PRAME gene expression and its biological and clinicopathological significance in gliomas.PRAME  is a biomarker studied in various human cancers. Little is known about the biological implications of PRAME in glioma. We aimed to perform a comprehensive analysis to explore We accessed the human cancer atlas (TCGA) database to collect glioma patients (n = 668) with primary tumors and gene expression data. Single nucleotide variants, copy number variation, DNA methylation data, and other clinicopathological factors were also extracted for the analysis.PRAME gene, respectively. The principal component analysis and pathway analyses showed that PRAME-positive gliomas (n = 498), which consisted of tumors with PRAME low expression and overexpression, expressed different oncogenic profiles, possessing higher activity of Hedgehog, P3IK-AKT-mTOR, and Wnt/β-catenin pathways (p<0.001). DNA methylation analysis also illustrated that PRAME-positive tumors were distributed more densely within a grade 4-related cluster (p<0.001). PRAME positivity was an independent prognostic factor for poor outcomes in a multivariate cox analysis adjusted for clinical characteristics and genetic events. Kaplan-Meier analysis stratified by revised classification showed that PRAME positivity was solely associated with IDH-wildtype glioblastoma, grade 4. Finally, PRAME-overexpressing cases (n = 14) had the worst clinical outcome compared to the PRAME-negative and PRAME-low cohorts (adjusted p<0.001) in pairwise comparisons.Overall, 170, 484, and 14 tumors showed no expression, low expression (FPKM≤1), and overexpression (FPKM>1) of the PRAME expression statuses may dictate different biological and clinicopathological profiles in IDH-wildtype glioblastoma. PRAME gene and PRAME protein can be practically evaluated by pigmented lesion assay (PLA) and immunohistochemistry (IHC), respectively . In this study, 476 tumors in DNA methylation space can be relatively divided into two unsupervised tSNE clusters, small  and large clusters (left and upper part) . The small cluster densely consisted of PRAME-low and PRAME-overexpressing samples while the larger cluster had a significant portion of PRAME-negative samples . The revised subtype , CIMP clusters , and IDH status  were strongly associated with these 2 clusters. Fig 2E shows a heatmap of distribution of PRAME expression status within CIMP clusters. There were significant difference in the distribution of PRAME expression status . This discrimination can be seen in LGm6-GBM (6/12 vs. 7/484 vs. 0/170), classic-like (1/12 vs. 63/484 vs. 4/170), and mesenchymal-like (2/12 vs. 87/484 vs. 9/170).In the study cohort, there were a total of 476 tumors with available data about 450K DNA methylation. We performed t-SNE dimension reduction to explore the differences in the distribution of Table 1 summarizes the clinicopathological characteristics of PRAME-negative and PRAME-positive gliomas. Clinically, patients with PRAME-positive gliomas were older (p<0.001). There were no differences in gender (p = 0.419) and race (p = 0.382). Comparisons of revised classification between PRAME-negative and PRAME-positive cohorts showed that IDH-mutant astrocytoma, grade 2 (26.5% vs. 13.1%) and oligodendroglioma (35.9% vs. 21.7%) dominated PRAME-negative glioma while the incidence of IDH-wildtype glioblastoma, grade 4 (32.9% vs. 4.1%) was much higher in PRAME-positive glioma. These differences were significant (p<0.001). Regarding genetic abnormalities, PRAME-positive tumors more frequently acquired EGFR amplification , CDKN2A/B homozygous deletion , and 7 gain 10 loss chromosomal aberrations  than PRAME-negative tumors. Conversely, IDH1/2 mutations  and ATRX mutations  were significantly more common in PRAME-negative cases.PRAME was a general marker of prognosis in the entire studied cohort . Stratified by the new WHO classification, there were no significant results in IDH-mutant astrocytoma grade 2 , grade 3 , and grade 4 . However, PRAME positivity was of prognostic significance in IDH-wildtype glioblastoma grade 4 . There was no obvious survival difference between PRAME-positive and PRAME-negative oligodendrogliomas. We also compared the survival outcomes of IDH-mutant/1p19q codeletion and IDH-mutant/non-1p19q codeletion  but PRAME positivity was not related to the prognosis. Table 2 shows multivariate analyses adjusted for clinical characteristics, whole genome sequencing (WGS) , whole exome sequencing (WES) (TERT promoter mutaton), and copy number variation information . The prognostic effect of PRAME positivity was significant and independent to clinical characteristics , WGS , and CNV  features.In survival analysis, PRAME overexpression when FPKM > 1, which was observed in only 14 gliomas within the entire studied cohort. Table 3 shows the clinicopathological characteristics of these 14 patients. Notably, only 1 case (7.1%) of this cohort was IDH-mutant. Next, we performed KM analysis to compare the overall survival of PRAME-negative (n = 170), PRAME-low (n = 481), and PRAME-overexpressing (n = 14) cases (PRAME-low and PRAME-overexpressing comprise PRAME-positive cohort) . To avoid false-positive results, pairwise log-rank test comparisons were conducted, using Benjamini-Hochberg correction to calculate the adjusted p-values. Even with a small sample (n = 14), PRAME-overexpressing glioma showed a significantly worse outcome than PRAME-low (adjusted p<0.001), and PRAME-negative (adjusted p<0.001). 2-year overall survival rates of PRAME-negative, PRAME-low, and PRAME-overexpressing cohorts were 92.8% (95%CI = 88.1% - 97.8%), 64.0% (95%CI = 59.2% - 69.1%), and 13.8% (95%CI = 2.6% - 73.3%), respectively.First, we defined PRAME is associated with cytotoxic T-cell activation and killing in glioblastoma Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Partly**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: In this manuscript, Khang Le et al. explore the publicly available human cancer atlas to reveal the role of PRAME gene expression and its biological and clinicopathological significance in gliomas and established it as a potential prognostic biomarker for glioma.The study has enormous translational importance, as they justified the prognostic signature in potential study cohorts for further clinical applications.The authors should address the following issues to consider this manuscript, primarily standard. However, the description is sometimes concise, and the authors should provide more details.Minor comments:1.The general description of pertaining to glioma vs glioblastoma should be mentioned inintroduction section of the manuscript.2. The author should discuss the role of IDH1-mutation in the prognosis of glioma.3. Should make a diagram of signaling pathways that are upregulated and downregulatedin PRAME-positive gliomas and PRAME-negative gliomas.Reviewer #2: In this manuscript, the authors investigated and claimed that PRAME positivity is an independent prognostic factor for poor outcomes. They suggested that its over-expression can indicate different biological and clinicopathological profiles, such as age, grade, and EGFR amplification, in adult glioma. High PRAME positivity was found to be solely associated with wtIDH glioma, glioma DNA methylation landscape, and poor outcome. While PRAME is an important molecular and clinicopathological marker for different cancers including adult glioma, the reviewer believes that the present work lacks novelty as similar conclusions have already been established in previous publications. Furthermore, the present work lacks a mechanistic approach, and several aspects of the manuscript still need to be properly addressed."Major concerns:1. It has already been established in previous publications that PRAME is expressed in adult glioma and is associated with poor prognosis.    The present study primarily utilized public databases, and no immunohistochemical data were provided to support their claim. Please provide reasons to support the uniqueness and novelty of this article compared to previous publications.2. According to the WHO 2021 guidelines, wtIDH gliomas are clustered together in grade 4, including diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, and glioblastoma NOS. The present study claims a positive correlation between PRAME and wtIDH glioma, but it doesn't clearly explain the correlation between PRAME and these wtIDH gliomas. It is important to mention the expression status of PRAME in these specific groups of gliomas.3. PRAME is highly immunogenic and serves as a robust target for immunotherapy. Certain drugs, such as Decitabine, can increase PRAME expression to enhance T-cell-mediated toxicity against GBM, which contradicts the findings of the present study. . The present study lacks an exploration of the immunological aspects of PRAME in gliomas. It is important to investigate the correlation between different immunological populations, pro-inflammatory signaling cascades, and PRAME.4. Among 665 patients, 170 patients were PRAME-negative, and 495 patients were PRAME-positive. Moreover, within the PRAME-positive population, 481 had low PRAME expression, and only 14 had high PRAME expression. The study compared the PRAME-positive and -negative populations for various biological and clinicopathological parameters. However, the study didn't explain the biological and clinicopathological differences between the low and high PRAME-expressing populations and their relationship with patient survival. It would be worthwhile to compare the biological parameters between the 481 low and 14 high PRAME-expressing sample sets. Additionally, the number of samples in the high PRAME-expressing set is too small to draw conclusions. Increasing the sample size for the high PRAME-positive population is advisable.5. In the Results section, the authors mentioned that PRAME-positive tumors expressed higher activity in the PI3K-AKT-mTOR, P53, and IL2-STAT5 pathways in gliomas. However, Fig 1C doesn't correlate with this statement, as it shows upregulation of KRAS, TGFb, and Notch signaling pathways in PRAME-positive tumors. Additionally, in Fig 3A, 3B, and 3C, it seems that the PRAME-positive population has longer survival than the PRAME-negative population, although statistical significance is lacking possibly due to the small sample size. Similarly, in Fig 4 and Supplementary Fig A & B, the PRAME low-expressing population tends to have longer survival than the PRAME-negative population. In Fig 3D, PRAME-positive mutant IDH1/2 shows higher survival than PRAME-negative wt-IDH1/2 population. Please provide a proper explanation of the data.6. It has already been established that PRAME overexpression can inhibit the growth of breast cancer and leukemia. However, PRAME overexpression in glioma is associated with poor outcomes. The present study compared the hallmarks of apoptosis between PRAME-positive and -negative adult glioma but found no differences in apoptotic hallmarks between the two groups. Please explain these findings in detail with underlying mechanisms. Also, the specific role of PRAME in retinoic acid signaling in glioma malignancy is unknown. Since the present study lacks a mechanistic approach, it is important to consider the correlation of PRAME expression with retinoic acid signaling status in wtIDH gliomas.7. Histone methylation or acetylation status, along with other epigenetic modifications, are important hallmarks of glioma development and progression. The present study did not establish any correlation between PRAME expression and histone methylation or acetylation status in glioma. Additionally, what is the correlation between G-CIMP and PRAME in glioma?8. Radioresistance and chemoresistance are major obstacles in the successful treatment of adult glioma. Therefore, it would be advisable to investigate the correlation between PRAME expression and treatment resistance properties in adult gliomas.Minor concerns:1. Please add the references at the end of the third sentence in the Introduction section.2. Please mention the figure/table numbers in the Results section at the end of the statements (a) "The association between PRAME expression and WHO grades was statistically significant" and (b) "Conversely, IDH1/2 mutations (87.9%) and ATRX mutations (39.8%) were significantly more common in PRAME-negative cases" under the sub-heading entitled "PRAME-positive gliomas had more advanced grades and adverse outcomes."3. Properly mention the title of the X-axis in Fig 3A-D.4. On the title page, it should be ORCID, not ORCHID. Please correct this typo error.5. Please thoroughly check the manuscript for grammatical and typographical errors.6. Please rephrase the second sentence of the Discussion section, as it appears confusing to readers."**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  17 Jul 2023To the editors: Thank you for processing our manuscript. In this revised version, we made modifications to the author board and analyses. Although there many alterations added, there are 3 main changes.First, we regret to inform you that we removed our co-author, Kathryn Eschbacher, from the author board. It should be noted that there was no conflict of interest in this project and this removal was made solely based on the personal reasons of this author.Secondly, we re-run the analyses by using our published preprocessing pipeline [PMID: 37253389] and additionally used data cbioportal for a higher quality of data. The results were similar but the data presentation can be different due to different random seeds of analysis. All the results related to Figures and Tables were revised accordingly.Thirdly, we modified Table 2, focusing on 4 distinct multivariate analyses of PRAME status adjusted for clinical characteristics, genetic mutations (WGS-available data), TERT promoter mutations (WES-available data), and copy number alterations . The reason for these modifications is due to the heterogeneity of data availability of different data types . Stratified survival analysis of PRAME status by new classifications was done independently by Kaplan-Meier analyses. Reviewer #1: In this manuscript, Khang Le et al. explore the publicly available human cancer atlas to reveal the role of PRAME gene expression and its biological and clinicopathological significance in gliomas and established it as a potential prognostic biomarker for glioma. The study has enormous translational importance, as they justified the prognostic signature in potential study cohorts for further clinical applications.Thank you for reviewing our manuscript.The authors should address the following issues to consider this manuscript, primarily standard. However, the description is sometimes concise, and the authors should provide more details.Minor comments:1. The general description of pertaining to glioma vs glioblastoma should be mentioned in introduction section of the manuscript.We added a few sentences describing the context of WHO classification regarding glioma and glioblastoma in Introduction as follows:“Adult gliomas are a heterogeneous and common group of brain cancers with unclear cell-of-origin [5]. The biological profile of gliomas has been studied with respect to histology, epigenetic, genetic characteristics, cell-of-origin, and tumor microenvironment . There are highly diverse oncogenic mechanisms contributing to gliomagenesis and tumor progression, including Wnt/𝛽-catenin [8], PI3K/Akt/mTOR [9], TGF-𝛽 [10], and mesenchymal transition, among many others [11]. Important genetic abnormalities affecting the prognosis of glioma patients consist of IDH1/2 mutations, CDKN2A/B homozygous deletion, EGFR amplification, TP53 mutations, ATRX mutations, TERT promoter mutations, and 7 gain 10 loss chromosomal abnormalities [12]. The recent World Health Organization (WHO) classification of Tumours of the Central Nervous System (CNS) emphasizes that glioma can be divided by IDH mutation and 1p/19q codeletion status. IDH-wildtype astrocytoma has more advanced clinicopathological progression and tumor with grade 4 is referred to as the “glioblastoma” category. Glioblastoma is diagnosed by the absence of IDH mutation and one of the high-grade features, including high-grade morphology, TERT promoter mutation, 7 gain/10 loss chromosomal abnormality, or EGFR amplification. However, the prognostic factors of glioma are still under investigation.”2. The author should discuss the role of IDH1-mutation in the prognosis of glioma.Thank you for your comments.We added more details about IDH mutation in Introduction to clarify the background in the previous comment.3. Should make a diagram of signaling pathways that are upregulated and downregulatedin PRAME-positive gliomas and PRAME-negative gliomas.We added Figure 1D that describes pathways that are down-regulated or up-regulated regarding the PRAME expression status.  Reviewer #2: In this manuscript, the authors investigated and claimed that PRAME positivity is an independent prognostic factor for poor outcomes. They suggested that its over-expression can indicate different biological and clinicopathological profiles, such as age, grade, and EGFR amplification, in adult glioma. High PRAME positivity was found to be solely associated with wtIDH glioma, glioma DNA methylation landscape, and poor outcome. While PRAME is an important molecular and clinicopathological marker for different cancers including adult glioma, the reviewer believes that the present work lacks novelty as similar conclusions have already been established in previous publications. Furthermore, the present work lacks a mechanistic approach, and several aspects of the manuscript still need to be properly addressed."Thank you for reviewing our manuscript.Major concerns:1. It has already been established in previous publications that PRAME is expressed in adult glioma and is associated with poor prognosis.    The present study primarily utilized public databases, and no immunohistochemical data were provided to support their claim. Please provide reasons to support the uniqueness and novelty of this article compared to previous publications.Thank you for bringing these studies to our attention. We believe that the purpose of the aforementioned studies is different from our present work. We focused on the biological and clinicopathological investigation of PRAME expression in a more comprehensive way, including epigenetic, genetic, and gene expression. We added a paragraph to discuss this issue as follows:“The significance of PRAME expression has been mentioned previously. Wu et al [20] developed a PRAME-containing formula for risk score, which was inferred from the regression model. This score quantified the risk of Karnofsky performance score, but not the prognosis itself. Therefore, the inference of PRAME prognostic value is plausible but weak. The other study by Zhang et al [21] mainly compared the PRAME expression between different types of brain tumors, including subtypes of astrocytic and non-astrocytic tumors. However, it can be difficult to conclude that PRAME expression is an independent prognostic factor by the current evidence. On the other hand, the goal of our study is to focus on the biological and clinicopathological characteristics of PRAME. Multiple analyses were performed to provide more concrete proof of the significance of this gene in glioma.”2. According to the WHO 2021 guidelines, wtIDH gliomas are clustered together in grade 4, including diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, and glioblastoma NOS. We believe that Glioblastoma, IDH-wildtype is defined when there is one or more of the following features: microvascular proliferation, necrosis, TERT promoter mutation, EGFR gene amplification, and 7+/10- chromosome copy-number changes according to WHO classification 5th edition. Therefore, astrocytic tumors with IDH wildtype status and no other feature are ill-defined and should be classified as Astrocytoma, NOS. Therefore, we revised the classification in a more detailed way, following our recently published paper [PMID: 37253389].We added details about our revised data processing pipeline as follows:“Data processinghttps://www.cbioportal.org) datasets that are related to TCGA-GBM and TCGA-LGG projects, including (1) Brain Lower Grade Glioma , (2) Glioblastoma Multiforme , and (3) Merged Cohort of LGG and GBM . This difference led to a slight inconsistency in the total number of glioma patients.” The Human Cancer Atlas (TCGA) database consists of many datasets. We extracted cases from the TCGA-GBM and TCGA-LGG projects. Only cases with available gene expression profiles (GEP) and primary tumors (no recurrent or metastatic tumors) were included in the study. To adapt to the new WHO classification, cases with grades 2/3 in the previous studies  were reclassified into grade 4 as follows: (1) the presence of both IDH1/2 mutation and CDKN2A/B homozygous deletion, or (2) the absence of IDH1/2 mutations and the presence of at least one of the following abnormalities: TERT promoter mutation, EGFR amplification, and 7 gain 10 loss chromosomal abnormality. Other histopathological grading features such as microvascular proliferation and pseudopalisading necrosis were assumed to be included in previously evaluated grade IV gliomas of the original studies . Tumors with the absence of IDH1/2 mutations and no other high-grade morphological and genetic features mentioned above were reclassified as Astrocytoma, Not Otherwise Specified (NOS). Tumors with the presence of both IDH1/2 mutations and 1p/19q codeletion were categorized as Oligodendroglioma. Mixed glioma was re-distributed into new categories based on IDH1/2 mutation and 1p/19q codeletion status. This reclassification was published in our previous paper [15]. The difference in data processing between this study and our previous one was that we used cbioportal for cancer genomics  to illustrate the PRAME expression status in each revised category of glioma.3. PRAME is highly immunogenic and serves as a robust target for immunotherapy. Certain drugs, such as Decitabine, can increase PRAME expression to enhance T-cell-mediated toxicity against GBM, which contradicts the findings of the present study. . The present study lacks an exploration of the immunological aspects of PRAME in gliomas. It is important to investigate the correlation between different immunological populations, pro-inflammatory signaling cascades, and PRAME.Thank you for your information.We added a section of analysis named “Analysis of the association between tumor microenvironment (TME) and PRAME expression” as follows:“Analysis of the association between tumor microenvironment (TME) and PRAME expressionGiven that PRAME is associated with cytotoxic T-cell activation and killing in glioblastoma [15], we compared the immunologic cell population between PRAME-negative and PRAME¬-positive gliomas. GSVA of cell type-specific gene sets [16] was performed to calculate the activity (ES) of 17 cell types, including B cells, T cells, T helper, Th1, Th2, TFH, Th17, Treg, CD8 T cells, T gamma delta, cytotoxic cells, NK cells, dendritic cells, eosinophils, macrophages, mast cells, and neutrophils . There were increased activities of T cells (p<0.001), Th2 (p<0.001), Th17 (p<0.001), cytotoxic cells (p=0.028), macrophages (p<0.001), and neutrophils (p<0.001) in PRAME-positive gliomas while there was reduced activities of TFH (p<0.001) and CD8 T cell (p<0.001).”We also added a corresponding discussion paragraph:“PRAME can also be associated with glioma TME. A previous study showed that Decitabine can increase PRAME expression and, thus, enhance the T-cell-mediated cytotoxicity, which makes PRAME an interesting target for immunotherapy [15]. However, TME of cell line can be difficult to interpret because the stromal or microenvironment context of cancer in vivo is different from that of cell line condition. Our study showed that PRAME higher expression was related to increased cytotoxic cell, macrophage, and neutrophil activity but it was also associated with many immune modulating cells such as Th2, Th17, and TFH. Therefore, PRAME expression is in a complicated relationship with many immunologic cell populations, not only cytotoxic T cells.”4. Among 665 patients, 170 patients were PRAME-negative, and 495 patients were PRAME-positive. Moreover, within the PRAME-positive population, 481 had low PRAME expression, and only 14 had high PRAME expression. The study compared the PRAME-positive and -negative populations for various biological and clinicopathological parameters. However, the study didn't explain the biological and clinicopathological differences between the low and high PRAME-expressing populations and their relationship with patient survival. It would be worthwhile to compare the biological parameters between the 481 low and 14 high PRAME-expressing sample sets. Additionally, the number of samples in the high PRAME-expressing set is too small to draw conclusions. Increasing the sample size for the high PRAME-positive population is advisable.Thank you for your suggestion. However, it is quite difficult to define a cutoff of PRAME overexpression. This is emperically determined by the data at hand and defined by >1, >3, or >5 FPKM. For example, a previous study used FPKM>1 to define higher expression [PMID: 27577089]. Therefore, we believe that it is better to follow these recommendations. In our data, a vast number of cases had PRAME read count<1. Therefore, >1 FPKM is an appropriate cutoff for determining high expression or overexpression.As the reviewer mentioned, the sample size of PRAME-overexpressing cohort was small to create reliable results. Therefore, to avoid multiple hypothesis problem, we only performed survival analysis for this cohort. We additionally applied Benjamini-Hochberg correction for the pairwise comparison in this survival analysis to solidify the result. The analysis of other biological parameters, which requires a lot of hypothesis testing, can be performed but it can lead to false positive results and misleading conclusions. Therefore, we believe that it is better not to over-do the investigation of the PRAME-overexpressing cohort.5. In the Results section, the authors mentioned that PRAME-positive tumors expressed higher activity in the PI3K-AKT-mTOR, P53, and IL2-STAT5 pathways in gliomas. However, Fig 1C doesn't correlate with this statement, as it shows upregulation of KRAS, TGFb, and Notch signaling pathways in PRAME-positive tumors. Additionally, in Fig 3A, 3B, and 3C, it seems that the PRAME-positive population has longer survival than the PRAME-negative population, although statistical significance is lacking possibly due to the small sample size. Similarly, in Fig 4 and Supplementary Fig A & B, the PRAME low-expressing population tends to have longer survival than the PRAME-negative population. In Fig 3D, PRAME-positive mutant IDH1/2 shows higher survival than PRAME-negative wt-IDH1/2 population. Please provide a proper explanation of the data.Thank you for pointing out this observation. In Figure 3A, PRAME-positive and PRAME-negative glioma patients had difference in survival patterns (p<0.001). We concur with you that the PRAME-negative cohort survived better in the >10 years of follow-up. Unfortunately, we do not have a clear explanation for this observation. Although it seems that PRAME-negative cohort deceased sooner than PRAME-positive cohort in the period of >10 years of follow-up, there may be many confounding factors because of the long follow-up time. Another confounding factor is sample size. The size of PRAME-positive cohort was much larger than PRAME-negative one. Therefore, the positive cohort was likely to have more outliers than the negative cohort. Generally, first years of follow-up are more reliable prognostic indicator compared to longer term of follow-up. To avoid this confusing phenomenon, we limited the x-axis from 0 to 10 years of follow-up, which eliminates such observation of the outliers.6. It has already been established that PRAME overexpression can inhibit the growth of breast cancer and leukemia. However, PRAME overexpression in glioma is associated with poor outcomes. The present study compared the hallmarks of apoptosis between PRAME-positive and -negative adult glioma but found no differences in apoptotic hallmarks between the two groups. Please explain these findings in detail with underlying mechanisms. Also, the specific role of PRAME in retinoic acid signaling in glioma malignancy is unknown. Since the present study lacks a mechanistic approach, it is important to consider the correlation of PRAME expression with retinoic acid signaling status in wtIDH gliomas.Thank you for your suggestion. We experimentally run pathway analysis to investigate the activity of retinoic acid signaling (the full gene set of this pathway is in the link below). The enrichment score (ES) of PRAME-negative and PRAME-positive glioma were compared by two-sample independent t-test, which was similar to other pathways. There was no significant difference between PRAME-negative and PRAME-positive samples (p=0.446). Therefore, the role of PRAME in glioma may not be related to its suppression of retinoic acid signaling. However, we believe that it is better not to include this analysis into the manuscript because this analysis may not be relevant to the main stream of analysis and it can also cause confusion to readers.The link to retinoic pathway: https://www.gsea-msigdb.org/gsea/msigdb/human/geneset/REACTOME_SIGNALING_BY_RETINOIC_ACID.html7. Histone methylation or acetylation status, along with other epigenetic modifications, are important hallmarks of glioma development and progression. The present study did not establish any correlation between PRAME expression and histone methylation or acetylation status in glioma. Additionally, what is the correlation between G-CIMP and PRAME in glioma?Thank you. The lack of histone modification data is the limitation of the present study. We added a few sentences to the Discussion as follows:“However, there were limitations in the present study. First, selection bias was a potential problem because this study used a public database. Second, our findings of PRAME positivity in IDH-mutant glioma were not significant potentially due to small samples, and, thus, sampling error. Therefore, a larger study of PRAME expression on IDH-mutant glioma can be helpful to examine the biological and clinicopathological relevance of PRAME positivity in these brain tumors. Third, protein expression data was not fully available and, therefore, cannot be analyzed. PRAME gene expression may be different from PRAME protein expression, which can be practically evaluated by immunohistochemistry. Hence, immunohistochemical studies are required to validate PRAME prognostic significance at the protein level. Additionally, data about histone modification is not available in TCGA-LGG and TCGA-GBM projects. Therefore, we were not able to analyze the relationship between PRAME expression and this epigenetic regulation. Finally, to our knowledge, there was no available information of chemotherapy and radiotherapy resistance in TCGA datasets. Hence, we were not able to investigate the relationship between PRAME expression and treatment response.”In addition, we created Figure 2E to demonstrate the relationship between G-CIMP and PRAME expression in glioma.To avoid confusion, we eliminate the analysis of t-SNE methylation clusters. We instead used the published methylation CIMP clusters and performed chi-square test. We modified the methylation Results section as follows:“In the study cohort, there were a total of 476 tumors with available data about 450K DNA methylation. We performed t-SNE dimension reduction to explore the differences in the distribution of PRAME-negative and PRAME-positive glioma in DNA methylation hyperspace. This DNA methylation space can be interpreted as the reduced representation of the CpG methylation landscape. We also included the Glioma CpG Island Methylator Phenotype (G-CIMP), which was published in a previous paper [33941250]. In this study, 476 tumors in DNA methylation space can be relatively divided into two unsupervised tSNE clusters, small  and large clusters (left and upper part) . The small cluster densely consisted of PRAME-low and PRAME¬-overexpressing samples while the larger cluster had a significant portion of PRAME-negative samples . The revised subtype , CIMP clusters , and IDH status  were strongly associated with these 2 clusters. Figure 2E shows a heatmap of distribution of PRAME expression status within CIMP clusters. There were significant difference in the distribution of PRAME expression status . This discrimination can be seen in LGm6-GBM (6/12 vs. 7/484 vs. 0/170), classic-like (1/12 vs. 63/484 vs. 4/170), and mesenchymal-like (2/12 vs. 87/484 vs. 9/170).”8. Radioresistance and chemoresistance are major obstacles in the successful treatment of adult glioma. Therefore, it would be advisable to investigate the correlation between PRAME expression and treatment resistance properties in adult gliomas.Thank you for your comment. However, there was no information about chemotherapy and radiotherapy resistance in the TCGA datasets. Therefore, we listed your comments as a limitation of our study.Minor concerns:1. Please add the references at the end of the third sentence in the Introduction section.We added citations at the end of the third sentence in Introduction section.2. Please mention the figure/table numbers in the Results section at the end of the statements (a) "The association between PRAME expression and WHO grades was statistically significant" and (b) "Conversely, IDH1/2 mutations (87.9%) and ATRX mutations (39.8%) were significantly more common in PRAME-negative cases" under the sub-heading entitled "PRAME-positive gliomas had more advanced grades and adverse outcomes."Thank you. The referred paragraph was written as a description of Table 1 and it is mentioned in the first sentence of this paragraph:“Table 1 summarizes the clinicopathological characteristics of PRAME-negative and PRAME-positive gliomas.”3. Properly mention the title of the X-axis in Fig 3A-D.We changed the title of the x-axis from “Time” to “Follow-up Time (Years)”.4. On the title page, it should be ORCID, not ORCHID. Please correct this typo error.We corrected this error.5. Please thoroughly check the manuscript for grammatical and typographical errors.Thank you for your comments.6. Please rephrase the second sentence of the Discussion section, as it appears confusing to readers.We modified the sentence “In gene expression analysis, we explored PRAME read counts and found that PRAME expression was generally low in these brain tumors. A significant number of tumors showed no PRAME expression.” into “In gene expression analysis, there was a large number of gliomas showing no PRAME expression while a few numbers of tumors possessed high levels of PRAME expression. The remaining tumors generally showed low PRAME expression.”AttachmentResponse_R1.docxSubmitted filename: Click here for additional data file. 11 Aug 2023Molecular and clinicopathological implications of PRAME expression in adult gliomaPONE-D-23-03723R1Dear Dr. Kondo,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Syed M. Faisal, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 25 Sep 2023PONE-D-23-03723R1 PRAME expression in adult glioma Molecular and clinicopathological implications of Dear Dr. Kondo:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Syed M. 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From 2019 to 2020, the PDH/ED households improved food security by 12.6 pp and diversified crop production (bananas (9.7 pp), papaya (11.1 pp), carrots (3.8 pp) and lemons (5.9 pp)), and increased the proportion of annual income ≥60,000 Taka by 12.4 pp and last month income ≥5000 Taka by 7.8 pp, compared to PDH group . However, there was no impact on child nutritional status, morbidity, livestock ownership and total annual/last income. Incorporating an ED program into nutrition programming could benefit food security and dietary diversity in rural Bangladesh.Given the high prevalence of child undernutrition in Bangladesh, multi‐sectoral approaches involving livelihood promotion have potential to mitigate the burden of undernutrition. This study examined the impact of an economic development (ED) program providing poultry assets, gardening skills and saving training added to the Positive Deviant (PD)/Hearth program (PDH/ED), compared to PD/Hearth only (PDH). A total of 1029 children who attended PD/Hearth sessions in September–November 2018 at 6–13 months of age were enrolled in the cohort study in July–August 2019. The cohort, comprised of 532 children in the PDH/ED group and 593 children in the PDH group, was reassessed in November 2020. The program impact on child nutrition, food security, crop production, dietary quality and household income was estimated using a difference‐in‐differences approach accounting for the sociodemographic differences between PDH/ED and PDH groups. Compared to the PDH group, the PDH/ED group showed increases in child dietary diversity score (DDS) (+0.32), child minimum dietary diversity (13.7 percentage points [pp]), and maternal DDS (+0.28)  program receiving poultry assets, gardening skills and saving trainings added to the Positive Deviant/Hearth program (PDH/ED) in rural Bangladesh, compared to the PD/Hearth (PDH) only on child nutrition and health. Our results demonstrated that, compared to the PDH group, the PDH/ED group increased child and maternal dietary quality, improved household food security, diversified crop production and increased the proportion of households earning income above the extreme poverty line. Incorporating asset provision and saving training into nutrition programming would help households safeguard the dietary needs of women and young children.Future nutrition‐sensitive approaches aimed at improving nutritional status should identify the point along the impact pathway where the program impact is likely to be weakened and incorporate appropriate complementary measures accordingly.Program strengthening is needed so that mothers can use food resources and household income to increase the consumption of animal protein and micronutrient‐rich foods for child growth. Specifically, the study examined if the ED program added to the PD/Hearth program enhanced the impact on reducing child underweight and morbidity, improving child and maternal dietary diversity, ensuring household food security, increasing crop production activities, and improving income generation, compared to exposure to the PD/Hearth program only.22.1The BRDMCN program was implemented in three upazilas , Joypurhat, Panchbibi, and Dhamoirhat in the Rajshahi division from March 2018–December 2020  less than − 1.0 in the community. The screened mothers and children with WAZ score < −1 attended a 2‐week, community facilitator‐led Hearth session. Through these Hearth sessions, the community facilitators transferred knowledge of healthy behaviors based on Positive Deviant Inquiry findings, which included: breastfeeding, complementary feeding, hygiene practices, caring, health‐seeking, and family planning. The participating mothers prepared and fed their children complimentary meals using local and low‐cost ingredients that are dense in micronutrients . Following the Hearth sessions, community facilitators conducted 2–3 home visits over 2 weeks to support mothers to continue practicing what they learned from Hearth sessions at home. Community facilitators weighed the children who attended Hearth sessions at enrollment, 12 and 30 days, and 3 and 6 months after the Hearth session. A total of 11,984 children enrolled in PD/Hearth sessions in BRDMCN. A nested research study based on weight monitoring data of approximately 6500 PD/Hearth children indicated that PD/Hearth participation held a greater benefit for children enrolled at a younger age   from the first and second batches in September–November 2018  among children in the PDH/ED group compared with the PDH group following 2 years of program implementation (2018–2020). Secondary outcomes were the difference between the PDH/ED group and the PDH group in the proportion of child and maternal dietary diversity score (DDS)/minimum dietary diversity (MDD), household food security, household crop production/livestock rearing activities, annual and monthly income (Bangladesh Taka) and child morbidity from 2019 to 2020.2.5www.kobotoolbox.org) was used to administer a tablet‐based survey. Interviewers were recruited by the survey firm in 2019 and recruited by World Vision in 2020. Researchers conducted a 3‐day training, and pilot tests were administered before the household survey. In 2019, the interviewers collected sociodemographic data, anthropometrics, diet outcomes, food security, crop production, livestock possession and child morbidity data. Child height and mid‐upper arm circumference were not measured in 2020 due to the reinforced social distancing.The research team initially developed paper‐based questionnaires, which were translated into Bangla and transferred to an online survey tool. The questions were reviewed by researchers and WVB's nutrition and agriculture experts for content and appropriateness. The project team hired a survey firm in Dhaka to conduct the surveys and the research team trained the survey firm interviewers and partnered with the implementation team to supervise data collection in the field. A KOBO tool  module was used to assess household food insecurity   and lemon. Rice and beans are the type of crops commonly harvested in the study area. The items of mango, papaya, lemon, and DGLV were based on the assets provided through the ED program. We assumed that the households who received assets would expand their farming/gardening activities into other crop items. Mothers were asked if they owned any livestock and how many cows, goats, lambs, chickens and/or ducks the household currently owned. These livestock species were commonly found in the project area.Child morbidity 2 weeks ahead of the survey was assessed by caregivers’ recall of the following symptoms: (1) diarrhea (≥4 loose watery stools); (2) acute respiratory infection ; and (3) high‐grade fever.Mothers in the study were asked about total income in the past year and in the past month. These income variables respectively were categorized into binary variables using the following cut‐offs: ≥60,000 Taka ($720) versus <60,000 Taka and ≥5000 Taka ($60) versus <5000 Taka. The cut‐off of 5000 Taka for monthly income and 60,000 Taka for annual income was estimated based on an extreme poverty line of $1.90 per day in 2011 purchasing power parity  and percentages for all variables, including outcomes. Comparability of sociodemographic characteristics was tested between PDH group and PDH/ED group with data collected in 2019 using p‐value of 0.05 was considered statistically significant.A difference‐in‐differences (DID) analysis was used to estimate the program impact for 1 year from 2018 to 2020 on primary outcomes (child underweight and WAZ) and from 2019 to 2020 on secondary outcomes  between two different exposures (PDH vs. PDH/ED group). The DID approach based on linear regression included an interaction term for the time of survey  and program exposure . The impact estimation in the DID regressions was adjusted for child age, gender, maternal age, father's occupation and education, and household building materials that differed in PDH and PDH/ED groups in 2019. All DID models accounted for clustering within individuals using the “cluster” option to obtain robust standard errors. All data analyses were done using STATA version 17 SE , not being met after three times of household visits (n = 18), child death (n = 4), dual ID at Hearth session (n = 16), refusal (n = 1) and other reasons (n = 19). The mean (SD) age was comparable in both the PDH only and the PDH/ED group, 19.3 (SD: 2.4) months and 19.4 (SD: 2.5) months, respectively  but the number of follow‐up visits by community facilitators was slightly higher in the PDH/ED versus PDH (10.5 times vs. 9.2 times) (Table 3.3p < 0.001) and maternal level of education (p < 0.001), in addition to better household building materials  , while the majority of mothers were either <25 years or 25–35 years of age in similar proportions. In both groups, the majority had ownership of the house, 96.7% in the PDH group and 93.6% in the PDH/ED group. Compared to the PDH/ED group, the PDH group had better socioeconomic characteristics, such as a higher paternal level of education p < 0.00 and mate3.4z‐score; p = 0.56) and underweight  was not significant in the PDH/ED group compared to the PDH group from 2018 (before intervention participation) to 2020. A drastic change is notable in nutritional status during the follow‐up. The prevalence of underweight decreased from 83.6% to 18.6% in the PDH and from 92.8% to 24.7% in PDH/ED group in 2019 (Table The program impact on WAZ (0.026 p = 0.96) and mothers  (Table p = 0.01), MDD by 13.7 percentage points (pp) (p < 0.001), and the mother's DDS by 0.28 score (p = 0.03), compared to the PD program only, after adjusting for socioeconomic differences in 2019 between two groups. Higher increases, although not significant, in consumption were found in some food groups among children and mothers in the PDH/ED group  in the PDH/ED group in 2020, compared to the PDH group (Table p < 0.001), papaya , carrot  and lemon , compared to the PDH group. There was no significant difference in the amount of crops produced between two groups (Table p = 0.40) and a significant decrease in the possession of duck livestock  in the PDH/ED group compared to the PD group (Tables The proportion of food‐secure households increased by 12.6 pp (up Table . Crop prps Table . There wp Tables .p = 0.62) and in the past month income (p = 0.64) also did not show significant improvement in the PDH/ED group, compared to the PDH group (Table p = 0.008) in the PDH/ED group, compared to the PDH group. The PDH/ED program showed no significant differences in the impact on child morbidity, compared to the PDH group  can impact child nutrition, maternal and child diet, household food security and income generation, compared to the PD/Hearth program only (PDH). After a year of follow‐up, we found the PDH/ED group significantly increased the dietary diversity of children and mothers, promoted household food security, diversified crop production and increased the proportion of the past year income  and the past month income  compared to the PDH group. However, there was no significant impact on child nutritional status, household livestock management, or total income in the past year and past month.4.1The ED program improved food security, dietary diversity, crop production and household income earning above the poverty line, but had no impact on child underweight. This result implies that the impact of ED program operated along the anticipated impact pathway  consumption, a staple in South Asia, increased notably in both children  and women  in PDH/ED compared to the PDH group .Improvement in food security and household income led to improved dietary diversity among children and mothers. The program impact on food security (12.6 pp) was mainly derived from a substantial reduction in the food‐secure households among the PDH group (63.6% in 2019–52.7% in 2020), while the proportion of food‐secure households in the PDH/ED group remained at 54% during the same period Table . In addiThe proportion of households that earned annual income  and monthly income (≥5000 Taka) above the extreme poverty line increased in the PDH/ED group compared to the PDH group. This finding implies that the ED program could give greater financial benefits to the extremely poor than the PDH program alone. Additional evidence supporting the results is that annual income from selling ducks and eggs during 2019 was USD 29 (IQR: 12–56) among 1200 duckling beneficiaries , while those who were not provided with chicken adopted chicken rearing gradually (0%–46.6%) (Volpenhein et al., No lack of observed difference between groups in the number of poultry currently owned can be explained by a few reasons. The rearing scope of ducks varied seasonally and could be reduced in early winter due to scarcity of pond water and feeding resources and depending on the market price of duck (Khan et al., 4.5Our research reveals the high potential of the ED program to improve child nutrition along the impact pathway of nutrition‐sensitive agricultural programming SPRING, . Future The sustained impact of SBCC approaches including PD/Hearth is likely to be weakened under the COVID‐19 lockdown. However, programs ensuring food security such as the ED have the potential to complement the nutritional impact of SBCC activities. Many low‐income countries, such as Malawi, adopted food distribution and transfer payments to mitigate the direct impact early in the pandemic and to ensure that people had adequate food access (Jiang et al., 4.5.1Our large sample size, the presence of a comparison group, and the parallel approach in the evaluation process of an ongoing program contributed to the strength of our study. However, our study has its limitations.First, except for weight measurement at PD/Hearth sessions, other key data began to be collected in 2019, 1 year after program implementation, when the cohort was generated. The present study, therefore, did not have a “true baseline” as our first measured outcomes were most likely to have shown improvements since program implementation. This limitation influenced our ability to compare to the endline.Second, height and other anthropometric measurements that require physical contact were not collected due to social distancing restrictions due to COVID‐19. Weight‐for‐age measures were collected according to PD/Hearth guidelines; hence, we could not measure the long‐term program impact on stunting by 2020. Weight is more sensitive to environmental influence.Third, the study participants were not randomized into the intervention groups and were not comparable in socioeconomic status. The households in the PDH/ED group had poorer socioeconomic status as the asset transfer targeted the poor or ultrapoor. This limitation was adjusted for in estimating the program impact by adjusting for socioeconomic and demographic factors. However, it cannot totally exclude the possibility that the results may have been affected by socioeconomic status. Fourth, there is a probability of recall bias in the information collected on the amount of crop produced and monthly or annual income.5Our study found that adding an ED program to the PD/Hearth program benefits food security, dietary diversity, crop diversification and income among the poor with children at risk of undernutrition, compared to the PDH program only. Our study provides supporting evidence that asset transfer programs based on a graduation approach may also potentially offer some protection against economic shock but need to be strengthened to make an impact on nutrition outcomes.Yunhee Kang and Yoonho Cho designed the study and prepared the proposal. Md. Iqbal Hossain, Jaganmay P. Biswas, and Shinhye Min conducted field data collection and data management. Yunhee Kang analyzed data. Yunhee Kang and Indira Prihartono wrote the manuscript. IH, Jaganmay P. Biswas, Shinhye Min, HYK, Yoonho Cho, Seungheon Han, and Hee Sun Kim reviewed the manuscript and substantially contributed to the interpretation of results. Yunhee Kang had primary responsibility for final content. All authors read and approved the final manuscript.The authors declare no conflict of interest.Supporting information.Click here for additional data file.


Glycogen storage disease type 1a (GSD Ia) is an inborn error of metabolism caused by a defect in glucose-6-phosphatase (G6PC1) activity, which induces severe hepatomegaly and increases the risk for liver cancer. Hepatic GSD Ia is characterized by constitutive activation of Carbohydrate Response Element Binding Protein (ChREBP), a glucose-sensitive transcription factor. Previously, we showed that ChREBP activation limits non-alcoholic fatty liver disease (NAFLD) in hepatic GSD Ia. As ChREBP has been proposed as a pro-oncogenic molecular switch that supports tumour progression, we hypothesized that ChREBP normalization protects against liver disease progression in hepatic GSD Ia.G6pc knockout (L-G6pc−/−) mice were treated with AAV-shChREBP to normalize hepatic ChREBP activity.Hepatocyte-specific Hepatic ChREBP normalization in GSD Ia mice induced dysplastic liver growth, massively increased hepatocyte size, and was associated with increased hepatic inflammation. Furthermore, nuclear levels of the oncoprotein Yes Associated Protein (YAP) were increased and its transcriptional targets were induced in ChREBP-normalized GSD Ia mice. Hepatic ChREBP normalization furthermore induced DNA damage and mitotic activity in GSD Ia mice, while gene signatures of chromosomal instability, the cytosolic DNA-sensing cGAS-STING pathway, senescence, and hepatocyte dedifferentiation emerged.In conclusion, our findings indicate that ChREBP activity limits hepatomegaly while decelerating liver disease progression and protecting against chromosomal instability in hepatic GSD Ia. These results disqualify ChREBP as a therapeutic target for treatment of liver disease in GSD Ia. In addition, they underline the importance of establishing the context-specific roles of hepatic ChREBP to define its therapeutic potential to prevent or treat advanced liver disease.The online version contains supplementary material available at 10.1186/s40170-023-00305-3. G6PC1 (G6pc in mice), G6Pase-α) [Glycogen Storage Disease type Ia (GSD Ia) MIM#232,200) is a rare inborn error of metabolism (IEM) caused by mutations in the gene encoding for the catalytic subunit of glucose-6-phosphatase  is the major glucose-sensitive transcription factor in hepatocytes , and ChRThese previous studies from our laboratory and others suggest context-specific roles of hepatic ChREBP in advanced liver disease, in particular hepatocellular tumour susceptibility, which are of critical importance to establish the therapeutic potential of ChREBP for the treatment of liver disease in GSD Ia patients. In the current study we therefore investigated the impact of prolonged ChREBP normalization on liver disease progression in hepatic GSD Ia mice. Our data show that normalization of hepatic ChREBP activity sensitizes liver-specific GSD Ia mice to advanced liver disease development, DNA damage, cellular senescence, as well as hepatocellular proliferation and dedifferentiation, suggesting increased susceptibility for hepatocarcinogenesis.See G6pc-floxed Alb-Cre negative (B6.G6pclox/lox) and G6pc-floxed Alb-Cre positive (B6.G6pclox/lox.SAcreERT2/w mice) on a C57BL/6 J background were infected with shRNAs directed against ChREBP (AAV-shChREBP) or a scrambled control (AAV-shScramble (shSCR)) (1 × 1012 particles per mouse) by intravenous injection into the retro-orbital plexus under isoflurane anaesthesia [G6pc-deficient mice (L-G6pc−/−) and wildtype littermates (L-G6pc+/+). Nonfasted animals were sacrificed for tissue collection at 8AM at 10 or 25–26 days after the last treatment (dpt). Two shChREBP/L-G6pc−/− mice were euthanized at 21 dpt because a humane endpoint was reached, yet were included in the analyses. Absolute liver weight of the 10-day follow-up study cohort has previously been reported [Male adult esthesia . At 11–1See See See See 2, 3 mM Mg(Ac)2, and 0.1 mM EDTA, with fresh addition of 1 mM DTT and 0.1% Triton X-100). In short, a 50–100 µm filter was placed on a 50 mL Falcon tube, and liver powder was poured onto the filter. Liver powder was stepwise and gently homogenized in 1.5 mL lysis buffer and pushed through the filter using a 5 mL syringe plunger. The resulting 1.5 mL nuclear suspension was transferred to a 1.5 or 2.0 mL tube, and supplemented with ~ 25,000 control cells (diploid human GFP-positive cells). Nuclei were spun down at 500xg for 5 min at 4 °C and the pellet was resuspended in 300–1000 µL PBS/BSA with Hoechst/PI DNA dyes (10 μg/mL for both). Nuclei were filtered through 35 µm FACS tubes and analysed on the Canto FACS machine (BD Biosciences) for ploidy analysis.For analysis of hepatocyte ploidy, ~20 mg of frozen powdered liver tissue was used for nuclei isolation (as described ) in lysiP values < 0.001 , 0.001 to 0.01 , and 0.01 to 0.05  were considered significant.Data in figures is presented as dot plots with median ± interquartile range (IQR), unless stated otherwise. Data in tables is presented as median (range), unless stated otherwise. Data in heatmaps represent z-score normalized values. Statistical analysis was performed using BrightStat and GraphPad PRISM software. Differences between multiple groups were tested by a Kruskal Wallis H-test followed by post-hoc Conover pairwise comparisons. G6pc−/− mice, while the number of γH2Ax-positive hepatocytes tended to increase  induced , and hepatic glycogen contents were positively correlated  and Bsep (Abcb11)  across different study cohorts (n = 64). In the current study cohort, plasma bile acids levels also positively correlated with Ctgf mRNA levels ). Combined, these data indicate that prolonged hepatic ChREBP normalization in hepatic GSD Ia mice enhances YAP activity, which may be mediated by hepatocyte-autonomous effects, such as cellular glycogen accumulation, and/or by hepatic bile acid sensing.To further investigate the origin of the extreme liver enlargement observed in hepatic GSD Ia mice upon prolonged ChREBP normalization Fig. A, we nextes Fig. B. They ates Fig. B in parates Fig. C. Intereted Fig. D, S2F. I8b1 Fig. E. It fur11) Fig. E while i11) Fig. F. IntereG6pc−/− mice induced chromosomal instability (CIN) marker genes , and p21 (Cdkn1a), and a massive increase in hepatic p21 protein levels  and HNF4A expression are associated with hepatocyte dedifferentiation and advanced liver disease including liver cancer [G6pc−/− mice. Reporter transcription factors analysis revealed that ChREBP- and HNF4A-targeted transcriptomes showed parallel responses to hepatic G6pc deficiency and combined G6pc deficiency/Chrebp normalization  is in line with previous studies , 48, 49.In summary, we show that ChREBP normalization in hepatic GSD Ia induces hepatocellular stress, chromosomal instability, DNA damage, and cGAS-STING pathway activation and provokes hepatocyte damage and inflammation, cellular senescence, and hepatocyte dedifferentiation. We hypothesize that hepatic YAP is induced to remove the damaged cells and to stimulate hepatocyte regeneration in order maintain liver function . HoweverThis work was supported by a VIDI grant from the Dutch Scientific Organization, a grant from the Stichting Vrienden Beatrix Kinderziekenhuis , and a grant from the De Cock-Hadders Foundation. In addition, this work is supported by European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement PoLiMeR, No 812616. M.H.O holds a Rosalind Franklin Fellowship from the University of Groningen.Additional file 1: Supplementary Materials and Methods, Supplementary Figures, SupplementaryTables, Supplementary References.


Persistent impairments in quality of life, particularly in cancer-associated fatigue, are a major limitation for breast cancer survivors. As physical activity and mindfulness interventions have been shown to be effective, the question arose whether a supervised Argentine tango program may also reduce fatigue symptoms. Evaluation of the self-reported quality of life parameters revealed that quality of life, including fatigue, improved after six weekly one-hour tango sessions. The TANGO trial shows that an Argentine tango program may be an efficacious intervention to improve fatigue and quality of life in breast cancer survivors.d) effect sizes, and association factors were also calculated. Results: Superiority of the tango intervention over the waiting list control was found in terms of improvement in fatigue , especially cognitive fatigue. In addition, a superiority of the tango intervention over the waiting list was found in the improvement of diarrhea . A pooled pre-post analysis of the 50 participants completing the six-week tango program revealed a close to 10% improvement of fatigue (p = 0.0003), insomnia (p = 0.008) and further quality of life outcomes. Adjusted multivariate linear regression analyses revealed the greatest improvements for participants who were more active in sports. In particular, survivors who received endocrine therapies, were obese, or had no prior dance experience seemed to especially benefit from the tango program. Conclusions: This randomized controlled trial demonstrated that a six-week Argentine tango program improves fatigue in breast cancer survivors. Further trials are warranted to determine whether such improvements lead to better long-term clinical outcomes. Trial registration: trial registration number DRKS00021601. Retrospectively registered on 21 August 2020.Background: Persistent impairments of quality of life—in particular, cancer-associated fatigue—are a major limitation for breast cancer survivors. As physical activity and mindfulness interventions have been shown to be effective in reducing fatigue symptoms, we investigated the efficacy of a six-week Argentine tango program. Methods: A randomized controlled trial was conducted with 60 breast cancer survivors diagnosed with stage I-III tumors 12–48 months prior to study enrollment and who had increased symptoms of fatigue. The participants were randomly assigned with a 1:1 allocation to either the tango or the waiting group. The treatment consisted of six weeks of supervised weekly one-hour tango group-sessions. Self-reported fatigue and further quality of life parameters were assessed at baseline and six weeks post-baseline. Longitudinal changes, correlations, Cohen’s D ( Breast cancer remains the most common cancer in women worldwide , and appThe purpose of this clinical trial was to evaluate the efficacy of a six-week Argentine tango program on fatigue in breast cancer survivors. It was expected that fatigue would improve in the tango group compared to the waiting group. In addition, it was anticipated that the six-week tango program would have additional QoL benefits as well.This clinical trial was an investigator-initiated trial approved by the responsible local ethics committee  and has been registered . The study protocol was published . All eliThe detailed methods of the TANGO trial have been reported and published in the study protocol . BrieflyThe tango program consisted of six weekly one-hour Argentine tango sessions and was conducted by a professional teacher in small groups of three to eight participants. The tango sessions were conducted in alternating and rotating partner exercises so that all participants learned both the leading and the following roles. The tango exercises were oriented towards walking to music, promoting self-awareness, spatial awareness, and interaction. In brief, the six weekly structured group sessions were comprised of breathing and relaxation exercises, balance finding, tango walking, and dancing elements as well as social interaction. Participants randomized to the waiting arm continued their routine activities during the six-week waiting period and subsequently received the same Argentine tango program as the participants in the tango group.Adapting to the then prevailing COVID-19 pandemic conditions, the tango sessions were conducted in compliance with the national security and hygienic regulations at that time. The number of sessions each participant actually attended was recorded. The dance program was led by a tango teacher with extensive experience, and the safety of the participants was warranted. The participants were encouraged to report any adverse events, discomfort, or problems they experienced. Post-exertional symptom exacerbation after exercise may occur , which wDemographic, behavioral, and medical information was collected with the help of clinical interviews, and self-reported fatigue—as well as QoL—was surveyed prior to and after the intervention. The primary outcome measure was the German version of the Cancer Fatigue Scale (CFS-D), consisting of a 15-item questionnaire on 3 subscales  with a possible range of 0 to 60 (maximum fatigue) . The secd) , and significance  were calculated. For pre-postanalysis of the entire tango group, adjusted multivariate linear regression analyses were performed to identify influencing factors and to address potential sources of bias and confounders. Predicting variables (with regard to T0) were age (in years), the respective QoL scale values at T0, the UICC cancer stages , the intake of endocrine therapy , participation in sport activities , possible dance experience , the menopausal status , and BMI .All statistical analyses were performed using Excel 2010  and the R software , a language and environment for statistical computing. One-way ANOVA was performed to determine the between-group mean differences. 95% confidence intervals (CI), Cohen’s D effect sizes  or did not meet all the inclusion criteria. In total, 87 interested candidates were referred to the study physician for a clinical interview, and 60 eligible participants were enrolled and randomized—following the randomization sequence—in the temporal order of the study inclusion and were allocated 1:1 to the tango (n = 30) or the waiting (n = 30) group . Of thesp = 0.03) over waiting, with a medium effect size for the CFS-D total scale  and especially for the cognitive fatigue subscale   .Boxplot analyses illustrate the between-group differences for the CFS-D total and the cognitive fatigue subscale .p = 0.02) with a medium effect size was found for the improvement of the symptom diarrhea  (d = −0.41) and sleep latency (d = −0.25); the EORTC QLQ-C30 subscales global health status (d = 0.48), physical (d = 0.48), cognitive (d = 0.33), and social (d = 0.41) functioning scales; and the symptom loss of appetite (d = −0.43)  . Additio= −0.43) .A total of 50 participants completed the six-week tango program and evaluations of the outcome measures were assessable . Pre-posp = 0.0003), social functioning (p = 0.02), insomnia (p = 0.008), and diarrhea (p = 0.01)  .p = 0.03) (p = 0.04) and similarly an improved emotional functioning  were found (p = 0.006) and fatigue . On the other hand, another association was found between pre-existing dance experience with a lower improvement of fatigue  and cognitive functioning  , cancer stages, respective baseline scale values at T0, endocrine therapy, sport activities, and prior dance experiences. All participants survived the study period, and none developed recurrence or metastases. For the entire study cohort, no mild, moderate, or severe adverse events were recorded. No case of symptom exacerbation was reported in this trial.A total of 56 participants received the tango program, and 50 of them (89%) completed the tango lessons . In totap = 0.01) and more receipt of chemotherapy (p = 0.05) in dropouts (In dropouts .The present randomized controlled trial is to our knowledge the first efficacy study to examine an Argentine tango program for reducing fatigue symptoms in breast cancer survivors. Superiority of the tango intervention over waiting was found for the improvement of fatigue. Medium effect sizes were observed for fatigue, especially cognitive fatigue. Furthermore, pre-postanalyses of the entire tango study cohort revealed a 10% improvement in fatigue, insomnia, and other QoL parameters. Moreover, multivariate regression analyses indicated that a higher level of sport activity was associated with an increase in the effectiveness of this tango program. The presence of obesity or the use of endocrine therapies appeared to be associated with an increased effectiveness of this tango program. Furthermore, the positive effect of the tango intervention was increased for those without previous dance experience. It is assumed that the cognitive gain through the learning experience and novelty may be relatively greater for participants who have never danced before, and thus, the effect sizes obtained for QoL improvements are greater.Compared to the published effects of physical exercise interventions on fatigue, the effect sizes found here are in accordance to the summarized effects in meta-analyses for supervised interventions for cancer survivors with cancer-related fatigue ,31. AddiMind–body therapies and physical activity  as well Based on current guideline prescriptions, breast cancer survivors are often advised to undergo adjuvant endocrine therapy to prevent cancer recurrence , and accIncreased fatigue levels and cognitive disorders are common among breast cancer survivors, especially if treated with chemotherapy . It was Notably, the most efficacious effects of the tango intervention appeared to be the improvement of diarrhea. Diarrhea is a well-known side effect of many oncological treatments and is experienced by a third of breast cancer patients . AccordiThe mechanism by which Argentine tango improves QoL can be explained by the fact that it has a specific effect on improving neurological and psychological functions . In addiLimitations of this study include the use of a waiting but not an additional active waiting group, which precludes the analysis of group effects. An additional limitation is a potential recruitment bias. Furthermore, the recruitment and implementation of the study occurred during the COVID-19 pandemic. Accordingly, the tango sessions had to be conducted under the locally prescribed hygiene conditions and protective regulations.The strengths of our trial are the randomized controlled trial design, high adherence to the intervention despite the COVID-19 pandemic situation, and an adjusted multivariate regression analysis—reducing potential confounding biases and identifying positive prognostic variables such as BMI, intake of endocrine therapy, exercise practice, and prior dance experience—which maximizes the generalizability of our findings.The present randomized controlled trial demonstrated that a six-week Argentine tango program improved the fatigue and the quality of life of breast cancer survivors with cancer-related fatigue. The results suggest that this program has the potential to improve fatigue in particular in breast cancer survivors taking endocrine therapy, showing obese BMI, and/or with no dance experience. Larger trials are warranted to substantiate the results of this study and to determine whether this tango program is useful for preventing fatigue and improving long-term clinical outcomes.


Smilax china L. polysaccharide (SCP) on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. Smilax china L. polysaccharide was isolated by hot water extraction, ethanol precipitation, deproteinization, and purification using DEAE-cellulose column chromatography to yield three polysaccharides: SCP_C, SCP_A, and SCP_N. Acute colitis was induced by administering 3% (w/v) DSS in drinking water for 7 days. Sulfasalazine, SCP_C, SCP_A, and SCP_N were administered by gavage for 9 days. SCP_C, SCP_A, and SCP_N could significantly improve symptoms, as evidenced by the declining disease activity index (DAI), decreased spleen weight, increased length of the colon, and improved colonic histology. Moreover, SCP_C, SCP_A, and SCP_N increased serum glutathione and decreased the levels of pro-inflammatory cytokines, malondialdehyde, nitric oxide, and myeloperoxidase in colon tissues. Additionally, SCP_C, SCP_A, and SCP_N modulated gut microbiota via ascending the growth of Lachnospiraceae, Muribaculaceae, Blautia, and Mucispirillum and descending the abundance of Akkermansiaceae, Deferribacteraceae, and Oscillibacter in mice with UC. The results suggested that Smilax china L. polysaccharide ameliorates oxidative stress, balances inflammatory cytokines, and modulates gut microbiota, providing an effective therapeutic strategy for UC in mice.This work aimed to investigate the preventive effect of  Inflammatory bowel diseases (IBDs), which include Crohn’s disease (CD) and ulcerative colitis (UC), are relapsing and chronic intestinal inflammations that lead to mucous ulceration and tissue damage and increase the risk of colon carcinogenesis . As a chBacteroidetes and Firmicutes and an increase in Proteobacteria _fissicatena_group and Akkermansia were positively correlated with MPO, DAI, and TNF-α. GCA-900066225, Eubacterium]_coprostanoligenes_group, Harryflintia, Lachnospiraceae_UCG-006, and Ruminiclostridium_6 were positively correlated with TNF-α.Spearman’s correlation analysis was used to determine the relationship between UC-related parameters and particular microorganisms B. MarvinSmilax china L., were isolated and purified, and their chemical structures were determined by HPGPC, HPAEC-PAD, UV -Vis, and FTIR. The in vivo anti-inflammatory effect of polysaccharide was investigated, and an acute colitis model was induced by 3% DSS. The basic symptoms observed in this model, including body weight loss, diarrhea, and bloody stools reflect the degree of severity. Supplementation with SCP_C, SCP_A, and SCP_N decreases DAI scores, alleviates colon inflammation and oxidative stress, and modulates the gut microbiota, thereby ameliorating DSS-induced colitis. However, the structure–activity relationship of SCP_C, SCP_A, and SCP_N in the treatment of UC needs further study. Our findings indicate that SCP_C, SCP_A, and SCP_N have strong potential for preventing and treating UC.In summary, different polysaccharides, namely, SCP_C, SCP_A, and SCP_N from