LINC00511/hsa-miR-573 axis-mediated high expression of Gasdermin C associates with dismal prognosis and tumor immune infiltration of breast cancer

Breast cancer (BC) is considered the second commonest human carcinoma and the most incident and mortal in the female population. Despite promising treatments for breast cancer, mortality rates of metastatic disease remain high. Gasdermin C (GSDMC) is an affiliate of the gasdermin (GSDM) family, which is involved in the process of pyroptosis. Pyroptosis is implicated in tumorigenesis, but the role of GSDMC in cancer cells is yet to be fully elucidated. In this study, we investigated the role and mechanism of GSDMC in breast cancer. We conducted a pan-cancer analysis of the expression and prognosis of GSDMC utilizing multidimensional data from The Cancer Genome Atlas (TCGA). We investigated GSDMC expression levels in 15 BC tissues and matched adjacent normal tissues by immunohistochemistry (IHC). Further verification was performed in the Gene Expression Omnibus (GEO) database. We discovered that elevated GSDMC expression was considerably linked to a worse prognosis in breast invasive carcinoma (BRCA). Next, we identified noncoding RNAs (ncRNAs) which contributing to higher expression of GSDMC by a series of expression, survival, and correlation analysis. We finally identified LINC00511/hsa-miR-573 axis to be the most promising ncRNA-associated pathways that account for GSDMC in BRCA. Furthermore, we demonstrated the significant correlations between GSDMC expression and immune infiltrates, immune checkpoints, and immune markers in BRCA. This study illustrated that ncRNAs-mediated upregulation of GSDMC linked to dismal prognosis and also exhibited a correlation with tumor immune cell infiltration in BRCA. It is anticipated to offer novel ideas for the link between pyroptosis and tumor immunotherapy.

www.nature.com/scientificreports/ studies have shown that GSDMA, GSDMB, GSDMD, GSDME, and GSDMF perform a crucial function in cell death, inflammation, and autoimmunity 19,24 . But the biological function of GSDMC has not been identified 25 . GSDMC is also known as leucine zipper-containing extranuclear factor 26,27 . Several investigations show that ultraviolet (UV) radiation increased the expression of GSDMC, and GSDMC may have an instrumental function in the triggering of ERK and JNK pathways which result in UV-induced MMP-1 expression 28,29 . Some scholars suggest the expression levels of GSDMC may be associated with the development of lumbar spinal stenosis. Past research reports have indicated that GSDMC could have an important function during tumorigenesis including cell proliferation in colorectal cancer as well as enhanced metastatic prospects in melanoma cells 28,30 . Recent studies demonstrate that the transcription of GSDMC is enhanced by PD-L1 interacts with p-Stat3 as well as its nuclear translocation under hypoxia 12 . The metabolite α-KG induces death receptor 6-activated caspase-8 which activates the GSDMC-dependent pyroptosis pathway in cancer cells, causing tumor necrosis 15 . Nevertheless, the exact functions of GSDMC are still inadequately investigated and need to be further elucidated.
In this research, we systematically examined the GSDMC expression and its link to the prognosis of pantumors utilizing multidimensional data from the TCGA and GEO databases. Validation immunohistochemical experiments were performed. Next, we identified microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) that accounts for GSDMC in BRCA. Furthermore, we demonstrated the associations between GSDMC expression and immune infiltrates, immune checkpoints, and immune markers in BRCA. Finally, we employed the cBioPortal online tool to evaluate modifications, mutations, and pathways of GSDMC in BRCA. This research illustrated that ncRNAs-mediated upregulation of GSDMC was linked to dismal prognosis and also exhibited a correlation with tumor immune cell infiltration in BRCA. Figure 1 exhibited the overall design, workflow and results of this study.
Next, we validated the link between the levels of GSDMC expression and patient prognosis in multiple cancer kinds via the GEPIA database and KM plotter database (Figs. 5, 6, Figs. S2, S3). In GEPIA, we discovered that elevated mRNA expression levels of GSDMC were linked to worse OS in BRCA (OS, HR = 1.4, P = 0.024), KICH (OS, HR 6.9, P = 0.034), LIHC (OS, HR 1.5, P = 0.034) and with poorer DFS in KIRP (DFS, HR 2.2, P = 0.011), and PAAD (DFS, HR 1.8, P = 0.008) (Fig. 5A,E,H,K,N). On the contrary, elevated mRNA expression levels of GSDMC were linked to improved prognosis in LGG (OS HR 0.6, P = 0.0054; DFS, HR = 0.72, P = 0.037) and better DFS (DFS HR 0.36, P = 0.00091) in CESC (Fig. 5D,I,J), but showed no significant correlation in other tumors (Fig. 5B,C,F,G,L,M, Fig. S2). Then we utilized the KM plotter to explore the link between the levels of GSDMC expression and patient prognosis in various pan-cancer types. As depicted in Fig. 6 (Fig. 6E), but showed no significant relationships with RFS in COAD (RFS, HR 1.1 (0.4-3.04), P = 0.85) (Fig. S3). Taken together, the combination of OS, RFS, DSS, DMFS, and PPS and concern of bias, our findings illustrated the expression levels and prognostic value of GSDMC in several kinds of cancers, GSDMC might perform as a negative prognostic biomarker in BRCA patients. However, much further research is needed to investigate the link between the expression of GSDMC and cancer patient prognosis in other kinds of cancers, including PAAD, COAD, KICH, etc.
Protein expression analysis and prognosis analysis of GSDMC in BC. Subsequently, IHC was performed to validate the expression of GSDMC in 15 pairs of BC tumor tissues and corresponding adjacent normal tissues. IHC staining analysis exhibited that GSDMC was mainly localized in the cytoplasm of cancer cells, and brown staining indicated positive staining (Fig. 7C,D). Weak to no expression of GSDMC were observed in the normal tissues (Fig. 7A,B). Statistical analysis revealed that GSDMC was also expressed significantly highly in BC tissues than in the adjacent non-tumor tissues (P < 0.001) (Fig. 7E). Survival analysis showed that high protein expression of GSDMC had worse PFS in BC, however, there was no statistically significant (P > 0.05) (Fig. S4A).
GSDMC expression and prognosis analysis of BC in GEO database. Then, we utilized GEO database to perform expression and survival analyses of GSDMC in BC. Expression analysis indicated that mRNA  www.nature.com/scientificreports/ expression levels of GSDMC were significantly higher in BC tissues than in normal control tissues in GSE29431 and GSE31448 (P < 0.05) (Fig. 8A,B). Survival analysis of GSE42568 exhibited significant relationships between high expressions of GSDMC and worse OS in BC patients (P < 0.05) (Fig. 8C). Whereas high mRNA expressions of GSDMC showed no significant relationships with RFS in BC (P > 0.05) (Fig. 8D). Thus, further experimental validation is needed.
Prediction and analysis of potential miRNA candidates of GSDMC. ncRNAs are well-recognized for regulating gene expression at almost every step [31][32][33] . In order to determine whether GSDMC was regulated by various ncRNAs in BRCA, we forecasted potential candidate miRNAs that might bind to GSDMC and ultimately identified 15 miRNAs. We used various target gene forecasting website, comprising of miRDB, miRmap, TargetScan, miRcode, miRWalk and DIANA-microT to establish a miRNA-GSDMC regulatory network (Fig. 9A). Because the action mechanism of miRNAs negatively modulates the GSDMC expression at the posttranscriptional level, there should be a negative relationship between GSDMC and miRNA in BRCA. So, we investigated the relationship between GSDMC and 15 miRNAs in BRCA via the TCGA database. As a result, we found that GSDMC was significantly negatively associated with hsa-miR-573 (MIR573) and positively linked to hsa-miR-548ao-5p (MIR548AO) in BRCA (P < 0.001, Fig. 9A, Table 1). Meanwhile, no statistical expression relations were observed between GSDMC and other miRNAs (Fig. 9A, Table 1). Then we explored the miRNA expression levels of hsa-miR-573 in 1109 tumor tissues and 113 adjoining tissues from BRCA in the TGGA dataset. The outcomes indicated that the levels of hsa-miR-573 expression were lower than normal tissue control in BRCA (Fig. 9B). Subsequently, we ascertained the link between hsa-miR-573 expression levels and BRCA patient prognosis in the TCGA database. We discovered that elevated miRNA expression level of hsa-miR-573 was considerably linked to improved DSS in BRCA (DSS, HR 0.62 (0.40-0.92), P = 0.039) (Fig. 9D). Meanwhile, the hsa-miR-573 expression level was linked to improved OS in BRCA, but it was not statistically significant (OS, HR 0.76 (0.55-1.05), P = 0.095) (Fig. 9C). Taking together survival analysis, expression analysis, and correlation analysis, we suggested that hsa-miR-573 might serve as potential regulating miRNA for GSDMC in BRCA.
Evaluation of potential candidate lncRNAs of hsa-miR-573. Then we forecasted upstream potential lncRNAs that interact with hsa-miR-573 by using DIANA-LncBase v.2. A total of 39 possible lncRNAs were selected as candidate lncRNAs in breast tissues and mammary gland tissues (Threshold > 7). A lncRNA-hsa-miR-573 regulatory network was visualized using Cystoscope software (Fig. 10A). The competitive endogenous RNA (ceRNA) hypothesis suggests that lncRNA reduces the suppressive miRNA-effect on target-mRNAs. Therefore, in the ceRNA network, lncRNA should be positively correlated with target mRNA while lncRNA should be negatively correlated with target miRNA. Correlation analysis of hsa-miR-573 expression and 39 lncRNAs was done in the TGGA breast cancer database. The results highlighted that only LINC00511 was negatively associated with hsa-miR-573, and positively associated with GSDMC ( Fig. 10B). Then we took expression analysis in the TCGA set. As a result, we discovered that the levels of LINC00511 expression were considerably upregulated in BRCA as opposed to normal controls (Fig. 11A). We used the GEPIA database to validate, GEPIA results were consistent with the aforementioned results (Fig. 11B). Subsequently, we assessed the prognostic values of LINC00511 in BRCA. We observed that elevated mRNA expression levels of hsa-miR-573 were considerably In GEPIA database, elevated LINC00511 expression was also substantially linked to worse OS (HR 1.7, P = 0.03) in BRCA (Fig. 11C). External validation was carried out using two GEO databases (GSE29431, GSE42568). In GSE29431, the levels of LINC00511 expression were considerably upregulated in BC as opposed to normal controls (P = 0.00076) (Fig. S4B). The survival analysis showed high expression of LINC00511 might increase the risk of death for BC; however, this was not statistically significant (P = 0.064) (Fig. S4C). Taking into account survival analysis, expression analysis, as well as correlation analysis, LINC00511 might be the key potential upstream lncRNA of the GSDMC/hsa-miR-573 axis in BRCA.
Association of GSDMC, hsa-miR-573 and LINC00511 expression levels and prognosis in patients with molecular subtyping of BRCA . Molecular subtyping provides precision treatment guidance in BRCA. Thus, we utilized KM plotter to assess the association between expression levels of GSDMC, hsa-miR-573, LINC00511 and prognosis in patients with differently molecular subtyping of BRCA (Fig. 12).
Interestingly, results showed that only in luminal B BRCA, high mRNA expression levels of GSDMC and LINC00511 were significantly associated with dismal prognosis (P < 0.05), as well as high mRNA expression   Table 2). Remarkably, in BRCA, the levels of GSDMC expression were considerably negatively linked to Th2 markers (GATA3, STAT6) (P < 0.01, Table 2). These findings supported that GSDMC was considerably related to immune infiltrating cells as well as the immune microenvironment in BRCA.
The relationship between GSDMC and immune checkpoints in BRCA . SIGLEC15  www.nature.com/scientificreports/ to immunological checkpoints that perform a vital function in tumor immune evasion. Taking into account that GSDMC might be the potential oncogene in BRCA, the relationship of GSDMC with PDCD1LG2, SIGLEC15, LAG3, TIGIT, CTLA4, CD274, PDCD1, and HAVCR2 were assessed. As a result, we found that the expression levels of GSDMC were a significant positive correlation with PDCD1LG2, TIGIT, LAG3, CD274, CTLA4, HAVCR2, and PDCD1 in BRCA (Fig. 13B). On the contrary, GSDMC expression was a significant positive correlation with SIGLEC15 in BRCA (Fig. 13B). These findings indicated that tumor immune evasion and antitumor immunity might be implicated in GSDMC facilitated carcinogenic processes of BRCA.

Discussion
Breast cancer is the considered highest incident cancer type and the first contributor to cancer-associated fatality in women worldwide 10,34 . Based on molecular phenotyping and genotyping, targeted therapies and immunotherapy of BC have rapidly evolved in recent years 1,5,9 . However, mortality rates of BRCA remain high and treatments are limited 2,35 .
Early studies have demonstrated that pyroptosis is correlated to tumors 14,36 . Pyroptosis can enhance antitumor immunity for its immunogenic nature 37 . Recently research demonstrates pyroptosis of tumor cells may overcome tumor cells' apoptosis resistance and promotes antitumor immunity 38,39 . However, the specific mechanism of tumors pyroptosis remains poorly understood.
GSDMC, as an affiliate of the GSDM family, is mainly expressed in the skin, spleen, trachea, intestines, bladder, and gastrointestinal 26,28,40 . Some studies suggest that GSDMC expression level is correlated with some tumors, such as lung adenocarcinoma, metastatic melanoma, esophageal cancer, and gastric cancer 29,[40][41][42] . The latest study indicates that GSDMC and PD-L1 can lead to necrosis of breast cancer tissue by switching apoptosis to pyroptosis in the hypoxic area 12 . But the molecular mechanism of GSDMC in tumors is poorly understood.
In this research, we assessed the mRNA expression levels of GSDMC in pan-tumors and matching non-cancer normal tissues utilizing TGGA and TIMER databases. Taken together, upregulation of GSDMC was found in UCEC, BRCA, READ, CHOL, LUSC, COAD, LUAD, KICH, LIHC, and KIRC. These data together with some studies mentioned above suggested that GSDMC might serve as the pivotal player in the carcinogenesis of these Table 1. The relationship between of GSDMC expression and potential candidate miRNAs expression in BRCA. *P < 0.05 (5e-02); **P < 0.01 (1e−02); ***P < 0.001 (1e−03).   www.nature.com/scientificreports/ kinds of cancer 27,28,40 . Next, we carried out a pan-cancer survival analysis of the expression of GSDMC utilizing the TCGA database, after which the GEPIA database and KM plotter were employed for validation. Finally, a combination of the expression and prognostic significance of GSDMC in several kinds of cancers, we found that elevated expression of GSDMC might play as an unfavorable prognostic biomarker in BRCA patients. IHC analysis of 15 pairs of BC tumor tissues and corresponding adjacent normal tissues also revealed significantly higher expression of GSDMC in BC tissues than normal tissues. Furthermore, GSDMC expression and prognosis analysis of BC in GEO database showed the same results. Thus, further experimental validation is needed to investigate the link between the expression of GSDMC and the prognosis of cancer patients in BC and other kinds of cancers, including PAAD, COAD, KICH, etc. It has been well documented that ceRNA theory explains interactions among mRNA and ncRNAs (miRNAs, lncRNAs, and circular RNAs (circRNAs)) 32,43 . LncRNAs affects the miRNA affinity of target mRNA by attaching to similar miRNA response elements, thereby regulating gene expression at the transcriptional level 31,33 . We predicted potential miRNA candidates of GSDMC via several target gene prediction websites, consisting of miRDB, miRmap, TargetScan, miRcode, miRWalk, and DIANA-microT, and finally found 15 miRNAs. We found that GSDMC was only significantly negatively associated with hsa-miR-573 (MIR573) and positively linked to hsa-miR-548ao-5p (MIR548AO) in BRCA (P < 0.001). Meanwhile, no statistical expression link was observed between GSDMC and other miRNAs. The observed findings also illustrated that the levels of hsa-miR-573 expression were lower than normal tissue control in BRCA. Next, our survival analysis showed that hsa-miR-573 acting as tumor-suppressive miRNAs in BRCA. Taking together correlation analysis, expression analysis, and survival analysis, we suggested that hsa-miR-573 might serve as the most potential regulatory miRNA of GSDMC in BRCA. Early research also showed that hsa-miR-573 was a negative regulator of cell proliferation, migration, and invasion of pancreatic cancer 44,45 .
Then we selected 39 potential candidate lncRNAs that interacted with hsa-miR-573 by using DIANA-LncBase v.2. Based on ceRNA hypothesis proposes, there ought to be a positive relationship between potential lncRNA and GSDMC and negative relationship between potential lncRNA and hsa-miR-573, and it should be oncogenic lncRNAs in BRCA. By correlation analysis, survival analysis, and expression analysis, LINC00511 was chosen as the key potential upstream lncRNA of GSDMC/hsa-miR-573 axis in BRCA. In early studies, LINC00511 was determined as oncogenic lncRNAs in several tumors, including gastric cancer, lung squamous cell carcinoma, cervical cancer, bladder carcinoma, glioma, and breast cancer [46][47][48][49][50] . Taken together, LINC00511/hsa-miR-573/ GSDMC axis was well identified as potential regulatory pathways in BRCA (Fig. 15).
It is well known that breast cancer is a highly clinical and molecular heterogeneous disease. For this reason, we performed prognostic analysis of GSDMC, hsa-miR-573 and LINC00511 in patients with differently molecular subtyping of BRCA. The results indicated that LINC00511/hsa-miR-573/GSDMC axis might act in luminal B BRCA. Additional research would be necessary.
As an affiliate of the Gasdermin superfamily, GSDMC participated in the modulation of epithelial cell immune-related activities. Therefore, for another crucial facet of this study, we explored the link between GSDMC and tumor immune infiltration in BRCA. The tumor immune cell infiltration is an indispensable component of the tumor immune microenvironment which is closely associated with tumor progression, clinical outcomes as well as immunotherapy responses 51,52 . Our research showed a considerable correlation between levels of GSDMC expression and immune cell infiltration of myeloid dendritic cells, CD4+ T cells, CD8+ T cells, and neutrophils in BRCA. Moreover, we discovered considerable positive associations between GSDMC expression and several immunological biomarkers of these infiltrated immune cells. Some reports also found that GSDMC performed an instrumental function in adaptive immune responses 30 . These results indicated that GSDMC assumed a vital  www.nature.com/scientificreports/ function in modulating immune cell infiltration in BRCA, with specifically powerful influence on CD4+ T cells, CD8+ T cells, myeloid dendritic cells, and neutrophils. Immunological and T-cell-infiltrated tumors respond favorably to inhibition of immunological checkpoints, and anti-tumor immune response may be amplified through blocking of immune checkpoints 53,54 . SIGLEC15, PDCD1LG2 (PD-L2), TIGIT, PDCD1 (PD-1), HAVCR2 (TIM3), CTLA4, LAG3, and CD274 (PD-L1) are transcripts related to immunological checkpoints that have a function in tumor immune evasion. As a result, we found that, besides SIGLEC15, the levels of GSDMC expression were considerably positively associated with the 5 immune checkpoints in BRCA. Hou et al. identified a non-immunological checkpoint role of PD-L1 and found GSDMC/caspase-8 cause tumour necrosis via mediate a non-canonical pyroptosis pathway in cancer cells. Our research could provide more clues for GSDMC/PD-L1 therapeutic strategies. All these outcomes illustrated that tumor immune evasion and antitumor immunity might be implicated in GSDMC mediated carcinogenic processes of BRCA.
In summary, we discovered that elevated GSDMC expression was considerably linked to a worse prognosis in BRCA. Next, we identified a LINC00511/hsa-miR-573/GSDMC axis as potential regulatory pathways in BRCA. For another important aspect of this research, our research demonstrated that GSDMC was a pivotal player in BC carcinogenesis via elevating tumor immune cell infiltration and the expression of immunological checkpoints. It is expected to provide new ideas for the link between pyroptosis and tumor immunotherapy. Nonetheless, this process requires much more fundamental research and extensive clinical trials.

Materials and methods
Data processing and differential expression analysis, survival analysis and correlation analysis. The UCSC Xena dataset was used to acquire TCGA and GTEx expression and clinical information (https:// toil-xena-hub. s3. us-east-1. amazo naws. com/ downl oad/ TcgaT arget Gtex_ rsem_ gene_ tpm. gz; Full metadata) 55 . Dataset ID: TcgaTargetGtex_rsem_gene_tpm. Raw counts of RNA-sequencing data (level 3) and matching clinical data contains 10,363 tumor tissues and 730 adjacent tissues from 18 types of cancer. In BRCA, we obtained 1109 breast cancer tissues and 113 adjoining tissues. Three independent BRCA gene expression profiles (GSE29431, GSE31448 and GSE42568) were downloaded from the Gene Expression Omnibus (GEO) database (https:// www. ncbi. nlm. nih. gov/ geo/) and processed for analysis 56 . Detailed information of datasets was listed in Table S1. All analytical methods were carried out utilizing the R software version v4.0.3. Expression analysis and Survival curves were drawn using the R packages "ggplot2", "survival", and "survminer". The Log-rank tests as well as the univariate Cox proportional hazards regression generated hazard ratio (HR) and P-values with a confidence interval (CI) of 95% in KM curves. The R package "ggstatsplot" was used to analyze two-gene correlations. To examine the link between quantitative variables, Spearman's correlation or Pearson correlation analysis was utilized.
Tissue samples. 15  Immunohistochemistry (IHC) analysis. Formalin fixed paraffin-embedded tissues (4 µm thick) were analyzed by IHC with GSDMC antibody (1:50; Affinity, China) and horseradish peroxidase conjugated secondary antibodies (Maxim, China). For IHC quantification, the sections were analyzed using DM2000 LED microscope (Leica, Germany) and three randomly selected areas were photographed. The diagnoses were confirmed by three pathology specialists. The integral optical density (IOD) was determined by the Image-Pro Plus 6.0 software (Media Cybernetics, USA).  GEPIA2 database analysis. Gene Expression Profiling Interactive Analysis (GEPIA) contains the RNA sequence expression information of 9736 tumors and 8587 non-tumor normal specimens from the TCGA and GTEx projects, which is used to analyze its standard processing pipelines 58 . GEPIA2 (http:// gepia2. cancer-pku. cn/) is an updated version of GEPIA. We used GEPIA2 to determine the connection between the mRNA expression of GSDMC and patient prognosis in pan-cancers. We also examined the expression of LINC00511 and hsa-   64 . For subsequent analyses, we chose miRNAs candidates which, as indicated above, were often found in over three systems. These projected miRNAs were therefore chosen as GSDMC miRNAs candidates.
Immune cell infiltration and immune checkpoints analysis. The TCGA database was utilized to retrieve raw counts of RNA-sequencing data (level 3) in which contains 1109 BC tissues and 113 adjoining tissues. We investigated immune cell infiltration and immunological checkpoints of GSDMC in BRCA utilizing R packages "immunedeconv", "ggplot2", "pheatmap", and "ggstatsplot" to produce accurate immune infiltration estimates. R foundation for statistical computation (2020) version 4.0.3 was utilized to implement all of the aforementioned analytic techniques.
TCGA data and cBioPortal analysis. The cBioPortal for Cancer Genomics supports analysis, visualization, as well as downloading of cancer genomics datasets (http:// www. cbiop ortal. org/) 66 . The BRCA dataset (TCGA, Firehose Legacy) which contains data of 1108 BRCA patients, was selected for GSDMC analysis via cBioPortal database. The genomic signatures comprised of mutations, putative copy-number alterations (CNA), mRNA expression z-scores (RNA Seq V2 RSEM), and protein expression Z-scores (RPPA) from GISTIC. The computation of co-expression was carried out as per the online instructions of cBioPortal.
Statistical analysis. We analyzed data by a log-rank test, such as fold-change, Hazard ratio (HR), and P-values. We measured the extent of correlation between particular variables via Spearman's correlation analysis or Pearson correlation analysis, with the r values to measures the relationship strength. P-Value or Log-rank P-value of < 0.05 was judged as having statistical significance.
Ethics approval and consent to participate. This study was approved by the Ethics Committee of Liuzhou People's Hospital (Reference No. KY2021-021-01), and was performed according to the Declaration of Helsinki. Written informed consent forms were obtained from all subjects. www.nature.com/scientificreports/

Data availability
The UCSC Xena dataset was used to acquire TCGA and GTEx expression and clinical information (https:// toilxena-hub. s3. us-east-1. amazo naws. com/ downl oad/ TcgaT arget Gtex_ rsem_ gene_ tpm. gz; Full metadata). Dataset ID: TcgaTargetGtex_rsem_gene_tpm. Raw counts of RNA-sequencing data (level 3) and matching clinical data contains 10,363 tumor tissues and 730 adjacent tissues from 18 types of cancer. All the datasets were retrieved from the publishing literature, so it was confirmed that all written informed consent was obtained. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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