statistical probability criteria where HAZ was set to missing if HAZ > 6 or <−6 and WHZ was set to missing if WHZ > 5 or <−5. Additionally, any record with missing parameters for the calculation of WHZ or HAZ was dropped from the analysis.Most variables were used as stored in the survey datasets and are presented in We calculated pooled and individual countries weighted prevalence of wasting, severe wasting and concurrence of wasting and stunting. We also calculated the burden (number of children affected) overall and for each country. To calculate the burden of wasting and concurrent wasting and stunting, we used the 0 to 59-month-old population for the corresponding country and survey year from the State of the World Children annual reports. The burden of wasting, severe wasting and concurrence of wasting and stunting were then estimated by multiplying the observed prevalence by the total population estimate for 0 to 59-month-old children.We used univariate and multivariate multilevel logistic regression to identify individual, household, maternal, and child characteristics significantly associated with wasting in the region. First, we developed three separate models for each level of characteristics, namely household model, mother model and child model. Subsequently, we constructed a combined model assessing all the covariates together. For all the models, we included all the variables available for analysis into the dataset and excluded only the covariates introducing collinearity. For all the combined models, we also excluded covariates associated with higher than 10% sample size reduction. No stepwise method was used, and significant and non-significant covariates are presented in the tables to show the effect of adjustment for all the covariates. http://dhsprogram.com/data/Access-Instructions.cfm) and from United Nations Children’s Fund (UNICEF) for Multi-indicator Cluster Surveys that serve as authorization to access the datasets for secondary analysis research. The authorization to use the Timor-Leste NFNS data was obtained from the National Office of Statistics, Timor-Leste.Ethical approval for the analyses presented in this paper was not sought as the paper is based on data obtained after completed the mandatory registration to DHS Macro . For severe wasting, the prevalence was 2.9 (2.3–3.6%) for the 0 to 23 months age group and 1.3 (0.9–1.8%) for the 24 to 59 months age group (p < 0.001). When the age was dichotomized to <24 months and ≥4 months, the pooled prevalence (95% CI) of wasting for the 0 to 23 months age group and for the 24 to 59 months age group were 12.0 (10.8–13.4%) and 6.3 (5.5–7.2%), respectively (p < 0.001) in all the countries for both wasting and severe wasting. The relative risk of a child being wasted in the 0 to 23 months age group in comparison to a child in the 0 to 59 months varied across countries with the highest observed relative risk in Timor-Leste (RR 95% CI = 2.13 (1.88–2.41%)) and the lowest in Thailand (RR 95% CI = 1.24 (1.05–1.45%)). For severe wasting, Timor-Leste again had the highest relative risk (RR 95% CI = 3.23 (2.35–4.45%)) while Vietnam had the lowest (RR 95% CI = 1.15 (0.63–2.10%).The prevalence of wasting and severe wasting observed in these age groups by country are presented in supplementary The share of the total number of wasted children was equal for children in 0 to 23 months and 24 to 59 months age groups in the pooled analysis . The couThe prevalence of concurrence of wasting and stunting varied widely from 1.05% to 5.30% and was the highest in Timor-Leste and lowest in Vietnam . For thep < 0.001) between the country’s prevalence of wasting and concurrence of wasting and stunting , Lao PDR (p = 0.035), in Myanmar (p = 0.016), Timor-Leste (p = 0.001) and Vietnam (p = 0.033) but not in Thailand (p = 0.457). Despite the statistically significant linear trend, the highest prevalence was not always in the 48 to 59 age group. The peak prevalence for concurrence was observed in 36 to 47 months age group in Cambodia, in 12 to 23 months age group in Lao PDR, Thailand and Timor-Leste and in 48–59 months age group in Myanmar and Vietnam. When we excluded children below 12 months of age in the analysis, the direct linear relationship for the 12–59 age group was observed only for Timor-Leste (p < 0.001). The test of the linear trend was not significant for Cambodia (p = 0.920), Lao PDR (p = 0.115), Myanmar (p = 0.993), Thailand (p = 0.571) and Vietnam (p = 0.168).The concurrence was the lowest in the 0 to 5 month age group in four of the six countries . This prThe burden of concurrence was equally distributed across the under-five age groups for Cambodia, Lao PDR, Myanmar and Vietnam with each 12-month interval encountering approximately 20% share of the burden . For Thap = 0.001). −2 and <25 kg m−2) differed significantly with that of a mother with low BMI or high BMI. The risk of being wasted was 69% higher for a child of a mother with low BMI < 18.5 kg m−2 (AOR (95%CI) = 1.65 (1.50–1.90%); p < 0.001) when compared to a child of a mother with normal BMI. A child of a mother with higher BMI than the reference had a lower risk of being wasted (AOR (95% CI) = 0.76 (0.63%–0.91)); p = 0.003) for a child of a mother with BMI ≥ 25 kg.m−2 and BMI < 30 kg m−2, and AOR (95% CI) = 0.55 (0.34–0.87%); p = 0.0.011 for a child of a mother with BMI ≥ 30 kg m−2. Maternal short stature, height 145 cm was not associated with a higher risk of wasting (AOR (95% CI) = 0.97 (0.83–1.15%); p = 0.767). In this model, including only data from Cambodia, Myanmar and Timor-Leste, mother’s level of education was no longer a predictor of child wasting, but mother’s age remained (data not shown).The mother’s characteristics model identified mother’s age and mother level of education as independent predictors of wasting among children . Maritalp = 0.004) or smaller (AOR (95%) = 1.56 (1.28–1.90%); p < 0.001) than average at birth. On the contrary, the risk of being wasted was reduced if the child was large (AOR (95%) = 0.69 (0.57–0.82%); p < 0.001) or very large (AOR (95%) = 0.54 (0.33–0.90); p = 0.017) compared to the average at birth according to mother’s recall. The variables of adjustment for this model were age, sex, vaccination status, antenatal care, history of fever and history of diarrhea. The child characteristics model identified child’s sex, age, history of fever and or diarrhea as independent predictors of wasting . Size atInterestingly, while children of the poorest households had an increased risk of being wasted compared to those from households of the average wealth category in the household characteristic model, there was no difference in the combined model between these two categories . AnotherIn contrast, the differences between the average wealth category and the wealthiest categories that were not significant in the separate model became significant in the combined model with children in the wealthiest categories having an increased risk of wasting . The comThe analysis presented in this paper, based on nationally representative data of six countries of Southeast Asia, shows that the prevalence of wasting is of medium public health importance in the region. However, translating prevalence into an absolute number of children under five that are wasted highlights that wasting should be considered a serious public health problem for the region. The analysis also revealed that the prevalence of concurrent stunting and wasting is relatively low at 1.65%, but the overall number of children affected was more than 250,000 in the six countries alone. These findings call for increased attention to this health condition, given the associated higher risk of death. The analysis also showed that the age groups at which prevalence of wasting peaks varies across countries in the region, four countries showed a peak prevalence of wasting in children 0 to 5 months and two countries had the highest prevalence among children 6–23 months. Finally, the combined multivariate modelling of households, maternal and child variables identified antenatal care by skilled staff, maternal BMI and child size at birth as independent predictors of wasting, mediated through prenatal growth restriction, the source of drinking water, and history of recent episode of fever and diarrhea, that operate through postnatal growth deterioration and place of residence, wealth level of the household, mother’s formal education level and child’s sex.Our analysis has demonstrated that a significant proportion of under five children in the countries surveyed, are still suffering from wasting despite the rapid economic growth and the tremendous improvement of food availability ,39. IndeOur analysis has also shown that using prevalence figures for wasting solely to determine the public health importance of wasting may be misleading, especially in countries with a large population. For example, in Vietnam, using a wasting prevalence of 4.1%, wasting would be considered of low public health importance. However, the country has a higher absolute number of affected children than both Cambodia and Timor-Leste, which have wasting prevalence of 10.0% and 10.6%, respectively. Similar findings have been reported in seven countries of Southeast Asia, including four of the countries we analyzed, namely Cambodia, Lao PDR, Timor-Leste, Vietnam and Myanmar, Indonesia and the Philippines . ConsequStunting is also a serious public health problem in Southeast Asia region, with most countries having a stunting prevalence of above 30% ,43,44 anAs with the prevalence of wasting, interpretation of this low concurrence of wasting and stunting prevalence should take into account the population size of Southeast Asia. Indeed, the prevalence of 1.65% obtained in our analysis translates into a quarter-million of under five children affected. These children need particular attention as studies have shown that they are at high risk of dying even when they are moderately wasted ,46. ThusLastly, the observed low prevalence of concurrent stunting and wasting at less than 2% has important policy and programmatic implications for the region. It suggests that in Southeast Asia region, the great majority of wasted children will not be reached by programs designed to combat stunting alone and those that only use height/length-for-age criteria for the selection of beneficiaries. Both conditions should be targeted specifically, although programmatically interventions can be combined to maximize the impact. Indeed, although limited, there is evidence that stunted and wasted children respond well to treatment of wasting and that recovery from wasting may be followed by linear growth acceleration ,48.Our analysis shows that in most countries reviewed the prevalence of concurrence was lower among infants below 12 months of age, similar to the observation by Khara et al. who observed increased concurrence prevalence after 12 months of age . These aOur analysis has shown that the source of drinking water had the strongest association with wasting in the full model, including all the available variables and data from all the six countries. This finding is consistent with the literature, including studies conducted in a different countries in Asia ,49,50. TSeveral maternal factors have consistently been identified as factors associated with increased risk of wasting in under-five children. Younger maternal age, height below 145 cm, low level of education and low BMI has been linked with increased risk of wasting in several studies ,61,62,63Size at birth was also an independent predictor of child wasting in a model including mothers’ BMI indicating that both variables had a direct and independent influence on the risk of wasting of under five children in the three countries that provided data for this analysis. A similar result has been reported by many other authors ,74. ThisOur analysis showed persistent wasting prevalence of >5% among children 24–35 months in comparison to the reference group. This shows that that post-natal factors contribute to the occurrence of wasting in the six countries, and that these age groups also require policy attention. This view is also backed by the fact that 50% of the absolute number of wasted children were from the age group above 24 months. While wasting prevalence is below 5% in children 0–23 months, the increase in risk of wasting is greater than 50% for all age categories less than 24 months, when compared to the 48–59 month group. This finding underscores the higher vulnerability of children below 24 months of age to wasting. In line with the now widely accepted concept of 1000 day window of opportunity, children in the 0–23 months age group should be prioritized for all nutrition interventions [A surprising finding from our analysis is the higher risk of wasting among children from urban households than those from rural households. Indeed, most studies have reported that rural children have a higher risk of becoming wasted than urban children ,41. FurtThe other variables associated with child wasting in our analysis are child sex, a recent episode of fever and a recent episode of diarrhea. For all these variables, our findings show a lower likelihood of being wasted for girls than boys and a higher likelihood of being wasted for those with a history of a recent episode of fever or diarrhea, consistent with the existing literature ,41,42,81The current global renewed interest in tacking undernutrition including wasting has not been sufficiently embraced by governments and other actors in the Southeast Asia region, yet it remains a serious public health problem ,83. InteLastly, the wasting prevalence of 8.9% we observed was encountered despite the rapid economic growth and food production increase experienced in the Southeast Asia region in recent years. This observation confirms that of several authors who previously reported that national-level indicators of economic situation and agriculture outputs can be misleading. An increase in macro-economic indicators such as the per capita gross domestic product may not have an immediate effect on nutrition indicators and the effect may remain limited years after the improvement of the income and food security national figures .The results presented and discussed in this paper should be interpreted taking into account the strengths and limitations of our study. The main strengths of this study are the use of data from the most methodologically robust national surveys and the use of a multicounty dataset combining data of six countries in the Southeast Asia region. However, we cannot assume that these countries are representative of the entire region, given that they were selected according accessibility of data. Another strength is that we used the most recent surveys available at the time of the analysis.Our study had several limitations. The first limitation arises from our study design  that does not allow to distinguish causality from association. Neither is it possible to distinguish direction of association. Second, we acknowledge that our datasets were not designed for looking at different age groups and data is only representative of the under-five age group. All analysis by age group should be looked at with caution. Third, is the unavailability of data on the head of the household characteristics, household food security level, sanitation type, mothers’ principal source of income, child exclusive breastfeeding history and the child dietary diversity score. The high proportion of missing data for some key variables such as size at birth, mother’s height and BMI and child’s history of cough is also an important limitation. Four, is the variation in years of surveys. Data of the oldest survey was collected in 2011 and the most recent in 2017. Despite these limitations, we believe that the findings are informative and should serve as a basis for updating strategies to combat wasting in examined countries and all countries of the region facing this serious public health problem.Wasting remains a serious public health problem in many countries of the Southeast Asia region. The global target to reduce the prevalence of wasting to below five per cent by 2025 may not be reached unless the policy environment is significantly improved to enable the scale-up of preventive and curative interventions with high impact on wasting incidence and nutrition outcomes. For a rapid decline in the prevalence of wasting, women of reproductive age (WRA) including adolescents, and children under five need to be targeted simultaneously with preventive and treatment approaches as both prenatal and postnatal factors are contributing to the burden of wasting in the region. Such integrated programming is likely to have an impact that goes beyond the control of wasting by reducing stunting and by contributing to the prevention of the looming non-communicable diseases pandemic. Our findings suggest that deliberate efforts must be made to address wasting despite the rapid economic growth and improvement in food security. Our recommendation is for countries in the region and their partners to immediately scale-up# interventions with proven efficacy to address wasting in children. Further studies should be undertaken to assess the causal pathways of wasting and propose the most appropriate strategies that support the elimination of wasting and other forms of malnutrition in the region.