Glioma is a fatal tumor originating from the brain, which accounts for most intracranial malignancies. Currently, Immunotherapy has turned into a novel and promising treatment in glioma patients. however, there are still few effective biomarkers to mirror the reaction to immunotherapy in patients with glioma. Therefore, we intended to elucidate the evaluable efficacy of SLC11A1 in glioma patients.In this study, samples from Shanghai General Hospital and data from TCGA, GEO, CGGA datasets were used to investigate and validate the relationship between SLC11A1 and the progression of glioma. We evaluated the predictive value of SLC11A1 on the prognosis of glioma with cox regression analysis. Then the relationship between immune infiltration and SLC11A1 was also analyzed. Ultimately, we performed the prediction on the immunotherapeutic response and therapeutic drugs according to the expression of SLC11A1.Expression of SLC11A1 increased with progression and predicted unfavorable prognosis for glioma patients. The hazard ratio for SLC11A1 expression was 2.33 with 95% CI (1.92-2.58) (P < 0.001) in cox analysis. And based on expression, we found SLC11A1 stratified glioma patients into subgroups with different immune activation statuses. Moreover, we observed that patients with higher SLC11A1 levels companied with better immunotherapeutic response, while those with lower SLC11A1 levels may respond better to temozolomide.This study provided evidence that SLC11A1 was a novel prognostic marker and immunotherapy response indicator for gliomas. In some cases, SLC11A1 could be an effective marker for identifying patients who might benefit from immunotherapy or chemotherapy. Glioma, the most common primary brain tumor, companied with poor prognosis in human adult . It is aThe use of cancer immunotherapy, such as immune checkpoint blockade (ICB), has been proven to benefit patients with gliomas. ICB inhibits tumor progression through the reinvigoration of tumor cytotoxic T cells. Although glioma is an immunogenic tumor characterized by high neoantigen levels, only a small subset of patients responds to ICB due to primary or secondary drug resistance . In lighA recent study showed that solute carrier family 11 member 1 (SLC11A1) has multiple effects on macrophage activation and exerts a vital role in immune response . SusceptOur study examined the expression patterns of SLC11A1 and its immunological function across a range of cancers. Anti-SLC11A1 therapy appears to be an appropriate treatment for gliomas. We also report that SLC11A1 expression promotes tumor progression and may serve as a biomarker for differentiating molecular subtypes of gliomas.Human samples were exempt from testing by Shanghai General Hospital’s Human Investigation Ethical Committee. The samples were recruited between January 2021 and January 2022 from the Department of Neurosurgery in Shanghai General Hospital. Among the 20 glioma patients  none had experienced chemotherapy and radiotherapy before. Each patient signed an informed agreement paper.Seurat4.0 . After imported into R, the filtered UMI count matrix was analyzed using the R package Seurat (A pan-cancer dataset from The Cancer Genome Atlas (TCGA) was analyzed with UCSCXenaShiny containing 33 subtypes of cancer and GTEx expression matrix data . All dateurat4.0 . Cell aneurat4.0  and markeurat4.0 . SpatialParaformaldehyde 4% and paraffin were added to the samples to fix them for 24 hours. After cutting the paraffin block into five millimeter-thick sections then blocked overnight at 4°C and stained with SLC11A1 . Using biotinylated rabbit IgG incubated with PBS after washing the sections. The sections were viewed under a microscope AX-80. The images were analyzed by Image J.Sections that were formalin-fixed and embedded in paraffin were deparaffinized, rehydrated, permeabilized, and rinsed. Antigen repair in citrate buffer was made for 15 min. Blocking was carried out in 5% BSA for 1 h at room temperature. Then sections were stained with SLC11A1 , CD68 , PD1 . 2% BSA/PBS was diluted in secondary antibodies, which were incubated for another 1 h. After stain with DAPI, microscope images were taken of the sections.Total RNA was extracted from human sample using TRIzol reagent. The reverse transcription is performed with FastQuant RT kit. Real-time PCR was carried out using SuperReal SYBR Green kit in Lightcycler 96). The primer sequences were listed as follow: SLC11A1 forward: GCACCTCCCAGAGAGACCT; reverse: GAGCAGCACCCAGAGAAGTT; PDCD1 forward: CAGTTCCAAACCCTGGTGGT; reverse: GGCTCCTATTGTCCCTCGTG; CA4 forward: CAAGTGCCTTCTGTGTGTGC; reverse: GAGCGGTGTTCAGGTCTTCA.TCGAmutations”  . In ordeGSVA” (http://timer.cistrome.org/). Expression and survival data from CGGA were merged and MCP-counter variables together with SLC11A1 were binarized using a median cut . For this study, we concatenated the binarized scores for the two variables of interest (Mono/Macro cells and SLC11A1), leading to four classes . The corresponding Kaplan–Meier curves for OS were then plotted and the p value of the corresponding log-rank test is calculated. This algorithm was previously described in Etienne et al’s study  signature were used to score the glioma samples. This signature was listed in pRRophetic package  and PRISM Repurposing dataset (19Q4). The  package  which hasurvival” and “survminer”. Meta-analysis was performed with R package “meta” . Based on the expression of SLC11A1, patients were classified into high-SLC11A1 or low-SLC11A1 subgroup. Then the log-rank test analysis indicated that patients with high expression of SLC11A1 in the Chinese Glioma Genome Atlas (CGGA), TCGA, Rembrandt and GSE16011 cohorts presented markedly poorer prognoses than those with low expression of SLC11A1  . Statistical analysis showed that high SLC11A1 expression related to older age, shorter survival time, higher tumor grade, GBM subtype, mesenchymal subtype, and wild type IDH, which further confirmed the findings from the TCGA analysis Table 1.We used the Cox regression model to perform univariate and multivariate Cox analyses for 1018 glioma patients on different clinical variables. According to the results of the univariate Cox regression analysis  is a strong candidate target for influencing autoimmune and infectious disease susceptibility . ResearcTherefore, our study was aimed at discovering how SLC11A1 expression relates to immune infiltration in gliomas and examining the molecular mechanisms by which SLC11A1 plays a role. We analyzed the DEGs between the different SLC11A1 groups (based on the CGGA dataset). The result indicates that 179 genes were upregulated, while 136 genes were downregulated. Then, the GO terms and KEGG pathways were annotated. In terms of GO biological processes, the enrichment expression of DEGs were focused on neutrophil activation involved in immune response, neutrophil degranulation, collagen-containing extracellular matrix, and leukocyte migration , we utilized glioma single-cell data derived from the GEO dataset GSE131928) (28 (9), uESTIMATE is a tool  used genImmunofluorescence experiments demonstrated that the increase in SLC11A1 expression was accompanied by increased malignant potential and increased macrophage and T cell infiltration. Consistent with the above results, as shown in In the next step, the immune cells infiltration levels was calculated by using several independent algorithms with TIMER website  based onTo identify the transcriptome results from public datasets, 20 patients were included from Shanghai General Hospital, and the association between the expression levels of PDCD1, CTLA4 and SLC11A1 by quantitative real-time PCR were investigated. The results indicated that SLC11A1 was positively associated with PDCD1 and CTLA4  based on the median SLC11A1 level. We utilized the same methodology to assess the sensitivity of different SLC11A1 subgroups to temozolomide. We demonstrated that patients with low expression of SLC11A1 showed significantly lower estimated AUC values of temozolomide . The Spearman correlation coefficient between expression of SLC11A1 and AUC value was tested to select compounds that are negatively correlated. Based on these analyses, we identified three CTRP-derived compounds  and ten PRISM-derived compounds . SLC11A1 was negatively correlated with all of these compounds, and the estimated AUC values were lower in the high-SLC11A1 group (p < 0.001); the most notable candidates included clofarabine (mean (IQR), low: 0.3850  vs. high: 0.3547 ) in the CTRP-derived compound group and AZD8330  in the PRISM-derived compound group ], which we also demonstrated in the present study, so to some extent SLC11A1 can reflect the number of macrophages in glioma. Moreover, increased macrophage infiltration suggests a suppressive immune microenvironment, which usually implies a poorer prognosis. We hypothesize that high expression of SLC11A1 could reflect a tumor microenvironment tending to be “cold”, which also provides an explanation for the association of high SLC11A1 expression with poor prognosis. Additionally, we observed that glioma patients with high expression of SLC11A1 were more sensitive to immunotherapy, while glioma patients with low expression of SLC11A1 responded better to temozolomide. Therefore, SLC11A1 may serve as an indicting factor for whether patients be treated with different treatment approaches (chemotherapy or immunotherapy). We yielded three CTRP-derived compounds  and ten PRISM-derived compounds . In previous study, fluvastatin inhibits the growth and alters the malignant phenotype of the glioma cell line, suggesting the potential use of this statin for the treatment of gliomas . InteresThe original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Shanghai General Hospital’s Human Investigation Ethical Committee. The patients/participants provided their written informed consent to participate in this study.HX, AZ, CF, and XW were involved in data analysis and interpretation. QZ, YL, and ZZ collected clinical data and tumor samples, and performed experiments. WW supported clinical data analysis. YX and ML designed the experiment, interpreted the data, and wrote the manuscript. All authors reviewed and approved the manuscript.This research received grants from 1) National Key R&D Program of China (2018YFA0108603); 2) Beijing Tianjin Hebei basic research cooperation project (19JCZDJC64600(Z)); 3) CAMS Innovation Fund for medical sciences(CIFMS) (2020-I2M-C&T-B-028); 4) Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences(2020-JKCS-026); 5) Cross Research Fund of Medicine and Engineering of Shanghai Jiao Tong University (YG2019QNA67).https://hiplot.com.cn) for providing technical assistance and valuable tools for data analysis and visualization.The authors would like to thank Qiyu Gong from Shanghai Jiao Tong University for her generous and outstanding help in this research design. We thank openbiox community and Hiplot team (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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


XIST-based degree of XCI skewing. We identify 62 genes, including 19 lncRNAs, with previously unknown patterns of escape. We find a range of tissue-specificity, with 11% of genes escaping XCI constitutively across tissues and 23% demonstrating tissue-restricted escape, including cell type-specific escape across immune cells of the same individual. We also detect substantial inter-individual variability in escape. Monozygotic twins share more similar escape than dizygotic twins, indicating that genetic factors may underlie inter-individual differences in escape. However, discordant escape also occurs within monozygotic co-twins, suggesting environmental factors also influence escape. Altogether, these data indicate that XCI escape is an under-appreciated source of transcriptional differences, and an intricate phenotype impacting variable trait expressivity in females.X-chromosome inactivation (XCI) silences one X in female cells to balance sex-differences in X-dosage. A subset of X-linked genes escape XCI, but the extent to which this phenomenon occurs and how it varies across tissues and in a population is as yet unclear. To characterize incidence and variability of escape across individuals and tissues, we conducted a transcriptomic study of escape in adipose, skin, lymphoblastoid cell lines and immune cells in 248 healthy individuals exhibiting skewed XCI. We quantify XCI escape from a linear model of genes’ allelic fold-change and  The difference in the number of X-chromosomes between mammalian males and females is compensated by a process known as X-chromosome inactivation (XCI), which turns off one of a female’s X chromosomes. XCI is incomplete: some sections of the silenced X chromosome escape inactivation. The ’escape’ is complex, and can vary across tissues and potentially across individuals. Because the X chromosome is enriched of genes with immune and neurological functions, this phenomenon has high biomedical relevance. We studied the extent to which escape occurs and varies across tissues and individuals in a large population of twins. We identify novel candidate escape genes, and genes whose escape is specific to a tissue or immune cell type. There is also substantial variability in escape across individuals. Using data from twins, which enable the assessment of the influence of genetics and environment on a trait, we found that both genetic and environmental factors influence escape. Our results allow detailed characterization of escape, and suggest that escape may influence disease risk and phenotype differences between the sexes, and within females. XIST spreads in cis along the inactive X chromosome (Xi) and promotes a progressive epigenetic silencing [The X chromosome is a paradigmatic genetic model . It carrilencing ,6,7. Howilencing ,9. Mary ilencing . Today, ilencing . Chromosilencing ,13; chanilencing ,14–16. DOur current knowledge of XCI escape in humans largely rely on conventional studies of male/female expression ratio, human/mouse hybrid cells, and epigenetic marks ,11,17,18+, T-CD8+ and NK cells), and individuals. We identify novel genes exhibiting tissue- and immune cell type-specific escape, and genes escaping XCI with high variability across tissues and individuals. We observe that escape varies across tissues and immune cells within an individual and across individuals, a phenomenon with high biomedical relevance. Using twins, we demonstrate that regulation of XCI escape has both heritable and environmental components, implying a complex interplay between genetic and non-genetic factors.Here, we characterize XCI escape using paired bulk RNAseq and DNAseq data in a multi-tissue dataset sampled from 248 skewed female twins of the TwinsUK bioresource . We inveXIST allele-specific expression (XISTASE) from paired RNAseq and DNAseq data [XISTASE calls for 522 LCLs, 101 whole-blood, 421 adipose, and 373 skin samples. In samples exhibiting skewed XCI  including 166 LCLs (32%), 26 whole-blood (26%), 57 adipose (14%), and 64 skin (17%) samples, the levels of escape of each X-linked gene were measured using a metric—herein referred to as Escape Score or ’EscScore’—derived from the gene’s allelic fold-change adjusted for the sample’s XCI skew (Methods). EscScore values range from 0  to 1  and active X (Xa)). We interrogated a total of 551 genes, of which 85% are protein-coding and 15% non-coding RNA genes. Based on a publicly available catalogue of XCI statuses [We quantified escape in multiple tissues concurrently sampled from female twins of the TwinsUK cohort . We deteseq data ,28,29. Fstatuses  . Among our escape calls, about 60 genes retain a Y-pseudogene or Y-homologue, and 13 are PAR-linked, supporting their escape [AL683807.1 (ENSG00000223511) escapes XCI. AL683807.1 is PAR1-linked, explaining its escape ability. We examined the chromosomal distribution of escape genes and confirmed a higher escape incidence on the short arm [Next, we conducted additional testing of EscScore, identified genes escaping XCI in our dataset, and benchmarked our call set with other studies. For X-genes annotated as silenced , the aver escape . For a mr escape ; (ii) Kar escape ; (iii) Sr escape ; (iv) Gar escape ; (v) Saur escape . We founhort arm  . We confirmed the escape status of CLIC2 in skin, as identified in GTEx [Presently, the extent to which tissue-specific escape occurs in humans is unclear. We used the gene’s median EscScore (across ≥3 tissue samples) as a measure for tissue-specific levels of escape. We found significant differences across tissues  an males . We also in GTEx , and alsWe investigated whether the escapees may interact with other factors and be involved in biological processes. To address this, we selected genes exhibiting escape in at least one tissue and conducted protein-protein interaction network analysis using a recent human protein interactome as reference . We founZFX and the histone demethylase KDM5C which is linked to intellectual disability and autism [KDM6A [FMR1, a gene linked to Fragile-X and learning disability [DMD [ASMTL which escaped XCI in all tissues with EscScore ranging from 0.6 to over 0.8. ASMTL’s behavior was also highlighted in GTEx [Understanding the extent to which escape varies across tissues within an individual is of high biological interest. We investigated this phenomenon in 6 donors, each exhibiting skewed XCI in LCLs, adipose and skin. This strategy was employed in the GTEx survey using a single skewed female donor . Within d autism ,34. We am [KDM6A ,35, FMR1sability , and theity [DMD . In para in GTEx . Aside fBTK, a gene involved in the control of lymphocyte maturation, and CD99L2, involved in leucocyte homeostasis. Both genes exhibited consistent EscScore across most donors in LCLs  also exhibited consistency across most donors in both LCLs and a solid tissue  showing consistent EscScore across individuals in at least 1 tissue . Represe in LCLs . A subsed tissue . Genes ed tissue  accounted tissue . Interes+ cells were available from both co-twins, while T-CD4+ and NK-cells from one co-twin. Per each immune cell type, when data were available from both co-twins, we calculated the average gene’s EscScore across the 2 co-twins as a proxy for immune cell type-specific escape. We observed differences between cell types in the average EscScore . The incidence of escape varied between cell types, being 15% in monocytes, 20% in B-cells, 22% in T-CD4+, 25% in T-CD8+, and 29% in NK-cells. Thus, in line with current knowledge, most X-genes are subject to XCI in immune cells. In parallel, our data indicate that escape is heterogeneous across immune cell types, with overall higher incidence in lymphocytes than monocytes. To investigate intra-lineage variation, we compared the EscScore(s) between lymphoid cell types and also found substantial differences (P≤0.01), indicating intra-lineage variation. Among the 53 genes with EscScore data available in all cell types  escaping XCI in at least three immune cell types. CASK is a novel candidate escapee. The genes PRKX, ZFX, JPX and DIAPH2 escaped XCI in all 5 immune cell types, in line with their behavior as constitutive escapees across tissues. For most of these genes, the escape status in immune cells is a novel finding. Interestingly, KDM6A exhibited highest EscScore in T-CD8+ cells, possibly because of its roles in T-cells control [+ and T-CD8 cells). Altogether, these data indicate that escape varies between immune cell types within an individual. Presumably, this heterogeneity is driven by mechanisms with immune cell type-specific effects.Females have a higher risk of autoimmune disease than males, and such risk may correlate with increased X-dosage ,39. Thisll types , we iden control . We iden control . We alsoρ′s t-test, P≤0.05; DZ = 27.1%; Av.Disc.RateMZ = 19.5%). These data suggest a significant genetic component of escape, in line with a previous report on MZ twins [ρmonocytes = 0.8; ρB-cells = 0.68; ρT-CD8+ = 0.6; P<1e-10). Genes with discordant XCI status were observed in all three immune cell types, and their prevalence differed between cells ranging from 6.4% in monocytes to 11% in B-cells, and 18% in T-CD8+ cells. Interestingly, the genes CA5B and ZNF81 exhibited discordant XCI between the two co-twins in both T-CD8+ and B-cells. In all other cases, discordant XCI events concerned distinct gene subsets in distinct immune cell types ; 10 dizygotic ), we quantified the concordance in the escape in LCLs between co-twins and compared such concordance between MZ and DZ twins. We correlated the EscScore (using ≥5 genes) between co-twins of each pair , and fouMZ twins ,28. In pll types . Taken tAL683807.1, which escaped XCI in LCLs, in line with GTEx data on the novel transcript ENSG00000223511.6, whose expression is substantially higher in EBV-transformed lymphocytes than all other tissues. The X chromosome is enriched for non-coding RNAs, yet their transcriptional modes and roles are unclear. Due to their unique ability to recruit factors and target a genomic address [In this study, we investigated escape in tissues and immune cells using paired transcriptomic and genotype data from nearly 250 female twins from the TwinsUK bioresource . The larBTK and CD99L2, which, in line with their roles in lymphoid cells, exhibited consistent EscScore in LCLs, but inter-female variability in adipose and skin. Thus, the escape behaviour could depend on the gene’s functional context. The tumorigenesis-related genes DDX3X and KDM6A both escaped XCI, in line with previous studies [KDM5C and the transcription factor ZFX, escaped in LCLs and solid tissues in the same individual. Other genes manifested more composite behaviour, with escape restricted to either LCLs or solid tissues in an individual, supporting tissue-specificity of escape. Altogether, these patterns highlight the complexity of escape, and anticipate its roles as phenotype modulator. The intra-individual analysis would benefit from a larger sample size, and additional tissue types available from each individual, allowing more comprehensive and precise inferring of the across-tissue escape variability within a subject. Furthermore, the variable nature of RNAseq total read depth introduces differences in allelic read depth across samples, which may contribute to the observed differences, and will impact the total number of genes within a donor with sufficient read depth to analyze escape. Despite the use of established methods, we acknowledge that phasing switch errors can occur over the chromosomal length, a problem that can be solved by the availability of parental genotypes  with constitutive or tissue-specific effects, modulate the escape dosages. We found genes with consistent and with heterogeneous EscScore across individuals. We reasoned that genes with key physiological roles may be subject to shared regulation across females. Examples are  studies ,30,44, a studies , and plaes (e.g. ).PRKX, DIAPH2, JPX, ZFX) escaped XCI in all cells, in line with their constitutive escape behavior across tissues. For ZFX, this aligns with data showing its involvement in networks for X-linked dosage regulation [TCEANC, TAB3, MIR222HG, ARMCX4, AC234775.3, NTX, SLC25A43, INTS6L, GAB3) exhibited escape restricted to a single immune cell type which was always of lymphoid lineage. We also found significant variation between lymphoid cells. These data indicate immune cell type-specific propensity to escape. Myeloid and lymphoid lineages are subject to distinct regulation during development. Integrated functional and in-silico approaches will be needed to fully address the possibility that cell type-specific factors establish distinct escape dosages across cells within an individual [The X chromosome plays key roles in innate and adaptive immunity ,31,40,45gulation . 17% of dividual . These ddividual , may altdividual –51. The The extent to which genetics and environment influence escape in humans is unclear. Concordance in methylation-based XCI status between MZ twins supported a dominant model of cis-acting influences . MZ twinThe present study contributes a detailed characterization of escape in humans using a large multi-tissue transcriptomic twin dataset and demonstrates extensive variability in escape between individuals and tissues. Given the paradigmatic roles of the X chromosome in epigenetics and clinical genetics, a full understanding of XCI escape has implications on epigenetic research and therapeutics. Therapeutics may include genetic counselling and design of treatments for X-linked conditions. Despite nearly 60 years after Mary Lyon’s landmark intuition on escape, a lot is yet to be learned. Future large-scale studies that combine biomedical records and functional assays will be critical to disentangle the breadth of variability of escape from XCI in humans and characterize its phenotypic impact.This project was approved by the research ethics committee at St Thomas’ Hospital . Volunteers received detailed information sheet regarding all aspects of the research, gave informed consent and signed an approved consent form prior to biopsy and to participate in the study. See The study included 856 female twins from the TwinsUK registry ,55 who pP-value <1e-6 were removed. Data quality checks were further performed with VCFtools[Details on 30X whole genome sequencing (WGS) sample and library preparation, clustering and sequencing have been reported elsewhere . The DNA VCFtools to check VCFtools.The Illumina TruSeq sample preparation protocol was used to generate cDNA libraries for sequencing. Libraries were sequenced on a Illumina HiSeq2000 machine and 49 bp paired-end reads were generated . Samples+, T-CD8+ and NK cells were purified using fluorescence activated cell sorting (FACS) from two monozygotic twins exhibiting skewed XCI patterns in LCLs. Gating strategy for cell sorting is described in Fig E in Monocytes, B, T-CD4To eliminate mapping biases in RNA-seq, the WASP pipeline for mappability filtering  in STARvWGS genomes were read-back phased using recent SHAPEIT2 implementation  that takWhere, for a biallelic gene:A = haplotype A;B = haplotype B;g,s = RNAseq allelic count at haplotype A of gene g in sample s;ACg,s = RNAseq allelic count at haplotype B of gene g in sample s;BCg,s = Total RNAseq allelic read depth at gene g in sample s;TCg,s = ACg,s + BCg,s.TCThe gene ASE values range from 0 to 1, with 0 and 1 indicating monoallelic expression and 0.5 indicating completely balanced haplotypic expression. From [To quantify gene silencing and gene escape, the effect size of allelic imbalance in expression for each gene in each sample was calculated as allelic fold change (aFC), that is the ratio between the allele with lower RNAseq count and the allele with the higher RNAseq count, as similarly used in a previous study :aFCg,saFC values range from 0 to 1, with 0 indicating monoallelic expression (full gene silencing) and 1 completely balanced haplotypic expression (full escape). For the purpose of our study, a gene’s aFC can be interpreted as Xi/Xa expression ratio. The Xi is assumed to be the allele with the lower RNA-seq count, while the Xa the allele with higher RNA-seq count.XIST allele-specific expression (XISTASE) was used as proxy for XCI skewing levels. XIST is uniquely expressed from the Xi, and thus the relative expression of parental alleles within XIST transcript is representative of XCI skewing in a bulk sample [XISTASE values  range from 0 to 1, with 0 or 1 indicating completely skewed XCI (100:0 XCI ratio), and 0.5 indicating balanced inactivation ratio. To be consistent with previous literature [XISTASE ≤0.2 or XISTASE ≥0.8 to have skewed XCI, and samples with 0.2< XISTASE < 0.8 to have random XCI. To have an absolute measure of the magnitude of XCI skewing levels in each sample, the degree of XCI skewing (DS) was calculated from the XISTASE calls. DS is defined as the absolute deviation of XISTASE from 0.5, and it has been similarly been used to assess XCI patterns and XCI status of X-genes [In each sample, the k sample ,29,70,71terature ,28,72, w X-genes ,73,74. IXIST gene expression levels and DS , there was no evidence of correlation between the newly derived EscScore(s) and the degree of XCI-skew in our dataset (ρ = 0). This indicates that our procedure of using residuals and normalization generates EscScore(s) robust to variation in XCI-skew across samples removing the dependence between aFC and XCI-skew. To further assess this latter in detail, we grouped our dataset into 3 bins of degree of XCI-skew, and then randomly sampled 200 X-linked genes to check their distribution of average aFC and average EscScore values at the different degrees of XCI-skew. We repeated the random sampling step 3 times (each drawing 200 X-genes) and confirmed that as opposed to aFC, the EscScore is robust to various degree of XCI-skew  and complete biallelic expression (full escape), respectively. Due to low number of skewed whole-blood samples with available data in other tissues, we excluded our whole-blood estimates from analyses of XCI escape. We detected average EscScore of 0.32, 0.36, and 0.37 across LCLs, adipose and skin samples, respectively, whose median is 0.36. We compared different EscScore cutoffs to the Balaton’s list  and founFor analysis of inter-individual variability of escape, genes classified as escapee in at least 1 tissue (median EscScore across skewed tissue samples ≥0.36) and with available EscScore data in ≥10 tissue samples were used. Consistent EscScore in a tissue across individuals was defined when in ≥80% of individuals the gene’s EscScore lay between ±1 standard deviation from the gene’s average EscScore in that tissue . Otherwiwww.interactome-atlas.org) [P-value ≤0.01.Genes with EscScore data available in all three tissues and escaping XCI in at least one tissue were analysed for protein-protein interaction (PPI) with a new genome-wide protein interactome database as reference (las.org) . PPI netlas.org) , and prolas.org)  for visulas.org) . A term S1 AppendixTable A. Summary statistics  of EscScore values of genes with different annotated XCI status according to the Balaton’s list. A) LCLs; B) adipose; C) skin. Each statistics is computed across ≥3 tissue samples. Table B. Statistical comparison between the EscScore(s) of different gene categories previously annotated. Escapees refer to genes annotated as fully or mostly escaping XCI. For each interrogated gene, the median EscScore value across ≥ 3 tissue samples was used for comparison. Tables report the p-value of the Wilcoxon test between two gene categories A) LCLs; B) adipose; C) Skin. Table C. Benchmarking different EscScore cutoffs against the Balaton’s list of XCI status. The EscScore cutoff of 0.36 resulted into both overall higher reproducibility of gene calls and lower discordance with previously annotated XCI status data. Table D. Fraction of X-linked genes exhibiting escape (EscScore ≥0.36 in a tissue in a donor) in 1, 2 or all 3 studied tissues in each of the 6 female donors exhibiting skewed XCI in all three studied tissues . Fig A. Scatterplot of degree of skewing (y-axis) and XIST gene expression levels  in LCLs, adipose and skin tissues. There is no evidence of significant association between the two variables in all three tissues (P>0.1). Fig B. Assessing the dependence between genes’ aFC and XCI-skew, and between EscScore and XCI-skew in our dataset. Our data were grouped into 3 (nearly same size) bins of average degree of XCI-skew (XCIskew Bin1 < XCIskew Bin2 < XCIskew Bin3). Random sampling of 200 X-genes was then performed three times . At each sampling, the average aFC and average EscScore values per gene (across samples) were calculated within each XCI-skew bin. The EscScore is robust to various degree of XCI-skew . Each dot represents a gene. Red and green dots are escapee and silenced genes, respectively. Fig D. Distribution of median EscScore values in each of the three studied tissues . Median values were calculated per gene across ≥3 skewed tissue samples. Fig E. Gating strategy for immune cell sorting. Gating strategy used to sort monocytes (CD14+), B , NK , T-CD4+  and T-CD8+ cells  from freshly isolated PBMCs from 2 monozygotic twins exhibiting skewed XCI in LCLs.see also . Fig C. (PDF)Click here for additional data file.S1 TableThe table lists the gene’s EscScore in each tissue, computed as the median EscScore across ≥3 samples. Column 2 indicates whether the escape status of the gene was either known (previously reported) or is a novel call.(TXT)Click here for additional data file.S2 TableThe table lists the gene’s EscScore in each tissue, computed as the median EscScore across ≥3 samples.(TXT)Click here for additional data file.S3 TableThis is a subset of (TXT)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(TXT)Click here for additional data file.S6 TableThe table lists the genes’ EscScore in each immune cell type.(TXT)Click here for additional data file.S7 Table+ cells in each individual are reported.Genes’ EscScore in monocytes, B-cells and T-CD8(XLSX)Click here for additional data file.S8 TableThe 6 donors examined for intra-individual analysis are annotated.(XLSX)Click here for additional data file.S1 FigPlotted are genes classified to have consistent EscScore across individuals. Each green dot is an individual.(PDF)Click here for additional data file.S2 FigPlotted are genes classified to have variable EscScore across individuals. Each green dot is an individual.(PDF)Click here for additional data file.


An almost universal phenomenon occurring during aging is a state of chronic, low-grade, sterile inflammation. Inflammation is a crucial contributor to various age-related pathologies and natural processes in aging tissues. Tumor necrosis factor-α (TNF-α), a master regulator of the immune system, plays an important role in the propagation of inflammation. Recent research has found correlations between hearing loss and markers such as TNF-α. However, the intrinsic molecular mechanism by which TNF-α influences aging individuals’ increased risk of hearing loss remains unclear. In this study, we found that TNF-α expression gradually increased with age in DBA/2J mice. We then used recombinant TNF-α to upregulate TNF-α levels in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and found that low concentrations of TNF-α could activate the nuclear factor kappa B (NF-κB) transcriptional response to mediate hair cell survival, while high concentrations of TNF-α could activate the Caspase-3 cascade to mediate hair cell apoptosis, which preliminarily confirmed that a TNF-α mediated signaling pathway plays an important role in the pathogenesis of age-related hearing loss. Presbycusis, also termed age-related hearing loss (ARHL), is progressive, irreversible, and symmetrical bilateral neuro-sensory hearing loss associated with aging caused by the limited repair capacity of sensorineural tissues in the cochlea . The occin vivo loss-of-function analysis demonstrated that TNF-α was a factor that worsened cochlear inflammation  and then relocated to the Affiliated Yantai Yuhuangding Hospital of Qingdao University for breeding in specific pathogen free animal rooms. The room temperature was maintained at 24 ± 1Auditory-evoked brainstem response was measured at various intervals  in the DBA/2J mice. Eight mice were selected for each week of age. A computer-aided evoked potential system  was used to test mice for ABR thresholds. The amplified brainstem responses were averaged and displayed on a computer screen. A plug-in earphone was inserted into the external auditory canal of DBA/2J mice to stimulate short pure tones at different frequencies . The frequency intensity started from 80 dB, and then gradually decreased. The minimum stimulus intensity of wave III was used as the auditory threshold .To test the function of the outer hair cells (OHC) of mice at 2, 4, 6 and 8 weeks, we used the IHS Smart EP 3.30 and USBez Software (Intelligent Hearing Systems) for distortion-product otoacoustic emission (DPOAE) measurement, which was conducted with pure tones from 6 to 35 kHz. Frequencies were acquired with an f2: f1 ratio of 1:22. The stimuli were presented from the lowest to the highest frequencies tested.Six mice were selected at the age of 2, 4, 6, and 8 weeks. The cochlea was excised, immersed in a fixative containing 4% paraformaldehyde in phosphate-buffered saline (PBS) solution for 1 day, and decalcified in 10% EDTA for 3–5 days. Sections (5 mm) mounted on glass slides were counterstained in hematoxylin and eosin (H&E). Hair cells, spiral ganglia neurons (SGNs), the stria vascularis (SV), and inflammatory cells were observed under a light microscope . The mean value in four sections from a basal turn of each animal’s cochlea was defined as the width of the SV. The width of the SV and the average SGNs density was quantified using ImageJ software .–ΔΔCT method (Actb (encoding actin) was the reference gene. The information on the primers used is shown in Samples of total RNA were reverse transcribed into cDNA using a Total-Transcriptome cDNA Synthesis Kit  according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction measurements were performed using the StepOnePlus fluorescence quantitative PCR instrument  and a SYBR Green qPCR Mix according to the manufacturer’s instructions. The data were analyzed using the 2T method , and theCochlear tissue was ground with Radioimmunoprecipitation assay (RIPA) buffer  containing phosphatase inhibitors  and phenylmethylsulfonyl fluoride (PMSF) (Beyotime). Total protein samples were separated by electrophoresis in 15% SDS polyacrylamide gels  and electrotransferred to a nitrocellulose membrane . The membrane was subsequently blocked with 5% non-fat milk for 1 h. Afterward, the membrane was incubated at 4°C overnight with the following primary antibodies: anti-NF-κB (1:2000 dilution) , anti-Caspase-3 (1:1000 dilution) .Next day, the membrane was washed three times with Tris-buffered saline-Tween 20 (TBST) buffer, incubated with corresponding horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature and then washed with TBST buffer three times. The immunoreactive proteins on the blots were visualized using the ECL reagent  and detected using a chemiluminescence imaging system . The protein bands were quantified by detecting the gray values using ImageJ software.2O2 for 10 min. The slides were then blocked with 3% bovine serum albumin (BSA) and 3% goat serum at 37°C for 1 h and stained with rabbit anti-cleaved Caspase-3 antibody (1:250 dilution)  or NF-κB antibody (1:50 dilution)  at 4°C overnight. Following a thorough wash with 1 × PBS for 5 min, the sections were immersed in Goat anti-rabbit IgG(H + L) HRP (1: 400 dilution) and Goat anti-mouse IgG(H + L) HRP (1: 400 dilution)  at 37°C for 30 min, and then counterstained with 4 Detection Kit  for 15 min and with Harris hematoxylin  for 1 min at room temperature. The slides were sealed using neutral gum and examined under a scanning confocal microscope.A time course of immunostaining of Caspase-3 and NF-κB was carried out on DBA/2J mice. The time point selected was 4 weeks. After dewaxing, the cochlear tissues were subjected to heat induced antigen retrieval in citrate buffer , washed three times in 1 × PBS at room temperature for 5 min, and permeabilized with 3% H2) in high-glucose Dulbecco’s Eagle’s medium  containing 10% fetal bovine serum  without antibiotics as described previously , incubated overnight for attachment to the substrate, and then treated with TNF-α at different concentrations . After TNF-α treatment , 10 μL of CCK-8 reagent was added to each well and incubated for 1 h. Absorbance at 450 nm was detected using a plate reader.Cell Counting Kit-8 (CCK-8)  reagent was used to examine cell viability according to the manufacturer’s instructions. HEI-OC1 cells were collected and counted, and the concentration was adjusted to 1.0 × 10House Ear Institute-Organ of Corti 1 cells were inoculated into 6-well plates, and the cell density was controlled at 30–50%. The cells were cultured in an incubator for 24 h. Liquid A comprised 1.25 pmol of different small interfering RNAs (siRNAs)  dissolved in 25 μL Opti-MEM solution . Liquid B comprised 0.75 μL of Lipofectamine 3000  was dissolved in 25 μL of Opti-MEM solution. Liquid A was mixed with liquid B gently and incubated at room temperature for 20 min. The 50 μL mixture was added to each well of HEI-OC1 cells, and continued in culture for 72 h. The information for the siRNAs used is shown in ANOVA was used to analyze the comparisons of ABR threshold and DPOAE amplitudes, the levels of gene transcription, the levels of protein expression, the widths of SV, the density of SGNs and the number of inflammatory cells were analyzed by t2-test. P < 0.05 was considered to be significant. Error Bars represent the standard deviation (SD) from the mean.Overall, the mean ABR  thresholPathological examination by H&E staining  revealedTfn-a (encoding TNF-α) and IL-6 increased progressively over time in DBA/2J mice , Casp3 (encoding Caspase-3), and Casp8 (encoding Caspase-8) at the age of 2, 4, 6, and 8 weeks. The results showed that the relative mRNA expression levels of Nf-kb and C-flip also increased, with the highest expression at 4 weeks and then began to decline. The relative mRNA expression levels of Casp3 and Casp8 were higher at 2 weeks of age and then decreased  for different times . Low concentration of TNF-α could increase NF-κB levels, while Caspase-3 and Caspase-8 levels barely changed. High concentrations of TNF-α increased both NF-κB and Caspase-3 levels in HEI-OC1 cells . CCK-8 aol group .In this study, we verified the characteristics of early onset hearing loss in DBA/2J mice. Hearing loss started in the high frequency region and was mainly manifested morphologically by hair cell loss, decreased SGN density, and a narrowed SV, which was basically consistent with previous studies. Thus, DBA/2J mice could be used as the animal model of senile deafness in this study.Aging and age-related diseases share some basic mechanistic aspects that largely converge on inflammation. During aging, chronic, sterile, low-grade inflammation contributes to the pathogenesis of age-related diseases. Several investigations have shown that chronic inflammation plays an important role in the initiation and progression of damage to the inner ear  and presThe inner ear was previously assumed to be an “immune-privileged” organ because of the existence of its tight junction-based blood-labyrinth barrier (BLB) in the stria vascularis . The barvia the activation and recruitment of immune cells through its receptor TNF receptor 1 (TNFR1), is associated with a variety of inflammatory diseases (The role of chronic inflammation in hearing loss in ARHL and the related mechanisms are still unclear. TNF-α, which plays an important role in the propagation of inflammation diseases . In genediseases . Engagemdiseases . NF-κB, Casp3 and Casp8 were higher at 2 weeks of age than at 4 weeks of age, and then decreased. In general, activation of TNFR1 does not induce death but instead triggers a robust pro-survival response. This response is suppressed unless some cell death checkpoints, which occur upon induction of pro-survival genes by NF-κB transcription factors, are disrupted. If NF-κB is blocked, the c-FLIP expression level diminishes and Caspase-3 undergoes auto-catalysis to initiate apoptosis. Based on the aforementioned results, we hypothesized that the downstream pathway activated by TNF-α signaling changed from the pro-inflammatory cell survival pathway to the cell destruction pathway before and after the death of inner ear cells.In our ARHL animal model, DBA/2J mice aged 8 weeks exhibited increased expression of the gene encoding the proinflammatory cytokine TNF-α compared with that at 2 weeks. TNF-α is mainly produced by monocytes and macrophages; therefore, these results were consistent with the presence of inflammatory infiltration. With the increase in TNF-α levels, the expression levels of the mRNAs encoding NF-κB and c-FLIP also increased, with the highest expression being observed at 4 weeks, after which it declined. The mRNA levels of Experimental studies have revealed that most cells survive at a low dose of TNF-α, while high doses of TNF-α induce apoptosis . By simuA cell exposed to an inflammatory stimulus has several potential fates: survival, production of inflammatory cytokines and possibly proliferation; non-inflammatory or even anti- inflammatory apoptotic cell death; or pro- inflammatory programmed cell death . In geneIn summary, TNF-α levels increased gradually with age in DBA/2J mice. Low concentrations of TNF-α can activate the NF-κB transcriptional response to mediate hair cell survival, while high concentration of TNF-α can activate the Caspase-3 cascade to mediate hair cell apoptosis. The TNF-α-mediated inflammatory signaling pathway plays an important role in the pathogenesis of ARHL.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.This animal study was reviewed and approved by the Institutional Animal Use and Care Committee of the Affiliated Yantai Yuhuangding Hospital of Qingdao University.TW: writing—original draft preparation. JZ and JQ: methodology and data curation. YuS and WG: find the relevant knowledge and provide experimental assistance. LC: conceived and designed the experiments. YaS and XS: writing—review and editing. All authors contributed to the article and approved the submitted version.


Normal pressure hydrocephalus (NPH) is a neurological condition characterized by a clinical triad of gait disturbance, cognitive impairment, and urinary incontinence in conjunction with ventriculomegaly. Other neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and vascular dementia share some overlapping clinical features. However, there is evidence that patients with comorbid NPH and Alzheimer's or Parkinson's disease may still exhibit good clinical response after CSF diversion. This study aims to evaluate clinical responses after ventriculo-peritoneal shunt (VPS) in a cohort of patients with coexisting NPH and neurodegenerative disease.The study has two components; (i) a pilot study was performed that specifically focused upon patients with Complex NPH and following the inclusion of the Complex NPH subtype into consideration for the clinical NPH programme, (ii) a retrospective snapshot study was performed to confirm and characterize differences between Classic and Complex NPH patients being seen consecutively over the course of 1 year within a working subspecialist NPH clinic. We studied the characteristics of patients with Complex NPH, utilizing clinical risk stratification and multimodal biomarkers.p = 0.005). Improvement in gait and urinary symptoms did not differ between the groups. 26% of the Classic NPH group showed global improvement of the triad, and 42% improved in two domains. Although only 8% showed global improvement of the triad, all Complex NPH patients improved in gait.There was no significant difference between responders and non-responders to CSF diversion on comorbidity scales. After VPS insertion, significantly more Classic NPH patients had improved cognition compared to Complex NPH patients (via a specialist NPH programme. Further characterization of distinct cohorts of NPH with differing degrees of CSF responsiveness due to overlay from neurodegenerative or comorbidity risk burden may aid toward more precise prognostication and treatment strategies. We propose a simplistic conceptual framework to describe NPH by its Classic vs. Complex subtypes to promote the clinical paradigm shift toward subspecialist geriatric neurosurgery by addressing needs for rapid screening tools at the clinical-research interface.Our study has demonstrated that the presence of neurodegenerative disorders co-existing with NPH should not be the sole barrier to the consideration of high-volume tap test or lumbar drainage  The pathophysiology of Normal Pressure Hydrocephalus (NPH) still provokes great debate. It is classically a characterized by a clinical triad of gait disturbance, cognitive impairment and urinary incontinence in conjunction with radiographic findings of ventriculomegaly . Other nThe co-existence of NPH with neurodegenerative diseases has been reported –6, and sIn this study, we present the results of a progressive multi-year change in practice within a subspecialist NPH programme, to understand and adapt to the needs of this uncertainty. Firstly, we performed a pilot study focused primarily on characterizing the risk stratification of patients presenting with NPH but with overlay from significant vascular and neurodegenerative disease. As these patients demonstrate features of Classic NPH but both their level of CSF responsiveness and degree of comorbidities need to be quantified, we have proposed the term “Complex NPH” to distinguish this cohort from more typical patients within the INPH spectrum. Key features of this pilot include (i) the use of global risk scoring and multi-modal MRI for the characterization of Complex NPH (ii), correlation of semi-automated quantitative MRI changes with responsiveness to CSF drainage and (iii) genotyping for assessment of neurodegenerative risk potential. Our aim was to produce a well-characterized dataset of biomarkers that may serve as a basis for the future evaluation and consideration of such patients as a separate cohort of complex NPH as distinct from Classic NPH, in which more standard levels of responsiveness to CSF diversion would be expected. Following work in the pilot, we then performed a retrospective study on 1 year of consecutive patients with NPH being seen via a subspecialist clinic. We examined this unfiltered patient group for subtypes of NPH presentation, shunting intervention in cohorts of Classic vs. Complex NPH and confirmed their outcomes at 2 years.The study has two components; (i) a pilot study was performed that specifically focused upon patients with Complex NPH and following the inclusion of the Complex NPH subtype into consideration for the clinical NPH programme, (ii) a retrospective snapshot study was performed to confirm and characterize differences between Classic and Complex NPH patients being seen consecutively over the course of 1 year within a working subspecialist NPH clinic. In the pilot study, we studied the characteristics of patients with Complex NPH, utilizing clinical risk stratification and multimodal biomarkers (including imaging biomarkers and genotypic risk). We had previously published the characteristics of white matter injury in this cohort using diffusion tensor imaging (DTI) . We founIn the pilot study, 13 patients diagnosed with Complex NPH undergoing the extended CSF drainage protocol under the NPH programme at the National Neuroscience Institute, Singapore between 2016 and 2017 were selected for the study. We recruited NPH patients with known significant comorbidities, such as cardiovascular risk burden or neurodegenerative disease overlay from all patients presenting with NPH and meeting clinical criteria for further testing. Within the NPH programme, patients who were deemed to have shown significantly positive responses to high-volume tap testing were offered shunt insertion without additional supplementary testing  in at the National Neuroscience Institute from October 2017 to October 2018. 141 patients were reviewed in out-patient clinic for probable NPH, idiopathic NPH and secondary NPH. 67 out of these 141 patients were responders who demonstrated a positive response to a lumbar tap test or lumbar drainage. Only 35 responders underwent ventriculo-peritoneal shunt (VPS) insertion. Among these 35 patients, 2 patients who had VPS insertion more than 10 years ago and 1 patient who had surgery in a private hospital were excluded.Clinical descriptions for all patients met criteria, according to published guidelines, for either probable or possible NPH . PatientIn our study, patients with probable NPH demonstrated two or more features of the classic clinical triad of gait disturbance, cognitive impairment and urinary incontinence. Where patients were deemed to have possible NPH, they exhibited symptoms of either (a) incontinence and/or cognitive impairment, but without significant gait disturbance or (b) gait dysfunction or cognitive impairment alone. As per guidelines, these symptoms in patients with probable or possible NPH were not thought likely to be entirely attributable to other neurological, psychiatric, or general medical disorders, even if such disorders were found to be co-existing . All patAPOE genomic analysis. Patients with a lumbar drain in-situ underwent a three-day drainage/ seven-day global assessment protocol, achieving ≥300 mls total CSF withdrawal whereas the patient undergoing serial taps via Ommaya reservoir had a modified protocol achieving ≥150–200 mls total CSF withdrawal to account for the tolerance needed for more rapid drainage and increased infection risk from repeated tapping.In the pilot, all participants underwent insertion of a lumbar drain to according to our previously published extended CSF drainage protocol . Two parIn the retrospective study component, a final set of 32 NPH patients was included in this study. Demographic and clinical data, including age, sex, comorbidities, presenting symptoms and clinical responses after VPS were collected retrospectively from clinical records. Tinetti gait and balance scores pre- and post-VPS insertion were collected (1 Complex NPH patient did not have pre- and post-VPS Tinetti). Mini-Mental State Examination (MMSE) scores pre-VPS are reported in this study (2 Classic NPH patients did not have pre-VPS MMSE), although post-VPS MMSE scores were excluded from analysis due to missing data.We categorized these patients into the proposed conceptual framework; (i) Complex NPH, comprising 13 patients had underlying neurodegenerative diseases which include Alzheimer's disease, Parkinson's disease or parkinsonism, and vascular dementia, and (ii) Classic NPH, the other 19 patients who did not have such clinical risk burden. The diagnoses of neurodegenerative diseases were established by either a neurologist or geriatrician prior to referral to the NPH programme under the Department of Neurosurgery.Three comorbidity scales were used to risk stratify the pilot study cohort—Kiefer's Comorbidity Index, the Cumulative Illness Rating Scale, and the Charlson Comorbidity Index; each was scored by two independent raters. Kiefer's Comorbidity Index (CMI) is an NPH-specific scale, with patients scoring >3 points less likely to be shunt-responsive –17. We fIn scoring the Modified Cumulative Illness Rating Scale (CIRS), we used guidelines adapted by Salvi et al.  for eldeIn the retrospective study, we explored the use of frailty indices to describe NPH cohorts. Frailty has been shown to be predictive of health outcomes and post-operative morbidity and mortality –22. FraiMR imaging data for the pilot study were acquired with a 3.0-T MR scanner , including 3D T1, T2 and FLAIR sequences. A few patients were downgraded to the 1.5-T scanner due to clinical MR safety concerns.Fazekas scoring for white matter intensities was performed for all pre- and post-lumbar drainage FLAIR MRI sequences and independently verified by a second rater. We rated the periventricular white matter and deep white matter scores based on the presence, size and confluence of white matter lesions . ScoringAPOE genotypes were determined using Taqman® allelic discrimination assays (for SNPs rs7412 and rs429358) and genotyping was carried out on a 7,500 fast real-time PCR machine (Applied Biosystems) using standard protocols as recommended by the manufacturer.Genomic DNA was obtained from leukocytes using the QIAamp® DNA Blood Midi kit . Inpatients underwent therapist-led examinations of the 10 m walking test, Tinetti gait and balance examination, and MMSE assessments, as per our protocol for the NPH programme . After CThirty two patients underwent programmable VPS insertion between 2014 and 2018. Programmable shunt valves (with and without antisiphon devices) were implanted. Shunt valve models were decided according to surgeon's preference; predominantly, Strata (Medtronic) and proGAV (Miethke) valves were used, following all manufacturers' recommendations. Post-operative imaging, comprising a CT brain and shunt series radiographs, was performed to confirm satisfactory placement of all shunt components . Shunt valve setting adjustments were performed to optimize desired settings to match best patient functional performance. Patients underwent physiotherapy-led clinical assessments and outcomes were recorded using published NPH grading scales. All patients were followed up in outpatient clinic for at least 2 years. In addition, confirmational report was obtained from the patients or their caregivers on any improvement or deterioration in the clinical symptoms i.e., gait disturbance, cognitive impairment, and urinary incontinence.t-tests. Categorical variables were analyzed with the chi-square test. All statistical tests were two-tailed, and significance level was set at 0.05.Statistical analyses were performed using SPSS Statistics Version 23.0 . Continuous variables were analyzed with We have previously published patient characteristics from this pilot cohort elsewhere . Followi10 of 12 patients completed the Tinetti assessment at baseline, with a mean score of 15.7 ± 6.99. 8 of 12 patients were able to complete a 10 m walking test at 0-, 48- , and 72-h CSF drainage. one patient missed their 48-h assessment and responsiveness was based on the assessment at 72 h only. The remaining three patients were not able to undergo either baseline or any gait assessments and their responsiveness was based on other domains tested. Median time for the participant group to complete a 10 m walk was 12.9 Sec (IQR = 11.8–28.5 Sec) at 0 h and 14.8 Sec (IQR = 12.8–19.1 Sec) at 72 h post-CSF drainage.Thirty two patients with NPH were included in this study−13 Complex NPH with overlay from neurodegenerative diseases and 19 Classic NPH without overlay from neurodegenerative or significant vascular risks. Among the 13 Complex NPH patients, 6 patients had parkinsonism or Parkinson's disease, 4 patients had Alzheimer's disease, 2 patients had vascular dementia, and 1 patient had mixed Alzheimer's and vascular dementia. There was no significant difference in the age and sex of Complex  and Classic NPH  groups.All patients presented with gait and balance issues, with mean Tinetti score of 14.2 ± 6.12 in Complex NPH patients and 16.9 ± 8.97 in Classic NPH patients. 77% of Complex NPH and 79% of Classic NPH had cognitive impairment, with mean MMSE score of 20.2 ± 6.61 and 21.6 ± 8.37 respectively. 69% of Complex NPH and 47% of Classic NPH reported urinary symptoms. Although differences in symptom presentation between the groups were not statistically significant, 8 of 13 patients (62%) in the Complex NPH group presented with the complete triad of gait disturbance, cognitive impairment, and urinary incontinence, as compared to 7 of 19 patients (37%) in the Classic NPH group.Main comorbidities present were in the domains of hypertension (83%), neurological (83%), endocrine/metabolic (75%), genitourinary (58%), musculoskeletal (42%), and cardiac (33%). 42% had a diagnosis of parkinsonism. We were unable to distinguish vascular risk characteristics unique to responders. Mean scores were 3.83 ± 2.52 using the CMI, 1.42 ± 1.08 using the CCI, and 14.3 ± 4.70 using the modified CIRS. No significant differences were observed between responders and non-responders on all comorbidity scales, although the responders tended toward higher comorbidity scores overall.The retrospective cohort mainly had comorbidities of hypertension (78%), hyperlipidaemia (59%), and diabetes (41%). The proportion of patients with comorbid conditions was not significantly different in Complex NPH and Classic NPH patient cohorts . For fraAll patients exhibited ventriculomegaly with Evans' and Bicaudate indices ≥0.30 and ≥0.25 respectively. The degree of ventriculomegaly did not distinguish between responders vs. non-responders; there were no significant differences post-drainage in either group. CSF drainage did not influence CSF peak flow rates consistently in either group. One patient was excluded from CSF flow analysis for technical reasons. However, CSF peak flow values showed an interesting dichotomy between groups. Non-responders demonstrated both low and ultra-high values at baseline (3–15 and 65–90 ml/min), whereas responders only demonstrated CSF peak flow in a clustered range , whilst the protective ε2 allele was found in a responder with the ε2/ε3 genotype (1 in 4 responders). disease . One nonWe have previously reported clinical outcomes for this pilot study cohort of patients with Complex NPH . In summPost-surgery, three patients had infections requiring shunt removal, and one patient underwent a shunt revision due to blockage of the shunt. Two patients passed away within 3 years of shunt insertion; one developed sepsis secondary to ventriculitis, and the other had a subdural haematoma after a fall .p = 0.005). Improvement in gait and urinary symptoms was not significantly different between the groups. 26% of the Classic NPH group showed global improvement of the triad, and 42% improved in two domains. Although only 8% showed global improvement of the triad, all Complex NPH patients improved in gait. Tinetti scores were significantly higher in both groups after VPS insertion (p <0.001 in Complex and p = 0.002 in Classic NPH).After VPS insertion, significantly more Classic NPH patients had improved cognition compared to the Complex NPH group   . Our datSuch findings are consistent with observations previously noted in NPH literature on comorbidities . The ISHSuch global risk scales also distinguished between comorbidity risks and responses to CSF drainage. Our study revealed that responders and non-responders had differing profiles of comorbidity risks. A higher overall burden of comorbidities significantly correlated with cognitive impairment in the dementing range at presentation. Certain domains appeared predominant within Complex NPH patients, i.e., vascular risk burden (cardiac disease and hypertension domains) and functional symptomatology . Hypertension and parkinsonism outweighed other comorbidities in predisposing to non-response following extended CSF drainage. Surprisingly, we found a heavy load of individual comorbidity risks in responders. These included dementia (other), cardiac disease and hyperlipidaemia , cerebrovascular disease (13% higher), as well as diabetes (three times as common). We therefore concluded that there was no evidence that individual comorbidity risks directly correlated with lack of response, but some risks appeared more important than others. Overall, as expected, most of the group were non-responders. The response rate in Complex NPH was lower than that expected of Classic NPH (approximately 33% vs. 60–70% responders) , 27. HowThe striking trends of comorbidity burden in responders may be due to the smaller sample of responders, leading to overrepresentation of risks. However, certain patterns merit further discussion. NPH patients are known to have a higher vascular risk burden than expected . HyperteWe also found that vascular imaging burden, namely periventricular and deep white matter hyperintensities, did not distinguish between responders and non-responders. White matter lesions did not improve following CSF drainage in either group. Whilst it could be argued such lesions should be permanent, other groups have demonstrated changes in white matter hyperintensities following CSF drainage in NPH . EquallyWe found that, in Complex NPH, CSF peak flow rates were dichotomized according to group. Non-responders demonstrated CSF peak flow rates at both extremes of low and ultra-high values. By contrast, CSF peak flow rates for responders clustered near the thresholds of 18–24.5 ml/min found in classic shunt-responsive NPH , 30. CSFOur retrospective study focused on comparing patients with Classic NPH vs. Complex NPH; the latter group comprise patients presenting with features of NPH, as well as a spectrum of neurodegenerative diseases including parkinsonism/Parkinson's disease, Alzheimer's disease, and vascular dementia. The patients in the group termed Complex NPH were older than the group with Classic NPH. This finding is consistent with current published literature , 3, 8, 9It is not surprising that the group without neurodegenerative disorders did better. However, it was striking that, despite older age and overlay from neurodegenerative disease, all patients with Complex NPH showed improvement in their gait. However, only 15% in this group showed improvement in cognition or urinary incontinence. The published literature regarding coexistence of Alzheimer's disease and NPH are few and their conclusions vary. Savolainen et al. postulated that the relatively high prevalence of Alzheimer's disease in patients with NPH may explain the unsuccessful recovery of many patients after shunt placement and hence recommended cortical brain biopsy . Convers99mTc- TRODAT-1 SPECT scans to look for dopaminergic degeneration in order to predict the surgical outcomes because this comorbidity was thought to neutralize the degree of improvement after surgery , although the APOE ε2 allele phenotype, thought to be a protective factor, was only exhibited in one responder. The presence of this protective allele in NPH and its contribution to CSF responsiveness is largely unknown in literature but we note our small sample size is a limitation in our further interpretation of these results.Indeed, some authors have suggested that NPH and Alzheimer's disease are closely connected and may simply occupy different extremes of a common spectrum of disease . The depThe myriad of neurodegenerative disease subtypes and presentations demonstrates the value of a more simplistic framework of NPH distinguishing Classic vs. Complex NPH cohorts. This is because the specific diagnostic criteria, or lack thereof, of neurodegenerative disorder is not as critical to CSF responsiveness as quantifying the “NPH component of disease” remediable to surgical intervention, regardless of such overlay. We apply the same rationale to the inclusion of significant comorbidities as criteria for Complex NPH; specifically scoring individual comorbidity and frailty burdens alone did not distinguish the two NPH cohorts, whereas regarding their clinical risk stratification as a whole was more effective in separating their differing thresholds of outcomes. In more recent work, we have suggested a further refinement of the term Complex NPH, by expanding on our definition for consistency , to addrOur study had several limitations. Firstly, our pilot study focused upon the assessment of global comorbidity risks in patients with complex NPH undergoing extended CSF drainage, without considering the wider implications of such findings either to longer-term shunting or the pathophysiology of NPH itself. Further work is needed to characterize the contribution of specific components of vascular risk burden , as well as individual neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. Functional outcome is difficult to test in such patients. The group size was small and reflects both the relatively low incidence of NPH, as well as the smaller subgroup of complex NPH within the overall spectrum of disease.Despite this, our study numbers do match previous published cohort sizes for extended lumbar drainage . Due to Indeed, our larger retrospective study demonstrated much lower complication rates specific to shunting interventions, although decline and mortality from overlay of other conditions were still important determinants of longer-term outcomes. In addition, as described above, our aim was to develop a more simplistic conceptual framework for rapidly screening NPH subtypes according to expectations of treatment outcomes. More precise tools for confirming and characterizing neurodegenerative disorders, such as the use of brain biopsy, CSF biomarkers and advanced imaging tools, such as SPECT and DTI, may offer more sophisticated ways of distinguishing these cohorts.Our study has demonstrated that the presence of neurodegenerative disorders co-existing with NPH should not be the sole barrier to the consideration of high-volume tap test or lumbar drainage via a specialist NPH programme. Further characterization of distinct cohorts of NPH with differing degrees of CSF responsiveness due to overlay from neurodegenerative or comorbidity risk burden may aid toward more precise prognostication and treatment strategies. We propose a simplistic conceptual framework to describe NPH by its Classic vs. Complex subtypes to promote the clinical paradigm shift toward subspecialist geriatric neurosurgery by addressing needs for rapid screening tools at the clinical-research interface.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the SingHealth Centralised Institutional Review Board. Written informed consent was obtained from participants of the pilot study. The ethics committee waived the requirement of written informed consent for participation in the retrospective study.NK, CL, and ETG contributed to the conceptualization and design of the study and wrote the manuscript. ETG, CL, AT, BLT, SL, and JK contributed to data collection and analysis. SK contributed to design of the imaging protocol as well as interpretation. AA–A and VN contributed to genomic analysis. RP, YJT, AN, EKT, ZC, MC, and JP contributed to the characterization of the NPH cohorts and provided feedback on the project. All authors contributed to the article and approved the submitted version.This study was supported by grants from the National Neuroscience Institute Center Grant (NCG CS03) and the National Medical Research Council (NMRC/TA/0024/2013 and MOH-CSAINV18nov-0005).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Methods: Using multi-omics data from two independent consortia, we analyzed data from dozens of tumor types to identify patient cohorts characterized by poor outcomes, genomic instability, and wildtype BRCA genes. We developed several novel metrics to identify the genetic underpinnings of genomic instability in tumors with wildtype BRCA. Associated clinical data was mined to analyze patient responses to standard of care therapies and potential differences in metastatic dissemination. Results: Systematic analysis of the DNA repair landscape revealed that defective single-strand break repair, translesion synthesis, and non-homologous end-joining effectors drive genomic instability in tumors with wildtype BRCA and BRCA-related genes. Importantly, we find that loss of these effectors promotes replication stress, therapy resistance, and increased primary carcinoma to brain metastasis. Conclusions: Our results have defined a new pan-cancer class of tumors characterized by replicative instability (RIN). RIN is defined by the accumulation of intra-chromosomal, gene-level gain and loss events at replication stress sensitive (RSS) genome sites. We find that RIN accelerates cancer progression by driving copy number alterations and transcriptional program rewiring that promote tumor evolution. Clinically, we find that RIN drives therapy resistance and distant metastases across multiple tumor types.In the past decade, defective DNA repair has been increasingly linked with cancer progression. Human tumors with markers of defective DNA repair and increased replication stress exhibit genomic instability and poor survival rates across tumor types. Seminal studies have demonstrated that genomic instability develops following inactivation of BRCA1, BRCA2, or BRCA-related genes. However, it is recognized that many tumors exhibit genomic instability but lack BRCA inactivation. We sought to identify a pan-cancer mechanism that underpins genomic instability and cancer progression in BRCA-wildtype tumors.  Large-scale sequencing efforts have enabled the discovery of genetic events that drive cancer development ,2,3,4,5.Decades of research has revealed processes dysregulated by cancers, summarized as hallmarks of cancer ,7. Of thMYBL2 expression is a robust marker of poor patient outcomes across tumor types and genotypes. Importantly, this MYBL2 High cohort is defined by genomic instability despite containing wildtype BRCA. Analysis of the DNA repair landscape revealed that the genetic basis of MYBL2 High disease are heterozygous losses of single-strand break repair, translesion synthesis, or non-homologous end-joining effectors. These genetic lesions subject MYBL2 High tumors to significant replication stress. Functional clustering of replication stress-sensitive sites revealed that elevated replication stress promotes copy number alterations that rewire transcriptional programs and impact hallmarks of cancer master regulators. Clinically, this phenotype identifies patients at risk for poor outcomes when treated with chemotherapy and irradiation. Furthermore, our results demonstrate that MYBL2 expression stratifies patient risk for distant metastases, especially to the brain. Our data defines a new pan-cancer class of tumors driven by replicative instability (RIN), unifying seemingly disparate cancers. Moreover, these results describe how RIN accelerates cancer progression by impacting several hallmarks of cancer.Previous work from our group revealed that elevated expression of the transcription factor MYB proto-oncogene like 2 (MYBL2) identified lung adenocarcinomas with genomic instability and wildtype BRCA . In thisMYBL2 High and MYBL2 Low cohorts using a quartile method; the top 25% of samples expressing MYBL2 were called MYBL2 High and the bottom 25% MYBL2 Low.Thirty-two tumor types curated by the TCGA and others were analyzed . Where mThe Kaplan-Meier estimator was used to estimate time-to-event distributions for OS, DSS, and PFS comparing MYBL2 High and MYBL2 Low quartiles. The log-rank test was used to test for significant differences between distributions using a two-sided test. OS denotes the time from initial diagnosis until death. DSS is the time from diagnosis until the time of death; patients who died from other causes were not included. PFS reflects the time from diagnosis until progression or death. For all survival analyses, patients who did not experience an event or were lost to follow-up were censored at time of last contact. Kaplan-Meier survival analyses were conducted using survival and survminer packages .For each pathway, we identified comprehensive lists of pathway effectors through extensive literature review ,23,24,25Correlations between WE values were calculated and visualized using stats and corrplot R packages .https://cancer.sanger.ac.uk/signatures/downloads/). Eighteen of the 78 SBS mutational signatures capturing likely artifacts were excluded. Statistical significance between average signature weights across samples was assessed using two-sided Student’s t-tests.COSMIC SBS v3.2 signatures were generated using deConstructSigs . For TCGEight gene ontology (GO) terms were identified that capture key processes involved in replication stress responses . Genes were pooled and redundant entries removed to generate a final list (n = 205). The RS score is the sum of gene log2 mRNA expression values, divided by the number of genes in the RS response gene list. Differences in medians were assessed for statistical significance using Wilcoxon signed rank tests .RS ScorThe MPS score is the difference between somatic mutation location and the gene start, divided by gene length.www.ensembl.org). Differences in MPS densities were assessed for statistical significance using Kolmogorov-Smirnov tests.Mutation locations were obtained from the TCGA public MAF file. Gene start and end positions were obtained from the Ensembl genome browser  was developed to map the origin of RNA-seq detected fusions. Tool rules were determined through literature review . T. T43]. Td GTF2H5 C. Also, ectively . SBS21 wco smoke . Importaough NER . This anMYBL2 High tumors experience elevated replication stress, we developed a novel metric called the replication stress score (RS score) (Methods). This metric captures all major pathways involved in sensing replication stress, protecting and processing stalled forks, and the rescue of replication . In. In50]. MYBL2 expression identified patients with poor responses to therapy, we analyzed 25 tumor types provided by the Oncology Research Information Exchange Network (ORIEN). Kaplan-Meier analysis demonstrated that MYBL2 High patients had significantly worse OS outcomes when treated with chemotherapeutics or irradiation across multiple cohorts . Analysis of COSMIC signatures confirmed resistant MYBL2 High tumors demonstrate footprints of defective SSBR and TLS  and ID-BRE (axillary lymph nodes) . When comparing h nodes) B. Howevetropisms . Using cMethods, C. These  samples D. These MYBL2 expression identified tumors with genomic instability, inefficient HR, and wildtype BRCA  E. The deMYBL2 expression and RIN are intimately linked. The association of MYBL2 with RIN is both direct and indirect. In normal cells, MYBL2 is transcriptionally and post-translationally regulated by the cell cycle [MYBL2 mRNA is a robust marker of cells arrested prior to mitosis. In this study and our previous work, we have demonstrated that MYBL2 expression is tightly associated with upregulation of DNA repair genes that sense replication stress [MYBL2 expression and activity are robust markers of RIN. While we find that MYBL2 expression robustly identifies patients with poor outcomes across tissue types, it should be noted that MYBL2 expression as a single marker did not stratify all known tumor types. Moving forward, it will be imperative to identify additional biomarkers that can identify this aggressive RIN phenotype that likely extends to other tumor types besides those described here , checkpoint signaling, and error-prone repair (MMEJ) pathways confer resistance to these therapies ,55. Becag others . DysreguLAG3 and TIGIT, despite showing no difference in PDL1 (data not shown). This raises the possibility that anti-LAG3 and anti-TIGIT inhibitors may be better suited for treating RIN+ tumors. One of our most important discoveries is that increased MYBL2 expression stratifies patient risk for brain metastases. While the average risk for brain metastases for all lung cancers is ~15%, we find that MYBL2 High LUAD patients have a risk of ~40% while MYBL2 Low have a risk of <10%  has been viewed as the major driver of genomic instability. Data presented in our manuscript support an additional model wherein thousands of intra-chromosomal gains and losses are driven by stalled or collapsed replication intermediates as opposed to chromosomal missegregation events. Collectively, our data demonstrate that there exists a pan-cancer class of tumors driven by replicative instability (RIN), unifying seemingly disparate tumors.Moving forward, further study of RIN is urgently needed. Immunohistochemistry markers need to be identified and validated. New mouse models and cell line systems are required to identify therapeutic vulnerabilities that can be explored in clinical trials. Any advances in targeting RIN have the potential to drastically improve patient outcomes across tumor types.


Within the multiple-metal model, Pb, As, and Zn were associated with increased TNF-α levels with effects dedicated to the mixture of 50%, 31%, and 15%, respectively. Grouped WQS revealed that the essential metal group showed a significantly negative association with TNF-α and kidney function. The toxic metal group found significantly positive associations with TNF-α, serum creatinine, and WBC but not for eGFR. These results suggested Pb, As, Zn, Se, and mixtures may act on TNF-α even through interactive mechanisms. Our findings offer insights into what primary components of metal mixtures affect inflammation and kidney function during co-exposure to metals; however, the mechanisms still need further research.Exposure to heavy metals could lead to adverse health effects by oxidative reactions or inflammation. Some essential elements are known as reactors of anti-inflammatory enzymes or coenzymes. The relationship between tumor necrosis factor alpha (TNF-α) and heavy metal exposures was reported. However, the interaction between toxic metals and essential elements in the inflammatory response remains unclear. This study aimed to explore the association between arsenic (As), cadmium (Cd), lead (Pb), cobalt (Co), copper (Cu), selenium (Se), and zinc (Zn) in blood and TNF-α as well as kidney function. We enrolled 421 workers and measured the levels of these seven metals/metalloids and TNF-α in blood; kidney function was calculated by CKD-EPI equation. We applied weighted quantile sum (WQS) regression and group WQS regression to assess the effects of metal/metalloid mixtures to TNF-α and kidney function. We also approached the relationship between metals/metalloids and TNF-α by generalized additive models (GAM). The relationship of the exposure–response curve between Pb level and TNF-α in serum was found significantly non-linear after adjusting covariates ( Heavy metals, for instance, arsenic (As), cadmium (Cd), and lead (Pb), are widely utilized in industry . Based oNevertheless, several metal/metalloids are essential elements that play key roles in metabolism and antioxidant reactions . Cobalt Tumor necrosis factor-alpha (TNF-α) is primarily released from macrophages . TNF-α hOccupational exposure to heavy metals during manufacturing processes is well documented in many industries ,30. WorkIn this study, we explored the associations among seven serum biomarkers for metals, including white blood cells, serum creatinine, TNF-α, and estimated glomerular filtration rate (eGFR). In order to evaluate the effect of single-metal and metal mixture exposures on outcomes, we applied four models: (i) the linear regression model, (ii) the generalized additive model (GAM), (iii) the generalized weighted quantile sum (WQS) regression , and age [The participants for this research were 462 workers enrolled in annual health examinations between 2009 and 2010 in the Kaohsiung Medical University Hospital in Taiwan. We excluded subjects with rheumatoid arthritis, cancer, and chronic kidney disease, those <20 or >80 years old, those who failed to offer specimens, those with eGFR <60 mL/min/1.73 m.993)age . R Stati.993)age , gWQS [4.993)age , and gro.993)age  packages® RECIPE  control Level I No. 8880 and Level II No. 8881, to ensure quality control and quality accuracy during analysis. With the SEMs, coefficients of variation were less than 3% for the measurements at high levels (Level II No. 8881) and were less than 5% for the measurements at low levels (Level I No. 8880). The serum from each subject was separated from coagulated blood samples. Serum concentrations of TNF-α were measured by the protocol of Human TNF-alpha Quantikine ELISA Kit measurement (R&D Systems).We collected blood samples at annual health examinations and stored them at 4 °C. We used dilution solution, blending solution with 0.2% ammonia solution, 0.1% Triton X-100, and 0.3% HNO3. All blood samples were analyzed via inductively coupled plasma mass spectrometry (ICPMS) of Thermo Scientific XSERIES 2 in the Kaohsiung Medical University Hospital. We tested the following metal and metalloid concentrations in blood: As, Cd, Pb, Se, Co, Cu, and Zn. The procedure for analyses was customized from the method of Ebba Barany in 2007 . We usedThe white blood count and serum creatinine levels were analyzed in the clinical biochemical laboratory in the Kaohsiung Medical University Hospital. All laboratories in the University Hospital were qualified by the Taiwan Accreditation Foundation, proving their quality assurance and quality control (QA/QC).Characteristics of participants were presented as number (%) or mean ± standard deviation (SD). Pearson correlations were followed to demonstrate the association between metals and metalloids levels and TNF-α. Multiple linear regression models were used to examine the association between serum TNF-α level and the seven elements. Model 2 was adjusted covariates for gender, age, BMI, alcohol consumption, and smoking status. The equation of the multivariable linear regression model was:We implemented GAM to survey the dose–response assessment between metals/metalloids and the ratio of TNF-α and white blood cells, adjusted for gender, age, BMI, alcohol consumption, and smoking status. In GAM analysis, the value of the effective degree of freedom (EDF) was greater than 1, which means a complicated relationship between response and predictor. We used smooth functions to predict and plot the relationships between metals/metalloids and the ratio of TNF-α and white blood cells in multivariable GAM.To identify the association of seven elements and explore the weights of the individual elements, we applied WQS regression. The typical WQS model is as follows:p-value of the weighted index by a holdout dataset in a generalized linear model are crucial parts of gWQS regression. To reduce collinearity and dimensionality, the weights of components are controlled to be 0 to 1. We can identify the prime contribution to the associations between mixtures of levels of As, Cd, Pb, Co, Cu Se, Zn, and TNF-α in serum.The WQS index represents the weight of each element component. We utilized gWQS regression to certify the association of the mixture with TNF-α, WBC, serum creatinine, the ratio of TNF-α and white blood cells, and eGFR. Estimating the weighted index of the components  in bootstrap samples of the random training subset and assessing the We also employed the grouped WQS regression to identify the health effects of different mixtures. As, Cd, and Pb are known nephrotoxicants . In thisp < 0.001), between Pb and TNF-α levels.The demographic characteristics of the study are shown in p < 0.001), Cd , Pb , Co , Cu , and Zn  levels in serum by multivariable linear regression. There were no significant associations with Se. Similarly, the associations for serum creatinine, eGFR, and each serum metal level were significant. Moreover, the associations between Cd , Pb , Se , Cu , and Zn  levels and WBC were significant. There were no significant associations with As and Co. The associations between metal/metalloid levels and TNF-α, WBC, serum creatinine, eGFR, and the ratio of TNF-α and WBC are shown in Adjusted for covariates, an increased risk of TNF-α was significantly associated with As , As , and the ratio of TNF-α and WBC, respectively. On the other hand, the evaluation of metalloids , Se , Zn ) and the ratio of TNF-α and WBC indicated significant non-linear relationships.Plots for the predicted smooth relationships of each metal/metalloid and the ratio of TNF-α and WBC are presented in p < 0.001), As , Cd  and eGFR, respectively. On the other hand, the evaluation of the metalloids , Zn , and Cu ) and the eGFR indicated significant non-linear relationships.Plots for the predicted smooth relationships of each metal/metalloid and eGFR are illustrated in p < 0.001) after adjusting for gender, age, BMI, alcohol consumption, and smoking status, as shown in In WQS regression analysis, our results showed that the WQS indices were positively associated with TNF-α. We observed that the toxic metal mixtures had significant effects on TNF-α levels , where Zn was highest (63%), followed by Co (26%) and Cu (7%), as shown in p < 0.001)  .p < 0.001)  . Pb, As,p < 0.001) after adjusting covariates, as shown in p < 0.001) (p < 0.001) were associated with serum creatinine due to Zn (79%) and Co (21%). Our results showed the essential metals mixture consisting of Zn (77%) and Co (23%) was significantly associated with eGFR  (p < 0.001), as shown in The WQS regression models for toxic metal and metalloid mixture indices both were associated with serum creatinine and eGFR but not WBC. We observed that toxic metals mixtures were significantly associated with serum creatinine  , primari< 0.001) . Within p < 0.001). The mixture of metalloids was positively significantly associated with WBC and serum creatinine, with the exception of TNF-α  and the ratio of TNF-α and WBC , eGFR .In our study, we found an association between mixtures of metals/metalloids, TNF-α, and renal function. We utilized different statistical strategies to examine the effects of serum metals and their mixtures. Based on the viewpoint of each element, the multivariable linear regression examined single metal/metalloids and adjusted covariates. This study also found that serum Pb, Se, and Co levels expressed a non-linear relationship with TNF-α and eGFR. Our main finding was the significant relationship between the weights of all types of mixtures and TNF-α and eGFR among workers in metal industries. In addition, the grouped WQS regressions provided quantifiable insight into the individual weights of the mixtures between essential metals and toxic metals. In grouped WQS regressions, the toxic metals significantly increased the level of TNF-α compared with the essential metals.Whether in the occupational area or general environment, an individual may possibly be exposed to multiple metals everywhere simultaneously ,45. HengWe observed a relationship between co-exposure to the toxic metal mixture  as measured in serum and lower eGFR. Sanders et al. found associations between metals  in urine with eGFR, but there was no significant difference between blood metal mixtures in the general population by WQS regression . ResearcThese findings for single metals were in line with previous studies. The levels of TNF-α were affected by occupational exposure of workers to Pb and Cd (median serum TNF-α: 56 pg/mL) versus workers who were not exposed (median serum TNF-α: 22.4 pg/mL) ,59. The A strength of our study was that the WQS regressions and grouped WQS regressions were used to examine the effect of metal/metalloid mixtures on inflammation and renal function in workers. In addition, seven metal/metalloids and TNF-α levels in serum were surveyed to comparatively and comprehensively express internal exposure. We acknowledge additional study limitations. We cannot determine causal relationships between the levels of serum for multiple elements and inflammatory biomarkers, serum creatinine, and renal function in this cross-sectional study. The lack of information on seafood consumption and supplements could lead to a gap in our results ; howeverThis study demonstrated that metal/metalloid mixtures in serum were associated with TNF-α levels and eGFR by GAM. Heavy metals are still one of the crucial environmental hazards associated with inflammation and kidney health. The effects of metal co-exposure are likely modified by the concentration and duration of exposure, genetic predisposition, and other individual factors. Further epidemiological studies in a framework with more inflammatory cytokines and biomarkers of oxidative stress and controlled environmental and individual factors would increase the clarity regarding the mechanisms of the metal/metalloid mixtures in occupational exposure.