Mga in two mouse models of non-small cell lung cancer using a CRISPR-based approach. MGA loss significantly accelerated tumor growth in both models and led to de-repression of non-canonical Polycomb ncPRC1.6 targets, including genes involved in metastasis and meiosis. Moreover, MGA deletion in human lung adenocarcinoma lines augmented invasive capabilities. We further show that MGA-MAX, E2F6, and L3MBTL2 co-occupy thousands of promoters and that MGA stabilizes these ncPRC1.6 subunits. Lastly, we report that MGA loss also induces a pro-growth effect in human colon organoids. Our studies establish MGA as a bona fide tumor suppressor in vivo and suggest a tumor suppressive mechanism in adenocarcinomas resulting from widespread transcriptional attenuation of MYC and E2F target genes mediated by MGA-MAX associated with a non-canonical Polycomb complex.MGA, a transcription factor and member of the MYC network, is mutated or deleted in a broad spectrum of malignancies. As a critical test of a tumor suppressive role, we inactivated  Malignant progression often results from the de-regulation of factors that drive normal developmental programs. Given the critical role of transcription factors in the control of development and in driving cellular functions, it is not surprising that transcriptional regulators can function as potent tumor suppressors or oncogenes. Arguably, one of the most well-characterized oncogenic transcription factors is MYC, which is also essential for normal development    as well Mga functions as a tumor suppressor in vivo, we utilized CRISPR-CAS technology to inactivate Mga in a KrasLSL-G12D/+ (henceforth termed Kras) driven mouse model of lung cancer  mouse model of lung cancer. We inactivated Mga in this model using the same lentiCRISPRv2 Cre approach described above and analyzed mice at a common time point 3 months later. At 3 months after intratracheal instillation of lentiCRISPRv2cre, KP-sgMga mice had a substantially higher tumor burden when compared to KP mice similarly treated with control vector  lung adenocarcinoma patients (TCGA) and in 233 of 3696 (6%) patients sequenced through the GENIE project . The majg cancer . Intratre vector  results ga locus . For surpatients . To furtl vector . This wal vector . Immunosl vector . Consistl vector . In addil vector . We confferation .Stag3, Sox30, and Tdrd1, previously reported to be directly regulated by MGA-PRC1.6 in ES and germline cells , several ncPRC1.6 complex repressed germ cell related transcripts, including king MGA , similarnd Itga1 . Gpc2 ana tumors . Gene sea tumors . Intrigua tumors . In addia tumors . Along wa tumors . We empland Gpc2  and ncPRnd Tdrd1  in a pannd Tdrd1 .Stag3 as well as multiple MYC targets in lung tumor initiating cells that overlap with previously reported MGA targets in ES cells as well as distinct gene signatures based on cellular context, such as Trp53 expression.Taken together, our data suggest that MGA regulates meiotic transcripts such as Mga-inactivated and control KP lines. Mga loss resulted in decreased protein levels of E2F6, PCGF6, and L3MBTL2  and several other proteins . InterroMga-inactivated and control (empty) KP tumor-derived cells lines. MGA, MAX, E2F6, and L3MBTL2 were seen to bind several thousand promoters in MGA-expressing KP tumor cells suggesting that the PRC1.6 complex plays a broad role in tumor cell gene expression , including at STAG3 and PODXL2 promoters  or containing sgMGA. We probed for MAX, E2F6, L3MBTL2, and MYC occupancy using CUT and RUN in the sgMGA and control lines . As for romoters . Concomiromoters . Taken tGiven that MGA-MAX heterodimers can arguably be considered as members of the MYC network, we assessed the contribution of MYC-MAX heterodimers to the transcriptional and phenotypic consequences of MGA loss of function. First, we examined TCGA data to interrogate co-occurrence of MGA and MYC paralog alterations in a panel of LUAD patient samples. Analysis of this data indicated that amplification of MYC paralogs is rare in LUAD whether or not MGA is inactivated . Moreover, TCGA samples did not indicate significantly altered MYC family RNA levels in MGA mutant tumors compared with wild-type MGA tumors . This isMYC in sgMGA-KP and control KP tumor lines and found suppression of proliferation in both cases   . These fMGA loss . The datMGA loss .MGA loss in patients. We then grouped patients into MGA WT and MGA altered groups, based on the presence of mutations and deletions at the MGA locus, to assess functional enrichment of expression of genes in MGA altered tumors. Interestingly, STAG3 appeared to be highly expressed (SD > 3 above mean) in a significant subset of MGA-altered cases (truncating mutations and deletions)  . Genes oletions) . Consisted cases . We thened cases .MGA loss leads to similar functional consequences in vitro, we used either shRNA- or CRISPR-based approaches to delete or knock down MGA in A549 and NCI-H23 lung adenocarcinoma lines  identified from our analysis in lung cancer models were significantly upregulated upon loss of MGA  at several thousand promoters . FurtherMga with deletions of ncPRC1.6 subunits Pcgf6 and L3mbtl2 in KP tumor cells. Deletion of either Pcgf6, L3mbtl2, or bothresults in expression profiles that are similar but not identical to the profiles generated by Mga deletion  or factors  associated with other regions of MGA  cells . A largeOur genomic occupancy studies also reveal that nearly all MGA-bound loci are associated with RNA polymerase II and that the genes upregulated upon MGA deletion display increased polymerase binding . This suBased on our results, we propose that MGA inactivation in pre-malignant settings destabilizes PRC1.6 and consequentially results in unoccupied E-boxes and E2F/Dp1 sites. A combination of invasion of E-boxes by MYC-MAX and/or binding of E2Fs at target loci may account for the net transcriptional activation of these genes and tumor initiation or progression . HoweverLastly, we note that, in both the lung adenocarcinoma cells and colon organoids, MGA deletion is also accompanied by downregulation of multiple genes. While some of this repression may be indirect, it is nonetheless apparent that direct MGA binding is associated with a subset of the genes downregulated upon MGA loss , which oIn summary, we demonstrate that MGA acts as a bona fide tumor suppressor in vivo and have uncovered a critical role for MGA and the atypical PRC1.6 complex in lung adenocarcinoma. We also provide preliminary evidence for a tumor suppressive role for MGA in colorectal cancer. Although our data is suggestive of a broad tumor suppressive role of MGA and PRC1.6 via antagonizing transcription of MYC and E2F targets, further studies will be required to elucidate the functional relevance of individual MGA-PRC1.6 targets such as STAG3 in tumorigenesis. Given the frequent inactivation of MGA across diverse cancer types, it will also be critical to further characterize MGA- and PRC1.6-mediated transcriptional attenuation in other malignancies with MGA alterations, such as colorectal cancer and diffuse large B-cell lymphoma. This is especially important as MGA, via atypical PRC1.6, likely regulates lineage-specific genes to mediate its tumor suppressor role in a context-dependent manner.Kras G12D LSL and Trp53 floxed alleles were maintained on a C57BL6 background. Heterozygous Kras G12D LSL/+ alone (termed Kras) or in combination with Trp53 fl/fl homozygous alleles (termed KP) were used for experiments. Mice 2 months or older were utilized for intratracheal instillation of lentiviral particles in a BSL2 facility  were designed and cloned into the lentiCRISPRv2cre vector . See KeyAll cell lines employed in experiments cited in this manuscript were either derived by the authors or obtained from verified sources and were routinely tested for and found to be free of mycoplasma. Human cell lines were deidentified. Mouse KP and KP-sgMga lung tumor cell lines were generated as described above and cultured in DMEM with 10% FBS. Human non-small cell lung cancer lines NCI-H23, NCI-H2291, NCI-H2347, NCI-H1975, and 91T were grown in RPMI-1640 with 10% FBS. Human squamous cell carcinoma line LOU-NH-91 was maintained in RPMI-1640 supplemented with 20% FBS. A549 human lung adenocarcinoma, DLD1 human colorectal adenocarcinoma, 293FT and 293TN cells were maintained in DMEM with 10% FBS.3 cells per well in a 96-well, 2D culture format, described previously (Organoid cultures were established as previously described . Brieflyeviously . Bright #1 sgMga#3 (mouse) and sgMGA (human) primers. Sequence information was input for ICE analysis (Synthego) to compute percent indel formation (https://ice.synthego.com), and Clustal Omega  was used to align sequences.To confirm indels, cell pellets and tumors were incubated in a Proteinase K containing digestion buffer overnight at 55°C. DNA was either isolated using 25:24:1 phenol:chloroform:isoamylalcohol followed by ethanol precipitation or extracted using the GeneJet genomic DNA purification kit (ThermoFisher). For human colon organoids, cells were dissociated into single cells and DNA was isolated using the Purelink Genomic DNA isolation kit (Invitrogen). PCR was performed using high-fidelity Phusion DNA polymerase (ThermoFisher) with primers flanking the region targeted by sgMga#1 and sgMga#3 (for mouse tumors and cell lines) and sgMGA (for human colon organoids). PCR products obtained were run on a 0.8% agarose gel and amplicons to confirm the presence of one amplicon corresponding to the correct size. PCR DNA was purified using a PCR purification kit (Qiagen) and sequenced using one of the sgMgaCells were cultured on 8-well chamber slides for immunocytochemistry. Briefly, cells were fixed in either 4% formaldehyde or 1:1 methanol:acetone, blocked, and incubated with primary antibodies followed by Alexa fluor conjugated secondary antibodies or Alexa 488-conjugated phalloidin (for Actin staining). Chambers were then removed and slides mounted using Prolong Gold anti-fade with DAPI. Immunohistochemistry was performed on 5 µm thick paraffin embedded mouse lung sections. Following heat-induced antigen retrieval, sections were incubated with primary antibodies followed by Alexa conjugated secondary antibodies. Sections were mounted in Prolong Gold anti-fade with DAPI (Invitrogen). Images were acquired using either TissueFaxs (Tissugnostics), Nikon E800, Deltavision Eclipse, Olympus Fluoview confocal, or Zeiss confocal microscopes. Image analysis and intensity measurements were performed using ImageJ.For protein gels, whole-cell lysates were prepared using RIPA buffer with protease and phosphatase inhibitors. They were then reduced in NuPage LDS buffer (Invitrogen), and a wet transfer was performed prior to western blotting. For histone blots, acid extracts were made following manufacturer (Abcam) recommendations. Refer to Key resource table for list of antibodies used.https://software.broadinstitute.org/morpheus).For all applications, RNA was isolated using Trizol (ThermoFisher Scientific) according to manufacturer’s recommendations or processed using the Direct-zol RNA Miniprep kit (Zymoresearch). For qPCR, cDNA was synthesized from 500 ng to 2 μg of total RNA using the Revertaid cDNA synthesis kit (ThermoFisher). A Biorad iCycler was using for SYBR green-based quantitative PCR. Refer to Twenty 15 cm plates of 293 T cells were transfected with pcDNA3-FLAG-His-MGA, MGA(967–1300), or MGA(2153–2856); 48 hr after transfection, the cells were lysed in TN buffer (10 mM Tris pH 7.4/150 mM NaCl/1% NP-40/1 mM AEBSF/10 μg/ml aprotinin/10 μg/ml Leupeptin/1 μg/ml Pepstatin A/20 mM sodium fluoride). The lysate was incubated with Ni-NTA agarose (Qiagen) and FLAG-His-MGA and its interacting proteins were collected by centrifugation, washed three times with TN buffer, and eluted with 50 mM sodium phosphate buffer pH 8.0/150 mM NaCl/250 mM imidazole. The eluted sample was immunoprecipitated with anti-FLAG antibody , the immunoprecipitate was eluted with FLAG peptide (Sigma-Aldrich), and the eluted protein sample was processed with an Amicon Ultra 0.5 3 k centrifugal filter device (Millipore) for concentration and buffer exchange to 50 mM Tris pH 8.5. Proteins were digested at 37°C overnight with trypsin  in the presence of 10% acetonitrile. The resulting tryptic peptides were analyzed by HPLC-ESI-tandem mass spectrometry on a Thermo Fisher LTQ Orbitrap Velos Pro mass spectrometer. The Xcalibur raw files were converted to mzXML format and were searched against the UniProtKB/Swiss-Prot human protein database (UniProt release 2016_04) using X! TANDEM CYCLONE TPP . Methionine oxidation was considered as a variable modification in all searches. Up to one missed tryptic cleavage was allowed. The X! Tandem search results were analyzed by the Trans-Proteomic Pipeline, version 4.3. Peptide/protein identifications were validated by the Peptide/ProteinProphet software tools . The masMga-inactivated line and one control line. Chromatin IPs were done using antibodies against MGA, MAX, MYC, L3MBTL2, and E2F6. Libraries were generated using the NEB Ultra II kit , followed by 50 × 50 paired-end sequencing on an Illumina HiSeq 2500 instrument. For human cell lines, CUT and RUN was utilized to determine genomic occupancy. Cells were bound to Concanavalin A beads , followed by permeabilization using a digitonin containing buffer. One million cell aliquots were then incubated overnight with antibodies against MGA, MAX, MYC, and L3MBTL2. An automated CUT and RUN protocol was followed for library preparation . Classification of somatic MGA mutations as homozygous or heterozygous was obtained from the ABSOLUTE-annotated MAF file containing the output from the ABSOLUTE method  were compared between MGA-mutant and non-mutant samples using unpaired two-tailed t-tests.Gene expression data of MYC-family genes in lung adenocarcinomas from the TCGA PanCanAtlas dataset were downloaded from For 2D growth curves, cells were seeded in 96-well flat bottom dishes (Corning) and imaged at fixed time intervals using either an Incucyte S3 or Incucyte zoom (Essen Bioscience). Percent confluence was used as a measure of cell growth. For 3D spheroid based assays, 5000 A549 cells were plated per well in ultra-low attachment plates and centrifuged at low speed (800 rpm) for 9 min. Phase-contrast images were acquired using an inverted scope. Spheroid area was calculated using ImageJ. For wound healing assays, cells were plated to reach full confluence prior to the start of the assay. Imagelock plates (Essen Bioscience) were utilized. Cells were pre-treated with mitomycin C (2.5–5 µg/ml) to retard cell growth, and scratch wounds were made using a Woundmaker (Essen Bioscience). Wound closure was monitored at fixed time intervals, and end point wound width or confluence was used as metrics for cell migration. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. 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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 The reviewers agreed that the paper provides strong in vivo data for a tumor-suppressive role for Mga in lung carcinogenesis. The authors are convincing that MGA is important in oncogenesis. We note here that MGA is highly understudied (~200 publications) in and of itself despite its involvement with the MYC network for oncogenesis . Given a protein of 3000 amino acids, the number of potential protein partners and PTMs that might modify its tumor suppressor functions are staggering. However, the reviewers also noted that a previous paper has addressed the same topic and the novelty of the data presented here needs to better explained and additional experiments are needed to strengthen and expand the new aspects.There are several loosely articulated stories here, but the reader does not come away with a clear take-home message. No particular oncogenic pathway is intensively explored or elucidated. MGA seems to be quite a general tumor suppressor, so is there a reason to focus experiments on lung vs. colon adenocarcinomas, or are these convenient models to explore general MGA action? The PRC1.6 story is interesting, but it is not integrated into a general description of MGA action. We think the authors buried the lede here; the take home message should be that there are multiple, perhaps independent mechanisms for tumor suppression embedded in MGA; these mechanisms should be clearly enumerated and the evidence for each supported. As presented, there is little attempt to separate or coordinate the various anti-oncogenic functions and contributions of the different domains of MGA-T-box, bHLH, or DUF480. Just considering the bHLH domain, there are multiple possibilities for MGA to disturb the MYC-network and suppress oncogenesis. The actin protrusions seem to be related to DUF480 but unrelated to PRC1.6; they probably relate with the observations about migration, wound healing and EMT . I would suggest a serious re-write to identify, organize and reinforce their story(ies).1. It seems that the investigation of publicly available datasets is essentially identical to the Schaub et al.. analysis and not new data. If the authors want to maintain this, they would need to better explain what is new. One important piece of information that seems to be missing is whether the mutations are homozygous or heterozygous. So data on MGA and MYC protein expression in human tumors would greatly strengthen this part.Inspections of the MGA mutations on cBio portal reveals broad tissue specificity of tumors, well beyond lung and colon adenocarcinomas-in view of this range there is really no need to justify the choice of tumors, only to be wary whether differences in expression of MGA targets truly reflect MGA versus the tissue as the source of a non-pathogenic difference. Looking at the distribution of the mutations within MGA, I disagree with the assertion that DUF480 is a hotspot. The only lukewarm to hot spot in the protein seems to be the bHLH region. The other mutations are rather evenly distributed throughout the protein. This does not mean that they are not pathogenic, more likely it indicates a broad range of contributory motifs and domains. I suspect that there is a lot of interesting and important biology throughout these 3000 residues.2. Conceptually, one would like to know whether tumor development in an MGA-delete situation depends on MYC. One would also like to know whether the polycomb complex that is assembled by MGA is tumor-suppressive. Therefore, the authors should perform a similar analysis as they did for MGA (introduce sgRNAs into the lung models) and score the phenotypes they get. Both experiments could be done in cell lines established from this model and either in vitro  or upon re-transplantation. This would also prevent simply reporting negative results.3. The interpretation of the VENN diagram and the heatmaps in Figure 5A,B is somewhat uncertain. If one plots these for MYC, occupancy often simply parallels occupancy by RNAPII, so essentially being bound by MYC simply says the promoter is open/active. Is this the case for MGA and its complex partners? Or is there a specificity in binding? The authors should do RNAPII ChipSeqs in these cells, preferentially +/- MGA, and then show these alongside .Along these lines, it is hard to understand how one obtain the extreme p-values shown in figure 5E and 5H, I would challenge this. If the authors want to maintain this, they should not use ENCODe data, but simply determine what genes are active in the cells (e.g. what promoters are bound by RNAPII) and then use those as background list and calculate P-values for overlap between MYC, MAX and E2F6.Based on the description, the ChIPSeq analyses are not spike-normalized and I could not find information about the number of repeats. If it is n=1, the authors need to find a way to exclude that the differences are due to experimental variation.4. The authors use the term "Empty" vector for their sgMGA -CRISPR-cas-Cre lentivirus without the sgRNA. This is confusing-what they mean is minus-sg; the vector is hardly "empty". While it would have been nice to have a non-targeting sgRNA just to ensure that no part of the phenotype reflects off-target or non-specific effects of CRISPR-CAS or CRE-expression, at least they should make clear in the text what is the difference between the vectors.5. The methods describing the observation and quantitation of the actin protrusions are a bit sparse. The interpretation relating DUF480 with actin protrusions and with gene expression separate fromPRC1.6 is hard to follow and sparsely supported.6. Contrary to what is stated in the manuscript, to my eye MAX is reduced in LOU-NH-91.7. The KP mouse models are driven by activated RAS called RASV12G but not MYC. Is MYC amplified in these models? I was struck by the finding in Figure 5 and comment that whether MGA is present or not, MYC will drive tumor growth. Some lung adenocarcinomas acquire MYC amplification. Do the human lines used in the experiments have MYC amplified?It is unclear to me whether MAG loss and MYC amplification are mutually exclusive or correlated? The authors should comment as to whether MGA is required or not in MYC amplified tumors.8. Does MGA-MAX compete with MYC-MAX to bind the E boxes, and if so, would enforced expression of MYC in the tumors reverse the phenotype? It would be good for the authors to define MAG as the MAX-Gene associated protein.9. While the title states that "Loss of MGA mediated polycomb repression..", I would add atypical or non-canonical polycomb to the title to distinguish is from the other better known PCR2 and PCR1 complexes.10. The authors use PRC1.6 rather than PCFG6-PRC1 used by Llabata. The authors should make sure in the text that is refers to the same complex.11. Figure 4 panels 4B and 4, MGA antibody shows multiple bands. A cleaner blot will help. Is MGA expressed as spliced variants or is the antibody dirty? The reviewers agreed that the paper provides strong in vivo data for a tumor-suppressive role for Mga in lung carcinogenesis. The authors are convincing that MGA is important in oncogenesis. We note here that MGA is highly understudied (~200 publications) in and of itself despite its involvement with the MYC network for oncogenesis . Given a protein of 3000 amino acids, the number of potential protein partners and PTMs that might modify its tumor suppressor functions are staggering. However, the reviewers also noted that a previous paper has addressed the same topic and the novelty of the data presented here needs to better explained and additional experiments are needed to strengthen and expand the new aspects.There are several loosely articulated stories here, but the reader does not come away with a clear take-home message. No particular oncogenic pathway is intensively explored or elucidated. MGA seems to be quite a general tumor suppressor, so is there a reason to focus experiments on lung vs. colon adenocarcinomas, or are these convenient models to explore general MGA action? The PRC1.6 story is interesting, but it is not integrated into a general description of MGA action. We think the authors buried the lede here; the take home message should be that there are multiple, perhaps independent mechanisms for tumor suppression embedded in MGA; these mechanisms should be clearly enumerated and the evidence for each supported. As presented, there is little attempt to separate or coordinate the various anti-oncogenic functions and contributions of the different domains of MGA-T-box, bHLH, or DUF480. Just considering the bHLH domain, there are multiple possibilities for MGA to disturb the MYC-network and suppress oncogenesis. The actin protrusions seem to be related to DUF480 but unrelated to PRC1.6; they probably relate with the observations about migration, wound healing and EMT . I would suggest a serious re-write to identify, organize and reinforce their story(ies).The reviewers point out that our paper provides strong and compelling in vivo data for a tumor-suppressive role for Mga in carcinogenesis. While they note that MGA is relatively understudied, they point out that the recent paper by Llabata et al. also addressed aspects of MGA’s tumor suppressive and DNA binding activities and the reviewers ask us to explain the novelty of our paper.As noted, a major focus of our paper is that it provides and validates a mouse model inwhich we delete MGA and demonstrate its tumor suppressive activity. Theexperiments in Llabata et al., including the biological assays and the ChIP-Seq,overexpressing MGA in cells which already express endogenouswere done by MGA. Therefore, all of their data monitor the consequences of overexpression ofMGA, a situation without clear biological relevance. In the experiments reported inour paper, we delete MGA. Therefore, our molecular data refer to a comparisonbetween MGA null and the same cells expressing endogenous MGA. This isimportant since MGA is a tumor suppressor and its loss of function is what is crucialbiologically, as we show here or the first time in our lung adenocarcinoma model.In addition, by deleting MGA we were able to show that its loss corresponds to anincrease in a core set of target genes previously associated with PRC1.6.Furthermore, we show that members of this core group are directly bound by the MGA-MAX PRC1.6 complex and are relevant to the proliferation of tumors that lack MGA .The PRC1.6 complex has been known to be associated with MGA since at least2012 as indicated in our references cited. Llabata et al. confirmed that result in somatic cells as do we, but we extend this data to show that PRC1.6 subunits are associated with MGA through the DUF4801domain of MGA. This is the first identification of the interface between PRC1.6 and MGA. It is important and relevant because multiple frame shift mutants in MGA have the consequence of deleting this region in a wide range of tumor types including lung cancers . In our revised paper we cite Llabata as well as earlier reports relating to MGA multiple times and describe in greater detail the differences with our data (line 91+).Regarding our choice of experimental systems (lung and colon) the reviewer is correct in suggesting that we were influenced by a combination of having available a well-established mouse model for lung adenocarcinoma as well as the ongoing organoid cultures from human colon. Both systems offer the possibility of assessing early events in oncogenic conversion. Furthermore, TCGA data indicates that LUAD is second (following UVEC) in terms of prevalence of MGA mutations. In the Introduction (line 58+) we cite the previous reports that led us to choose these models.The reviewers point out the potentially “staggering” complexity of MGA and the possibility that it has myriad activities potentially linked to functions within its three described regions  and suggest a re-write of our text to clarify what we know about the structure-function relationships related to MGA. In the revised paper we have reorganized and rewritten many sections to increase clarity and have underscored both in the results and discussion what functions can and cannot be attributed to the various regions of MGA. We can only agree that there is “… a lot of interesting and important biology…” associated with MGA and see our paper as providing a jumping off point for further exploration of this “highly understudied” tumor suppressor.1. It seems that the investigation of publicly available datasets is essentially identical to the Schaub et al.. analysis and not new data. If the authors want to maintain this, they would need to better explain what is new. One important piece of information that seems to be missing is whether the mutations are homozygous or heterozygous. So data on MGA and MYC protein expression in human tumors would greatly strengthen this part.Inspections of the MGA mutations on cBio portal reveals broad tissue specificity of tumors, well beyond lung and colon adenocarcinomas-in view of this range there is really no need to justify the choice of tumors, only to be wary whether differences in expression of MGA targets truly reflect MGA versus the tissue as the source of a non-pathogenic difference. Looking at the distribution of the mutations within MGA, I disagree with the assertion that DUF480 is a hotspot. The only lukewarm to hot spot in the protein seems to be the bHLH region. The other mutations are rather evenly distributed throughout the protein. This does not mean that they are not pathogenic, more likely it indicates a broad range of contributory motifs and domains. I suspect that there is a lot of interesting and important biology throughout these 3000 residues.MGA was barely mentioned in the Schaub et al. TCGA paper and was lumped together with MNT and the MXDs. In the present manuscript our point was to highlight the pervasiveness of MGA loss and focus on two tumor types where MGA loss of function is apparent and also include and compare TCGA and Genie-derived data.In response to the reviewer’s request for more information we have collaborated with Alice Berger and Sita Moorthi (now listed as co-authors) genomic analysis experts who have further analyzed the TCGA data and particularly focused on MGA mutations in lung adenocarcinomas (LUAD) that would be expected to lead to inactivation of the protein through loss of functional domains . The available data indicates that all of the truncating mutations in LUAD would be predicted to delete either the T-box, the DUF4801 region, or the bHLHZ or all three . It was based on this information that we decided to investigate the DUF region in the proteomics experiment (line 253+). Moreover, the available data indicates that 25% of the functional mutations are homozygous. We note that heterozygous mutations may be consequential as there is recent evidence from Okuda’s lab suggesting that, in germ cell development, an alternatively spliced form of MGA can act as a dominant interfering form . Please see our comments on (line 580) in the revised paper.Further analysis of the TCGA data revealed that only one of the LUAD samples with truncated MGA displayed amplification of MYCL and none showed MYCN or MYC amplification, while 3 tumors with MGA missense mutations showed MYC amplifications. Turning to RNA expression levels, the TCGA data show no significant difference in MYC family gene expression in MGA mutants compared to LUAD without MGA alteration . Lastly, LUADs with MGA mutations were mutually exclusive with gain or loss of MAX copy number or expression changes, as expected since MAX loss would be expected to inactivate MGA E-box binding. While some LUAD tumor lines have high MYC, in general MYC amplification is not strongly associated with LUAD, in contrast with SCLC where all 3 Myc family members are frequently amplified. This is described in the revised paper starting at line 324.2. Conceptually, one would to know whether tumor development in an MGA-delete situation depends on MYC. One would also like to know whether the polycomb complex that is assembled by MGA is tumor-suppressive. Therefore,the authors should perform a similar analysis as they did for MGA (introduce sgRNAs into the lung models) and score the phenotypes they get. Both experiments could be done in cell lines established from this model and either in vitro  or upon re-transplantation. This would also prevent simply reporting negative results.To address the reviewer's request we have now utilized CRISPR to inactivate Myc, Mga, L3mbtl2, Pcgf6, and L3mbtl2+Pcgf6 in tumor derived KP cell lines and verified relevant loss of expression changes in these genes . To assess potential effects on cellular behavior we carried out proliferation as well as invasion assays. MYC deletion results in growth arrest or apoptosis in the KP and the KP-sgMGA cells  consistent with the MYC and MAX inhibitor data already included in the paper . Deletion of MGA, or PRC1.6 complex subunits had no significant effect on proliferation rate relative to KP controls. In the spheroid assay the sgMGA cells showed marginally greater outgrowth than controls but the sgPRC1.6 had reduced outgrowth compared to controls. These very subtle effects on cell behavior are not entirely surprising given that the KP cells are already derived from advanced lung tumors at a late stage of tumor progression. The optimal experiment would be to generate the individual deletions in the initial murine model to address their role in tumor initiation – but given the time and depth of analysis required, we cannot realistically include it in this paper.Turning to the RNA-seq data we compared differentially expressed genes in KP cells upon individual deletion of Mga, L3mbtl2, Pcgf6, and L3mbtl2+Pcgf6 cells. In figure 6 we show heat maps and Venn diagrams demonstrating highly significant overlap among genes upregulated upon MGA loss and those upregulated by individual deletion of L3mbtl2 and Pcgf6. Most notably we find that genes associated with meiotic recombination and repair are among the most differentially upregulated upon Mga deletion as well as by deletion of Pcgf6 or L3mbtl2  as well as genes involved in EMT. These findings support the notion of a critical role for the PRC1.6 complex in gene regulation by MGA. At the same time it is clear that deletion of key PRC1.6 factors do not fully recapitulate the effect of Mga loss. This underscores the notion that other regions of MGA contribute to the deletion phenotype. This data has been included in revised Figure 6. We have now added a separate section devoted to what we understand about the role of MYC in MGA deleted tumors (starts at line 335 and in the Discussion starting at line 584+).3. The interpretation of the VENN diagram and the heatmaps in Figure 5A,B is somewhat uncertain. If one plots these for MYC, occupancy often simply parallels occupancy by RNAPII, so essentially being bound by MYC simply says the promoter is open/active. Is this the case for MGA and its complex partners? Or is there a specificity in binding? The authors should do RNAPII ChipSeqs in these cells, preferentially +/- MGA, and then show these alongside .Along these lines, it is hard to understand how one obtain the extreme p-values shown in figure 5E and 5H, I would challenge this. If the authors want to maintain this, they should not use ENCODe data, but simply determine what genes are active in the cells (e.g. what promoters are bound by RNAPII) and then use those as background list and calculate P-values for overlap between MYC, MAX and E2F6.Based on the description, the ChIPSeq analyses are not spike-normalized and I could not find information about the number of repeats. If it is n=1, the authors need to find a way to exclude that the differences are due to experimental variation.Assessing RNAPII, Myc and Mga occupancy is an excellent suggestion and is something that we had planned to do as a close follow-up to the present study. Nonetheless we have now carried out these experiments and included the data in our revised paper . The data shown are from ChIP-seq and the Venn diagram numbers are derived from the 2 experiments. We have included a new Venn diagrams showing the overlap between MYC, MGA and RNAPII as well as TSS +/-2kb line plots and box plots indicating the concurrence of RNAPII and MGA binding in the +/- MGA conditions. As evident from the data, there is a high degree of overlap between occupancy by MGA, MYC, and RNAPII. We also note a significant increase in RNAPII occupancy to genes upregulated upon MGA deletion . Downregulated genes and MYC bound genes display a modest decrease in binding . As requested, we deleted the Encode-based categories from Figure 5. These findings are also discussed in the results and in the Discussion section (line 584).4. The authors use the term "Empty" vector for their sgMGA -CRISPR-cas-Cre lentivirus without the sgRNA. This is confusing-what they mean is minus-sg; the vector is hardly "empty". While it would have been nice to have a non-targeting sgRNA just to ensure that no part of the phenotype reflects off-target or non-specific effects of CRISPR-CAS or CRE-expression, at least they should make clear in the text what is the difference between the vectors.We employed the term ‘empty’ to highlight the fact that it was indeed minus sgRNA, something that is not infrequent in the existing literature. Nonetheless we take the reviewer's point that it is ambiguous and have now ensured that it is clearly defined it in the text, as suggested.5. The methods describing the observation and quantitation of the actin protrusions are a bit sparse. The interpretation relating DUF480 with actin protrusions and with gene expression separate fromPRC1.6 is hard to follow and sparsely supported.We have removed the data on actin protrusions.6. Contrary to what is stated in the manuscript, to my eye MAX is reduced in LOU-NH-91.We pointed out the lack of a strong effect on MAX levels because previous work from us and others has indicated that MAX loss is associated with increased MYC protein degradation. Therefore, the presence of MAX would be consistent with an effect on MYC gene expression rather than protein turnover. In response to the reviewer’s comment, we have measured the levels of MAX by densitometry as shown in 7. The KP mouse models are driven by activated RAS called RASV12G but not MYC. Is MYC amplified in these models? I was struck by the finding in Figure 5 and comment that whether MGA is present or not, MYC will drive tumor growth. Some lung adenocarcinomas acquire MYC amplification. Do the human lines used in the experiments have MYC amplified?It is unclear to me whether MAG loss and MYC amplification are mutually exclusive or correlated? The authors should comment as to whether MGA is required or not in MYC amplified tumors.We have not directly measured MYC levels in untransformed and KRAS transformed lung epithelial cells. However, activation of the RAS pathway has been known for many years to result in increased MYC protein stability and this is likely to be the case in the KRAS tumors. Importantly, MYC mRNA, protein, and overall genomic occupancy are not measurably different in the KRAS/p53 tumor derived cells whether or not MGA is deleted. This is consistent with the TCGA data which do not show consistent amplification or elevated expression of MYC in LUAD that correlates with MGA inactivating mutations . In fact, amplification of MYC is not very common in LUAD (in sharp contrast to SCLC). Nonetheless there are some LUAD lines with amplified MYC (the human H23 line does have amplification of MYC while the A549 lacks MYC amplification). Our data indicate that the endogenous levels of MYC in both KP and KP-sgMga lines are required for proliferation and survival. It is conceivable that high MYC levels may trigger apoptosis in the absence of MGA. Having said all this it is important to note that the expression of MYC is critical for the growth of most cells including the KP and the KP-sgMGA cells . Our future experiments will address the role of MYC and other transcription factors as a consequence of MGA loss.8. Does MGA-MAX compete with MYC-MAX to bind the E boxes, and if so, would enforced expression of MYC in the tumors reverse the phenotype? It would be good for the authors to define MAG as the MAX-Gene associated protein.In our paper reporting the identification and initial characterization of MGA  we noted that equivalent amounts of the GST-linked bHLHZ regions of MYC and MGA bound roughly equal amounts of MAX and E-box containing DNA oligonucleotides. Rigorous binding affinities have not been determined and would certainly be worthwhile carrying out for MGA as well as other network factors. While not definitive in terms of binding strength, our ChIP analyses indicate that MGA and MYC and other network factors can occupy the same regions of DNA.In that same 1999 paper we identified MGA as one of a group of novel genes and proteins that heterodimerize with MAX. We named it MGA to: (i) distinguish it from the other group of MAX dimerizing proteins (MAD or MXD proteins) and (ii) to underscore that it is by far the largest of the MAX network proteins. We note in the present paper that it should be pronounced "mega" to reflect this fact (line 59).9. While the title states that "Loss of MGA mediated polycomb repression..", I would add atypical or non-canonical polycomb to the title to distinguish is from the other better known PCR2 and PCR1 complexes.Title: at the reviewer's suggestion we have changed the title. In addition we have now referred to this complex as ncPRC1.6 to make it clear throughout the paper that this is a non-canonical complex.10. The authors use PRC1.6 rather than PCFG6-PRC1 used by Llabata. The authors should make sure in the text that is refers to the same complex.In our opinion, Llabata's use of PCGF6-PRC1 is unusual and somewhat confusing. PRC1.6 or ncPRC1.6 has been consistently used to refer to this complex by multiple publications over the last decade and we prefer to use this nomenclature.11. Figure 4 panels 4B and 4, MGA antibody shows multiple bands. A cleaner blot will help. Is MGA expressed as spliced variants or is the antibody dirty?There are MGA splice variants and there are also likely to be non-specific bands in this blot.


The active hormones are not released in the extracellular space   is the lRDS can be an early phase of bronchopulmonary dysplasia (BPD), another lung complication of premature birth. BPD was originally described as a heterogeneous group of lung disorders associated with preterm birth and lung impairment due to mechanical ventilation . Today, in vitro exposure of fibroblasts to androgens decreases their ability to stimulate the maturation of PTII cells  biosyntheses belong in two pathways. The intra-gonadal endocrine production and the peripheric intracrine production. The endocrine pathway is carried by the ovaries and testes. Active sex steroids are synthesized from cholesterol and released in circulation to have their effects on target tissues. The intracrine pathway depends on intracellular activation of adrenal derived precursors , namely DHEA and androstenedione. These circulating precursors, when presented to tissues with the required enzymes, are converted into active androgens capable of having a direct effect on the cell in the presence of AR . Many tiin situ synthesis of active steroids in the cell where the steroids have their action . The rodent model was considered in the first place to make direct measurements. It is important to note that androstenedione is the main SSPs in rodents whereas in humans DHEA and androstenedione are the main precursors . In 198617β-hydroxysteroid dehydrogenases (17β-HSDs) , 27 3β-Hin vitro using A549 cells. This PTII like cell line was established from a lung adenocarcinoma of an adult male. They have been widely used to study androgen metabolism  cells to synthesize androgens from SSPs tabolism –34. As ntabolism  and are tabolism . Our restabolism . These cting DHT .We also aimed to describe the enzymatic activity regarding androgen metabolism in perinatal pulmonary fibroblasts using eight normal diploïd cells. Regarding androgen synthesis, none of them showed the capacity to form androgens from SSPs but the opposite reaction was observed. Indeed, the human lung fibroblast cells show a pattern of androgen inactivation where testosterone is mainly converted back into androstenedione and androstanedione without any formation of 5α-DHT. The enzymes responsible for androgen metabolism in lung fibroblasts are 17β-HSD type 2 and 5α-reductase type 1, the latter having a strong substrate preference for androstenedione, the product of 17β-HSD type 2. In addition, all lung fibroblasts studied have the ability to inactivate androgens into their 17-ketosteroid counterparts regardless of sex  or fetalin vitro but that male and female fibroblasts could modulate the exposure to androgen. To go further into this complex steroid metabolism within the developing lung, in vivo experiments were designed.From this point, we stipulated that androgen could be synthesized by normal male PTII cells 17β-HSD type 5 is expressed in PTII-like cell line A-549  in the m17β-HSD type 2 is expressed in mice  and humaIn summary, 17β-HSD type 5 participates in the production of androgens while 17β-HSD type 2 modulates the paracrine action of testosterone in lung fibroblasts. Thus, androgen synthesis must be a physiological feature in normal lung development and could play an essential role in cell reprogrammation when the emergence of mature PTII cells occur .As the mechanisms of androgen action require the presence of AR, we searched for the distribution of AR within the fetal lung of both male and female.The AR is expressed at similar levels in male and female mouse with no detectable modulation over time . In humaIn conclusion, both male and female express AR and are capable of synthesizing and inactivating androgen in the period overlapping the surge of surfactant. Thus, the lung is not passively exposed to circulating androgens, it is a dynamic tissue with a fine-tuning action on AR occupancy .To make progress, we needed a faster, more precise and reliable morphogenic analysis method. To do so, we developed an automated image analysis program to study the lung development with higher reproducibility, reliability and rapidity than manual analysis. Moreover, a greater portion of the lung and a larger number of samples can be evaluated with this novel method .With this algorithm, we were able to confirm an experimental model of BPD with hyperoxia. Indeed, newborn mice from postnatal (PN) 1 to PN 4 were exposed to 80% oxygen (hyperoxia) and compared with newborn exposed to 21% oxygen (normoxia). The mice exposed to hyperoxia presented drastic changes in density of closed area, a diminution of the relative frequency of closed area under 1,000 mm . These cTo evaluate the contribution of androgens during lung maturation, we treated neonate pups with flutamide during the junction between the saccular and the alveolar stage. Flutamide is a pure antiandrogen that binds to the AR but prevents androgens to have an AR mediated response in the cell . Both noIf removing androgen response is deleterious to the lung, adding them should reverse this effect. Surprisingly, mice exposed to 80% oxygen then treated with exogenous DHT, had similar morphogenic parameters as mice treated with flutamide alone . At 21% In 2004, we proposed that androgens should play a positive role in lung development for both sexes . Since tDuring development, the lungs and particularly the female lungs are not exposed to a significant amount of circulating androgens . NeverthThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Laval University Animal Care Committee.YT heads the laboratory. He has supervised the research work of the laboratory for the last 30 years. He supervised AM-L in the writing of this chapter. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Health and social care workers (HSCWs) are at risk of experiencing adverse mental health outcomes (e.g. higher levels of anxiety and depression) because of the COVID-19 pandemic. This can have a detrimental effect on quality of care, the national response to the pandemic and its aftermath.A longitudinal design provided follow-up evidence on the mental health  of NHS staff working at a remote health board in Scotland during the COVID-19 pandemic, and investigated the determinants of mental health outcomes over time.A two-wave longitudinal study was conducted from July to September 2020. Participants self-reported levels of depression , anxiety  and mental well-being  at baseline and 1.5 months later.The analytic sample of 169 participants, working in community (43%) and hospital (44%) settings, reported substantial levels of depression and anxiety, and low mental well-being at baseline . Although mental health remained mostly constant over time, the proportion of participants meeting the threshold for anxiety increased to 27.2% at follow-up. Multivariable modelling indicated that working with, and disruption because of, COVID-19 were associated with adverse mental health changes over time.HSCWs working in a remote area with low COVID-19 prevalence reported substantial levels of anxiety and depression, similar to those working in areas with high COVID-19 prevalence. Efforts to support HSCW mental health must remain a priority, and should minimise the adverse effects of working with, and disruption caused by, the COVID-19 pandemic. It has been shown that high levels of stress and anxiety among HCWs can decrease staff morale, increase absenteeism and negatively affect quality of care.6Much speculation, anecdotal reports and preliminary evidence have been circulated about how the public health crisis has affected healthcare providers who are directly involved in managing COVID-19.11 Mental well-being has been found to be protective not only against physical disease, but also against the negative effects of workplace stress, absenteeism and accompanying lower productivity.12 Furthermore, mental well-being has been shown to contribute to greater personal resilience, and there is evidence that it can be nurtured in individuals and systems.14 For HSCWs, mental well-being can assist individuals and systems to develop in a positive way, despite the very real adversity and stress that they are facing in working through this pandemic.Most studies on mental health functioning during the pandemic have focused on distress, but the absence of distress (i.e. no depression or anxiety) does not necessarily equate to healthy psychological functioning. We postulate that measuring indices of negative mental health outcomes among HSCWs is of benefit, but that we would do well to do this alongside a measure of positive mental health. The COVID-19 pandemic emerged at a time when there has been increased interest in applying positive psychology and the concept of mental well-being to healthcare workers.7,10 Most have concentrated exclusively on secondary care hospital staff, and there is minimal evidence looking at social care workers and primary care staff.15 This is of particular concern in the UK, where a large proportion of COVID-19 deaths have occurred in care homes. There is also a paucity of longitudinal and positive mental health data on HSCWs, which is important for tracking changes in functioning over time.10 To address some of these gaps in knowledge, this study leverages longitudinal cohort data to provide an assessment of both negative and positive mental health outcomes over time, in a rural National Health Service (NHS) board, looking not just at hospital-based staff, but also incorporating HSCWs from the community. First, we provide an overview of how HSCWs might be coping with working through the COVID-19 pandemic, by tracking mental health outcomes over time. Second, we investigate the sociodemographic determinants of mental health outcomes. This will serve to indicate if any groups of HSCWs should be targeted more specifically to support their mental well-being.There have been several cross-sectional studies during this pandemic that have studied HCWs and their mental health outcomes.All procedures involved in this study complied with the ethical standards of the relevant national and institutional committees. The study was approved by Health Research Authority, National Health System UK . All participants provided online informed consent before starting the online questionnaires. They were informed about the purpose and nature of the study, and they had the right to withdraw their data if they wanted to.N = 225 adult HSCWs to participate in this study. A total of n = 56 participants from time point 1 did not complete the survey at time point 2. In this study, the analytic sample included 169 participants for whom we had time point 1 and time point 2 data. Eligibility criteria were being a UK resident aged 18 years or over and working in NHS Highland as an HSCW during the COVID-19 pandemic.We recruited a sample of Participants were asked to complete a series of demographic and work-related items , followed by the psychological measures (time point 1). These participants were contacted via email to participate a second time (time point 2), which involved completing the same psychological measures.16) was used to measure depression. The nine items ask participants to consider how bothered they have been over the past 2 weeks for each statement (e.g. ‘feeling tired or having little energy’). The questionnaire score ranges from 0 to 27; each question is given a four-point response . The questionnaire has demonstrated diagnostic validity.16 This measure has been used extensively in the UK10 and internationally,17 to measure levels of depression in various population settings during the COVID-19 pandemic. The PHQ-9 was interpreted as follows: normal (0–4), mild (5–9), moderate (10–14), moderately severe (15–19) and severe (20–27) depression. The cut-off score for detecting symptoms of clinical depression was 10.The Patient Health Questionnaire-9 . Each item is scaled from 0  to 3 (‘Nearly every day’), with a total score range of 0–21. The questionnaire has demonstrated diagnostic validity,18 and a number of studies have used the GAD-7 to measure levels of anxiety in various UK and international population settings during the COVID-19 pandemic, including those involving front-line healthcare staff.15 The GAD-7 was interpreted as follows: normal (0–4), mild (5–10), moderate (11–15) and severe (16–21) anxiety. The cut-off score for detecting symptoms of clinical anxiety was 10.The seven-item Generalised Anxiety Disorder-7 (GAD-7)19 and has been used internationally20 and in the UK21 to measure the mental well-being of HSCWs during this pandemic. The WEMWBS was interpreted as follows: the cut-off point of ≤40 is indicative of probable depression, and 41–44 indicates possible depression. Scores of 45–59 represent average mental well-being and scores of ≥60 indicate high mental well-being.Mental well-being was measured with the Warwick–Edinburgh Mental Well-being Scale (WEMWBS). The scale consists of 14-items used to measure subjective well-being and psychological functioning. The wording of each item is positive, and aimed at addressing positive aspects of mental health. Responses are completed with a five-point scale from 1 (‘None of the time’) to 5 (‘All of the time’); the total score ranges from 14 to 70. The WEMWBS has been validated for use in the UK,Both ‘clinical’  and ‘non-clinical’ (administrative) staff were recruited from NHS Highland. Recruitment was supported by NHS Highland Human Resources, general practice managers, NHS Highland social care managers and heads of departments in primary and secondary care, who included links to our study in emails and newsletters. A secondary level of recruitment was conducted on social media: a page for the study was made on Twitter, Facebook and LinkedIn. Interested individuals were directed to a secure data collection website via a weblink, where they first reviewed the study information and gave electronic consent to participate in the study before they completed an online questionnaire. Participant well-being was aligned with ethical committee recommendations of adding an ‘SOS’ button on every page of the online questionnaire, which included local and national mental health support resources, together with the researchers’ contact details. This study was a part of the Scottish Government's Rapid Research into COVID-19, and time restrictions limited recruitment activities to the funding timeframe.22 Time point 2 was approximately 6 weeks later, which occurred from 31 August to 12 September 2020 and coincided with the start of the second wave of the COVID-19 pandemic in Scotland  in response to a rise in cases and hospitality venues.22We collected data at two time periods. The first assessment (time point 1) took place from 15 July to 13 August 2020, with most responses (67%) collected between 15 July and 31 July 2020. During time point 1, there was a significant easing of lockdown measures in Scotland see . During land see , which sThe outcome variables were the three psychological measures described above, measured at time point 1 and time point 2. The independent (or predictor) variables were the demographic and background variables that were measured only at time point 1. The numbers of individuals were tabulated according to the independent variables, and cross-tabulated between the independent variables where the pairwise combination was deemed of interest.Our primary interest was in changes in the psychological scores and in providing a parsimonious model for those independent variables that best predicted each of the changes in the dependent psychological variables. There were three stages to this.24 First, we looked at P-values for univariate models for the changes in each of the three dependent psychological measures (in isolation) with respect to each of the 12 independent variables separately. By looking at the change in the psychological scores, we not only focus on the factor of interest (change), but we also avoid, in a very simple way, the probable difficulty of ‘temporal autocorrelation’. Autocorrelation is the correlation within an individual of their time point 1 score with their time point 2 score. Autocorrelation risks ‘pseudo-replication’, which is when statistical power is erroneously inflated by incorrectly considering correlated replicates as being independent replicates.Stage 1 involved testing each independent variable in isolation as a predictor of the change in each of the three dependent psychological variables.P-values , among those variables with a P-value of <0.05 in their corresponding univariate model from stage 1, and acknowledging that there is an increased risk of a type 1 error, given the number of tests. At this stage we preferred to err on the side of inclusivity. For justifying the retention or otherwise of each of these variables in the multivariable model we used a nested comparison (model with and without the particular independent variable), using an F-test with a threshold of the same arbitrary but commonly used P-value of 0.05.25 Once all of those selected variables had been tested, we then moved onto any remaining variable from the list of those that were considered to be of particular clinical interest24 , and used forward stepwise selection followed by backward stepwise regression to achieve our ‘parsimonious’ model.Stage 2 involved building a parsimonious model of the change in psychological measures dependent on the independent variables, using the results from stage 1. We used forward stepwise regression to add, in sequence, any independent variable according to their Stage 3 is the ‘enriched’ model. In addition to this ‘parsimonious’ model, we wished also to provide an enriched model consisting of the parsimonious model plus all other variables that were considered to be of clinical importance, so that their inclusion was also of interest . These variables were job type, age, being female, previous psychiatric disorder and hours of work. Missing data within the statistical modelling and the statistical nested comparisons of models was approached as follows: when a relevant independent variable had some values that were missing, those observations that were missing in that variable were removed for both the model with that particular independent variable and for the model without, to ensure that the data on which the model was applied was the same in both cases.https://doi.org/10.1192/bjo.2021.1079), with a postgraduate degree or higher (62%) and worked mostly as nurses (28%), doctors (23%), allied health professionals (12%), administrative staff (9%), healthcare assistants (5%) and other HSWCs (18%) (Supplementary Table B1). In terms of setting, community, including primary care and general practices (43%), and hospital (44%) accounted for most of the sample (Supplementary Table E2).A detailed demographic overview of our sample is provided in Supplementary Appendix 1. Participants were mostly female (88%), over 40 years of age (77%) , whereas nurses were more likely to be working in hospitals (Supplementary Table E2). The majority of the participants were not working directly with COVID-19 (76%), and doctors were more likely to be working with COVID-19 than nurses (Supplementary Table E3). Although the majority of participants worked 30–40 h per week (59%), doctors were the most likely to work more than 40 h per week (Supplementary Table B4). The majority of the sample reported that they were disrupted by COVID-19 either moderately (38%) or majorly (39%), with only 2% reporting no disruption (Supplementary Table C1). Work setting did not appear to be associated with the probability of having been disrupted moderately or majorly by COVID-19 (Supplementary Table C1). In our sample, we found no strong associations with participants reporting having previously been diagnosed with a psychiatric disorder (22% of our sample) and other variables (Supplementary Table B2).The original scores of the outcomes are presented as medians with confidence intervals in 16 at time point 1, and it remained constant at 30.2% at time point 2. Of note here is that a further 35.5% of the sample reported symptoms in keeping with mild depression (scores ranging from 5 to 9 on the PHQ-9) at time point 1 and 37.9% at time point 2.18 at time point 1, increasing to 27.2% at time point 2. Of note here is that 30.2% of the sample reported symptoms in keeping with mild or subclinical anxiety (scores ranging from 6 to 10 on the PHQ-9) at time point 1 and 31.4% at time point 2.In our sample, 20.1% reported scores in keeping with probable anxietyOn the WEMWBS, our sample reported average (45%) and high (5.9%) mental well-being at time point 1, with a slight decrease in average (43.2%) and high (5.3%) mental well-being at time point 2. At time point 1, 31.9% of participants reported scores in keeping with probable depression (scores of ≤40 on the WEMWBS), and 17.2% of participants reported scores in keeping with possible depression (scores of ≤44) on the WEMWBS. At time point 2, these reported scores remained constant for probable depression (31.4%), with a slight increase in possible depression (20.1%). In our sample, mental well-being was strongly negatively correlated with depression and anxiety .Table 215 Univariate associations between the risk factor (independent variables) and the change in the dependent variables  can also be found here. The results of the 36 univariate models (36 pairs = 12 predictors×3 outcomes) are presented in Supplementary Appendix 2, Table 3. Two out of the 36 pairs had P-values of <0.05. These were working with COVID-19 and being disrupted by COVID-19 .Summary statistics for the main risk factors are provided in Supplementary Tables A–F1 within the appendixes. A pairwise cross-tabulation is presented for combinations of demographic variables that were of prior interest (Supplementary Tables A–F1).The forward stepwise regression led to the choice of our parsimonious models –6. The rThe selected multivariable model of change in well-being scores over time is presented in P = 0.01).An ANOVA test of this model with and without the change in the ‘disruption resulting from COVID-19’ variable confirmed that, even with the addition of these additional predictors, the variable ‘disruption resulting from COVID-19’ was statistically significant .The selected parsimonious model for depression is presented in P = 0.07), and none of the tested independent variables were associated with significant changes in anxiety. These data suggested that anxiety was not statistically significantly influenced by or associated with time or any of the measured variables.The overall change in anxiety between time point 1 and time point 2 was not significantly different from zero  in mental well-being. Working with COVID-19 was an important factor in change (increase) in depression measures between time point 1 and time point 2.10 Evidence from previous epidemics,26 together with pre-pandemic data, indicate a high and persistent burden of psychological distress among HSCWs,8 placing them at risk for exacerbated adverse mental health outcomes during this pandemic.5 Our findings corroborate the existing literature by reporting substantial levels of probable depression and anxiety in our HSCW cohort over time. Our results add to the existing literature by indicating that HSCWs working in areas outside of COVID-19 hotspots experience levels of adverse mental health outcomes in keeping with those working in COVID-19 hotspots. Previous studies have identified determinants of mental health outcomes,10 and our results add longitudinal evidence that groups at increased risk of adverse mental health outcomes are those working directly with COVID-19 and those whose work roles have been disrupted by the pandemic.The aim of this study was to examine the mental health functioning of rural Scottish HSCWs during the COVID-19 pandemic over time. To this end, we tracked changes in mental health outcomes over two time points and explored the determinants of those outcomes in a cohort of HSCWs working in the Scottish Highlands. Other large-scale studies in areas with high COVID-19 infection rates have generally reported an increase in the prevalence of adverse mental health outcomes  in this population during the COVID-19 pandemic.10 that persisted over time. Although there was no statistically significant change in participants’ levels of anxiety, depression and mental well-being between two time points in summer and autumn of 2020, we found category changes of clinical interest in our cohort's levels of anxiety  and mental well-being (decreasing from average to low). Participants meeting the cut-off for probable anxiety increased from 20.1% at time point 1 to 27.2% at time point 2.Our analytic sample reported relatively high levels of anxiety and depressive symptoms28 Public Health Scotland published a rapid review of the impact of the COVID-19 pandemic on population mental health.29 Although the review found little good-quality longitudinal evidence to suggest changes in the prevalence of population-level mental health outcomes caused by the pandemic, it identified health and social care staff as an at-risk group of adverse mental health outcomes. Although direct comparison of the mental health of our cohort and the general Scottish population during the pandemic is currently limited, our results are suggestive that our cohort's levels of depression were broadly in line with those seen internationally for healthcare workers during the pandemic.17Levels of self-reported symptoms in line with probable depression did not change between time points 1 and 2 . These findings were corroborated in our cohort's mental well-being scores, where we found that over 30% of HSCW who responded to our survey reported low levels of mental well-being consistent with probable clinical depression over both time periods. This finding suggests that levels of probable depression and mental well-being for HSCWs in the Scottish Highlands during the COVID-19 have been worse than in the general population of Scotland during the pre-pandemic period.17 conducted a systematic review and meta-analysis on the mental health impact of COVID-19 on health workers, the general population and patients at high risk of COVID-19.17 They reviewed 62 studies published between 1 November 2019 and 25 May 2020 and included 162 639 participants from 17 countries. The pooled prevalence of anxiety was 33% and the pooled prevalence of depression was 28%. Studies from China, Turkey, Spain, Italy and Iran reported higher than pooled prevalence of anxiety and depression among health workers compared with the general public.17 However, this finding has not been replicated across all contexts; in Germany, for example, Skoda et al found that healthcare professionals showed less anxiety and depression than non-healthcare professionals.30 Respondent data from the Scottish Highland sample suggest levels of depression broadly in line (slightly higher) with the international pooled prevalence and slightly lower than the average for anxiety. This study also adds to current literature, confirming that anxiety and depression symptoms are a concern for the healthcare service in the Scottish Highlands during this pandemic. In Scotland, there has been a steady increase in the proportion of the adult population who report two or more symptoms of depression since the Scottish Health Survey began reporting data on this measure (from 8% in 2010–2011 to 11% in 2016–2017).27Our data also permits comparison with the point-prevalence levels of depression and anxiety among HSCWs during the COVID-19 pandemic nationally and internationally. Luo et al8 Of note is that baseline data was collected during a period of easing of lockdown restrictions, and follow-up was completed at the start of the second wave of infections, when lockdown measures were reintroduced in Scotland. The timing of data collection is likely to have had an effect on mental health rates. Our results, together with previous studies,31 suggest that changes in mental health outcomes during the COVID-19 pandemic might depend on the timing of assessments within particular contexts, as well as the population groups assessed. Other UK studies have found that the general population reported worse levels of psychological health during the initial ‘shock’ of the pandemic, followed by consistent trends toward pre-pandemic levels of depression and anxiety.31 Although we did not collect data on this sample from before the COVID-19 pandemic, we speculate that front-line staff may find it particularly difficult to return to pre-pandemic levels of psychological health. In contrast to what was found in the UK general population, our study reported sustained substantial levels of depression and anxiety, and low levels of mental well-being over time. The prolonged second wave in the UK, high levels of hospital admissions, persistent social distancing regulations, and the added pressure of providing a nationwide vaccination program together with managing increasing pressure from non-COVID-19 health issues on the health service leads to concerns about worsening mental health for our HSCWs.This paper also adds, to a limited degree, a longitudinal perspective, demonstrating that our cohort's symptoms remained fairly stable over time and that there is a general trend toward worsening mental health outcomes with regard to anxiety and mental well-being. These findings are of concern, as sustained symptoms of low mental well-being and anxiety are more likely to lead to long-term psychological disorders, and are likely to contribute to absenteeism and low morale in the workplace.A further aim for this study was to identify determinants our population's mental health outcomes over time. The two variables that were statistically significant risk factors over time, and when accounting for confounding factors, were disruption caused by COVID-19 and working directly with COVID-19. This appears to confirm concerns that the pandemic itself is contributing to poor mental health among HSCWs.15 This may be exacerbated by a fear of infection (to self and others), which has also been found to be a risk factor in previous studies.15 Our study corroborated previous research, showing that working directly with COVID-19 was significantly associated with higher rates of depression. These findings could point toward the use of monitoring the mental health and providing additional psychological support for departments that work directly with COVID-19.15Working directly with patients with COVID-19 has been found to be a risk factor for poor mental health outcomes in previous studies.32 In Scotland there have, in the same time period, been 245 744 cases at a rate of 4498 per 100 000 population,32 and in the UK as a whole there have been 4.54 million cases at a rate of 6824 per 100 000 population.33 Unlike our cohort, the majority of studies thus far have studied urban, secondary care populations of HCWs in areas of high COVID-19 rates.15 In a recent study, Lamb et al examined the mental health in a large sample of healthcare staff working in areas with high COVID-19 rates.10 Our study's findings suggest levels of depression and anxiety similar to those reported in COVID-19 hotspots.10 This suggests that, although direct contact with patients with COVID-19 is a risk factor, there are likely to be other, indirect factors contributing to adverse mental health outcomes in this cohort outside of being a HSCW, and the likelihood thereof should not be negated in our study's results; for example, at the beginning of time point 1 (15 July) there were 772 new cases of COVID-19 in England, and at the end of time point 1 (13 August) there were 1138 cases. During time point 1 the average number of new COVID-19 cases in England per day was 835.33 In Scotland, at the beginning of time point 1 (15 July) there were 16 new cases of COVID-19, and at the end of time point 1 there were 52 cases. The average number of daily cases in Scotland during time point 1 was 28 .32 In NHS Scotland, the average number of daily hospital admissions attributed to COVID-19 during time point 1 was 3.0. In NHS Highland at the beginning of time point 1 there were zero new cases of COVID-19 recorded, and at the end of time point 1 there were two new cases. The average number of daily cases in NHS Highland during time point 1 was 0.5 .32 At the beginning of time point 2 (31 August) there were 1501 new cases of COVID-19 in England, and at the end of time point 2 (12 September) there were 2656 cases. During time point 2 the average number of new COVID-19 cases in England per day was 2912.23Although it is of interest that this study confirms direct contact with patients with COVID-19 as a risk factor for poor mental health outcomes in HSCWs, it is important to note that NHS Highland is a region with relatively low numbers and rates of COVID-19 infection, and the majority of our cohort (76%) did not work directly with COVID-19. From the beginning of the COVID-19 pandemic to 12 June 2021, NHS Highland has recorded 5419 cases of COVID-19 in total, at an infection rate of approximately 1614 per 100 000 population (the fifth lowest rate in the UK).32 In NHS Highland, at the beginning of time point 2 there were seven new cases of COVID-19 recorded, and at the end of time point 2 there were 15 new cases. The average number of daily cases in NHS Highland during time point 2 was 6.6 .32 Across NHS Scotland, the average number of daily hospital admissions attributed to COVID-19 during time point 2 was 10.32In Scotland, at the beginning of time point 2 (31 August) there were 144 new cases of COVID-19, and at the end of time point 2 (12 September) there were 207 new cases. The average number of daily cases in Scotland during time point 2 was 178 .Although there was an increase in COVID-19 incidence and hospital admission between time point 1 and time point 2 in the UK as a whole, in Scotland and in NHS Highland, the Scottish Highlands were not directly affected to a significant degree by clinical cases of COVID-19 during either period addressed in this study. NHS Highland serves a large but widely dispersed population, most of whom live far from the major metropolitan areas most affected by the pandemic. It may be that this relative degree of isolation represented a protective factor, although our paper was not designed to investigate this hypothesis. Given the relatively low local incidence and prevalence of COVID-19 during the study period, it may be that the increased incidence in poor mental health observed was also a result of the general societal impact of the pandemic.15 but that it was rather disruption resulting from COVID-19 itself that played a significant role in negatively affecting mental health. This is suggestive that systemic factors could have played a larger role in our cohort than individual factors, and has implications for policy, which often places emphasis on individual-level interventions.Self-reported subjective levels of disruption may go some way toward explaining the high levels of depression, increases in anxiety and decreases in mental well-being in our cohort. Our findings show that disruption resulting from COVID-19 was significantly associated with decreases in mental well-being over time. This suggests that the degree to which staff feel they are disrupted can impact their mental health – and that it is not necessarily correlated with actual exposure to patients with COVID-19 or levels of COVID-19 within the health board area. It is also notable that individual factors, such as gender, age or workload, did not have as great an effect on our cohort's mental health as reported in other studies,11 In addition, there has been some evidence from this current pandemic that higher levels of personal resilience were associated with lower rates of negative mental health outcomes in HCWs.15 Although future studies would do well to identify possible protective factors, and the interplay between mental well-being and resilience, emphasis on enhancing personal mental well-being should not divert responsibility onto an individual's to simply ‘cope better’ with a challenging working environment.Although this study did not identify independent factors protective of adverse mental health outcomes in our sample, we did observe mental well-being to be strongly negatively correlated with depression and anxiety at both times of measurement. Although mental well-being is seen as an umbrella concept incorporating various positive psychological constructs, it is its nurturing link to resilience that appears to be of importance for HSCWs during this pandemic. Recently, there have been calls to incorporate resilience training in medical education.34 described increased anxiety and depression scores among individuals who accessed high levels of pandemic-related media coverage, and it is possible that this may have contributed to our findings.Our study population were not working in a region with a high clinical burden of COVID-19 during the period under review. Healthcare workers in NHS Highland were nevertheless subject to lockdown measures, and most experienced disruption in their workplaces through service reconfiguration and infection control measures taken to help minimise transmission. Given the clinical need and common human impulse to access up-to-date information about the pandemic, it is likely that HSCWs in our study will have made use of COVID-19-related media, although this is not something we ascertained directly. Neill et al35 As such, the respondent sample represents approximately 2% of all staff employed by the local health board. Although 88% of respondents were female, this does not differ dramatically from the gender composition of the whole HSCW workforce in NHS Highland.35 The longitudinal aspect of the collected data was limited, and the identified trend toward worsening anxiety outcomes should be interpreted with caution. Although the maximum interval between measurements was 63 days, the follow-up period for most participants was approximately 40 days. Furthermore, those suffering from poor mental health may have been more likely to complete the surveys and thus potentially introduce self-selection bias into the findings. A further potential bias could be a result of attrition: participants who dropped out at follow-up, which could have affected the study's estimates. Participants were asked to self-report on their mental health, potentially introducing reporting bias. These potential biases, together with the small sample size and short follow-up period, places limitations on the generalisability of these findings. Additional longitudinal research that emphasises methodological rigor, including the use of standardised diagnostic interviews to establish mental health diagnoses, is necessary to better understand the mental health burden and identify those most at risk for adverse mental health outcomes in HSCWs.Findings from the present study must be interpreted in light of its limitations. NHS Highland provides care for a population of 320 000 people over a wide geographical area, and employs around 10 000 staff.13 Although individual-level interventions that foster mental well-being and resilience may be beneficial, there is a need for wider, structural adaptations if we are to support the mental health of our HSCWs effectively. This could lead to resilient working systems, not just resilient individuals.36 Rigorous further longitudinal data are needed to respond to the potential long-term mental health effects of the COVID-19 pandemic on HSCWs.In conclusion, our findings reveal that levels of anxiety and depression are a concern not only among HSCWs working in COVID-19 hotspots, but also for those in more remote settings like the Scottish Highlands. In contrast to what was observed in the general population, where studies found an improvement of mental health symptoms over time, our cohort's relatively high levels of anxiety and depressive symptoms persisted over time, raising concerns that this population may face immediate and ongoing adverse mental health consequences. Our findings suggest that although HSCWs with prolonged and high exposure to patients with COVID-19 need mental health support, it is also important not to overlook the negative mental health effects on all HSCWs. Mental health support is needed across different working contexts, and interventions to help staff cope with, understand and negotiate feelings of disruption may be beneficial.


There were 12 (10%) current smokers, an abnormal chest X-ray was found in 106 (92%) patients and distance walked in the 6-min walking test (6MWT) ranged from 30–750 m (mean 452 ± 120); 24 (21%) patients walked less than 400 m. Time taken to perform the post-TB assessment, including the 6MWT, ranged from 8–45 min (mean 21 ± 8 min). In 98% of the completed questionnaires, health workers stated that conducting post-TB assessments was feasible and useful. This study shows that post-TB assessments can be conducted under routine programmatic conditions and that there is significant morbidity that needs to be addressed.There is growing evidence that a substantial proportion of people who complete anti-tuberculosis treatment experience significant morbidity and mortality which can negatively affect their quality of life. It is suggested that national tuberculosis programs conduct end-of-treatment assessments, but whether this is feasible is currently not known. We therefore assessed whether tuberculosis program staff could assess functional and general health status of patients at the end of treatment in five TB clinics in four provinces in China. There were 115 patients, aged 14–82 years, who completed anti-tuberculosis treatment and a post-TB assessment. There were 54 (47%) patients who continued to have symptoms, the commonest being cough, dyspnea and fatigue. Symptom continuation was significantly more common in the 22 patients with diabetes ( Worldwide, tuberculosis (TB) remains a major global public health problem and one of the top 10 causes of death overall [Despite a reported treatment success rate of 85% for people newly enrolled on anti-TB treatment , a signiAvailable data also suggest that people successfully treated for TB have significantly increased mortality following treatment compared with the general population. In 2004, a study in rural Malawi revealed that 65% of registered TB patients had died within 7 years after anti-TB treatment [Given the current burden of post-TB disease, morbidity and mortality, patients with TB would merit being assessed for general health status at the end of anti-TB treatment. We have previously argued that an end-of-treatment assessment needs to be carried out within the context of a national TB program (NTP), using a simple check list that documents on going symptoms, co-morbidities, social determinants and lung function, with appropriate actions taken if disability is identified ,8. HowevTo address this knowledge gap and draw government attention to post-TB disease and quality of life of TB survivors, we carried out a simple and rapid post-TB assessment at the end of anti-TB treatment under programmatic conditions in TB centers in China. The specific objectives were to determine: (i) whether a simple questionnaire and protocol to evaluate symptoms, signs, laboratory investigations and functional status of TB patients who had successfully completed anti-TB treatment was feasible to be carried out by the established healthcare staff, (ii) the findings of the evaluation in terms of the general health status of the patients at the end of anti-TB treatment and (iii) how long it would take for the healthcare staff to complete the questionnaire and perform a six-minute walking test (6MWT).This was a cross-sectional study carried out within the routine health services in China, and patients were prospectively recruited.Five hospitals and outpatient clinics in mainland China were selected. They included the Infectious Disease Hospital of Heilongjiang Province and No. 8 Hospital of Xinjiang Medical University, representing hospitals in the urban areas; Shantou Institute of TB Control and Prevention, representing an urban outpatient clinic; Xingguo County Institute of TB Control and Prevention in Jiangxi Province and the TB Clinic at Shule County Hospital in Xinjiang, representing outpatient clinics in rural areas. The selection of the sites was based on broad geographical coverage, sufficient numbers of TB patients registered each year and willingness of staff to attend to the post-TB work without additional resources. All the sites that were approached agreed to participate. The location of the five sites is shown in Patients included persons aged 14 years and above who had been diagnosed and registered with TB and who had just completed standard anti-TB treatment in one of the five TB hospitals or clinics between April and June 2021 (the second quarter of 2021).A simplified evaluation form was developed based on the potential lung damage caused by TB, lifelong comorbidities and measures outlined in the WHO Policy on Prevention of Cardiovascular Disease . The conThe 6MWT was performed outdoors. For those hospitals or clinics with a large garden or open square, the attending physician or nurse directed the patients to walk around a track for 6 min using a mobile phone or simple watch to measure the time. For a clinic with no outdoor facility, the 6MWT was performed on the side of the street and patients were directed to walk back and forth on a measured track. Distance walked in meters was measured using a pedometer or a known length of track.We measured length of time in minutes for the health care staff to complete the questionnaire and perform the 6MWT. However, we did not include length of time to have blood taken for fasting blood glucose or receipt of results by the patient nor length of time for chest radiography or its interpretation by a radiologist. Finally, we invited the healthcare staff to tell us if the post-TB assessments were feasible and useful within their established working environment.p < 0.05).Individual patient data were received on a monthly basis and cross-checked by the head of each implementing site and the principal investigator (YL). Data were then entered to an Excel file and analyzed. Categorical data were presented as frequencies and proportions and continuous data as means and standard deviations (SD). Comparisons between groups were made using the chi-square test with levels of significance set at 5%  TB patients underwent post-TB assessment, but 13 (10%) were not included in the analysis due to incomplete data. There were, therefore, 115 patients who were assessed and had complete data. Their ages ranged from 14–82 years with a mean (SD) of 44 (±19) years. Details of the number of patients who completed treatment, were assessed and had complete data available in the questionnaire at the five health facilities are shown in p = 0.027) and in the 12 patients who had been previously treated for TB (p = 0.008). There were no differences in the proportion with symptoms between those from urban and rural communities (p = 0.19) and between those treated in hospitals or in out-patient clinics (p = 0.99): patients with drug-resistant TB were mainly treated in hospital, and they had no symptom differences compared with those who had drug-susceptible TB.The results of the post-TB evaluation are shown in p = 0.52), between those with drug-susceptible and drug-resistant or multidrug-resistant TB (p = 0.41) or between patients presenting to hospitals or outpatient clinics (p = 0.91).An abnormal chest radiograph at the end of treatment was found in 106 (92%) patients. Over one third of patients had either unilateral or bilateral abnormalities, and between 10–20% had shadowing, cavitation or scarring. There were no significant differences in the proportion of patients with chest radiographic abnormalities between those with and without diabetes . There were 24 (21%) patients who walked less than 400 m during the six minutes. Of these, there were 13 males and 11 females with an age distribution as follows: 14–29 years (6); 30–49 years (3); 50–59 years (4); 60–69 years (4); and 70–82 years (7). There were no significant associations between gender, age group, post-TB assessment symptoms, diabetes status or current smoking status and having a 6MWT less than 400 m (data not shown).The time taken to complete the post-TB assessment questionnaire, including the 6MWT, ranged from 8–45 min (mean 21 ± 8 min). The mean time to perform the post-TB assessment by staff in Shule County Hospital (mean 26 ± 12 min) was longer than the time in the other sites (mean 15 ± 4 min). These times did not include the performance of the blood-tests and receipt of their results or the chest radiographic examinations and their interpretation.This is the first report from China to assess the feasibility and findings of conducting post-TB assessments at the end of treatment under routine programmatic conditions. The established healthcare workers in China carried out the additional work within the routine TB services without any extra resources needing to be brought in. In 85% of patients completing treatment, a post-TB assessment was carried out and in the majority of these, questionnaires were completed and the data fully documented, with 20 min on average being needed to complete the task including the 6MWT. Almost all the interviewed healthcare workers felt that these post-TB assessments were useful.For many decades, most NTPs have traditionally focused on how to increase case detection and how to ensure high treatment success rates, and these aspirations have been fully in line with global strategies to combat and end TB ,11. DespOur findings, however, indicate that nearly half of the patients successfully completing treatment did not feel healthy and were symptomatic, with cough, shortness of breath and fatigue each occurring in 10–20% of those assessed. These findings are in line with recent studies from Nigeria and South Africa showing that a high proportion of TB patients completing treatment have poor health-related quality of life scores, particularly amongst those aged > 40 years and those with more marked radiographic abnormalities ,17. The In our post-TB evaluation, we investigated for comorbidities and enquired about determinants closely associated with TB. Diabetes mellitus increases the risk of TB by a factor of 2–3 times compared with the general population . Those wCigarette smoking, excess alcohol consumption and injecting drug use are all associated with an increased risk of TB ,25,26. WWhile there appears to be no association between hypertension and risk of TB , we feltOver 90% of our patients had some degree of chest radiographic abnormality when the chest radiographs were conducted at the end of anti-TB treatment. This proportion seems high, but our findings are in line with those reported from Mexico, Italy and Zimbabwe ,13. SimiIn the absence of traditional lung function equipment and pulmonary spirometry, we decided to measure the patient’s functional status by a 6MWT. A study in India attested to its simplicity and value in predicting mortality in patients with chronic lung disease . The IndThe strengths of our study were that we attempted to conduct post-TB assessments on consecutive patients completing anti-TB treatment within the routine programmatic setting in China. We used the established staff and no special resource package was introduced to support implementation. Our findings therefore reflect on, and have relevance for, routine TB care in China, demonstrating that it is feasible to conduct such evaluations and that these evaluations do not take excessive time.There were a number of limitations in the study. The study sample was small and we only selected five clinics. Unfortunately, we did not obtain the reasons for why some patients were not assessed and why 10% of those who were assessed had incomplete questionnaires. Having this information might have given us more insight into feasibility issues. We did not collect information about who had bacteriologically confirmed TB and clinically diagnosed TB, and this might have been useful in assessing post-TB treatment morbidity. In terms of co-morbidities, we did not include the measurement of HIV status and this was because HIV-prevalence in TB patients is <2% in China [Despite, these limitations, our study has a number of programmatic implications. Firstly, our post-TB assessment checklist needs to be used elsewhere in the country for more experience in the feasibility of its use and implementation. Attention needs to be paid to having all patients fully assessed on completion of anti-TB treatment, in order for TB patients to potentially benefit from further care. During this expansion, more thought is also needed about its various components. Are we collecting enough information on symptoms, determinants and co-morbidities? Is a chest radiograph needed at the end of anti-TB treatment? Should the 6MWT be better refined in terms of how it is conducted and in terms of generating national reference values for gender, age and height? How in the programmatic context does the 6MWT correlate with more sophisticated lung function tests, and is it a good indicator of the need for pulmonary rehabilitation? In all of these considerations, it is important to balance our wishes for comprehensiveness against the feasibility of busy front-line staff conducting these assessments under routine conditions.Secondly, while our study did not address or document the further care and assistance that is needed for patients who are still disabled and in need of help at the end of anti-TB treatment, this is an important issue in need of further work. Patients with comorbidities such as diabetes need to be referred back to appropriate care and those with determinants such as smoking need to be helped to quit. Patients with respiratory symptomatology and functional disability should be considered for pulmonary rehabilitation. A Ugandan study showed that a pulmonary rehabilitation package in adults with post-TB lung disease, which was supervised by physiotherapists using aerobic and resistance exercises which were performed later at home, was associated with significant improvements in quality of life, exercise tolerance and respiratory outcomes . PulmonaFinally, this first feasibility study in China paves the way for implementation of the “Fourth 90”, that states “ensuring that 90% of all people successfully completing treatment for TB can have a good health-related quality of life” . The “foThis study, carried out under routine program conditions in patients completing standard anti-TB treatment in five clinics in China between April and June 2021, showed that post-TB assessments were feasible to conduct, they could be conducted in an average time of about 20 min and the health care workers involved found them useful. About half of the patients who were fully assessed had on-going symptoms, the most common being cough, shortness of breath and fatigue. Over 90% had some chest radiographic abnormality at the end of treatment and one fifth had a 6MWT below 400 m. There is significant morbidity amongst TB patients completing anti-TB treatment and this needs to be addressed if we are to honor the Lancet Commission pledge of providing good quality patient-centered care.