Nrf2 protects against drowning-induced acute lung injury via inhibiting ferroptosis

Background Drowning-induced acute lung injury (ALI) has been a major cause of accidental death worldwide. Severe oxidative stress injury is the key factor in drowning-induced ALI. The latest evidences indicate nuclear factor (erythroid-derived 2)-like 2 (Nrf2) suppress Ferroptosis and maintain cellular redox balance. Here, we test the hypothesis that activation of Nrf2 attenuates drowning-induced ALI via inhibiting ferroptosis. Methods In this study, we employed Nrf2-specic agonist (dimethyl fumarate), Nrf2 inhibitor (ML385), Nrf2-knockout mice and ferroptosis inhibitor (Ferrostatin-1) to investigate the benecial roles of Nrf2 on drowning-induced ALI and the underlying mechanisms. Results In this study, we rstly showed that Nrf2 activator dimethyl fumarate could increase cell viability, reduced the levels of intracellular ROS and lipid ROS, prevented glutathione depletion and lipid peroxide accumulation, increased FTH1 and GPX4 mRNA expression, and maintained mitochondrial membrane potential. However, ML385 promoted cell death and lipid ROS production. Furthermore, Nrf2 knockout aggravated seawater drowning-induced ALI in mice. Conclusions In summary, these results suggest that Nrf2 alleviate drowning-induced ALI in MLE-12 cells and mice through inhibiting ferroptosis. in inhibiting senescence of myeloid-derived suppressor cells from lupus mice via Nrf2/HO-1 pathway. Free Radic


Introduction
Drowning is one of the main causes of accidental injury and death (1,2). It is estimated that more than 360,000 people die each year from drowning worldwide (3). Moreover, it is worth noting that acute lung injury (ALI) is one of the most common complications of drowning and can develop ultimately into acute respiratory distress syndrome (4,5). Drowning can cause damage to alveolar epithelial cells, causing hypoxia, hemorrhage and oxidative stress, and pneumonia (6,7). Oxidative stress plays an important role in drowning-induced ALI (4,8). Our previous studies have shown that heme oxygenase-1 alleviated drowning-induced ALI (9). However, the molecular mechanisms involved in HO-1 induction in seawaterinduced ALI was still unknown. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) has a key role in regulating cellular antioxidant stress and eliminating reactive oxygen species (ROS) (10). Recently research found that Nrf2 prevents oxidative stress-induced pulmonary diseases (11,12). However, whether and how Nrf2 protects against drowning-induced ALI remain unknown. The main features of Ferroptosis are overwhelming cellular ROS production and iron-based lipid peroxide accumulation (13,14). The latest research shows that inhibiting ferroptosis can alleviate the acute lung injury induced by lipopolysaccharide (15). However, the role of ferroptosis in ALI induced by seawater drowning is unclear. In this study, we employed Nrf2-speci c agonist (dimethyl fumarate), Nrf2 inhibitor (ML385), Nrf2knockout mice and ferroptosis inhibitor (Ferrostatin-1) to test the hypothesis that activation of Nrf2 attenuates drowning-induced ALI by inhibiting ferroptosis both in vivo and in vitro.

Cell culture and treatment
MLE-12 cells (mice lung epithelial cell line, obtained from ATCC, USA) were maintained in DMEM (GIBCO, USA) medium supplemented with 10% fetal bovine serum (FBS, GIBCO, USA) and 1% penicillinstreptomycin solution (GIBCO, USA) and placed in a cell culture chamber containing 5% CO 2 at 37 °C.

Cell viability assay
Cells viability was measured by using Cell Counting Kit-8 (CCK-8) kits. Cells were seeded at a density of 1 × 10 4 /well in 96-well plates. After washing the cells with phosphate buffered saline (PBS), the CCK-8 solution was added to the medium at a dilution of 1:10 and incubated at 37 °C for 2 hours. Absorbance was measured at 450 nm using a microplate reader (BioTek, Winooski, USA).

Determination of the levels of SOD, GSH, and MDA
The superoxide dismutase (SOD) activity, glutathione (GSH) and malondialdehyde (MDA) contents in cells and lung tissues were measured by a commercial assay kit (Nanjing Jiancheng Bio Co., Ltd., China) according to the manufacturer's instructions.

Collection of intracellular ROS images by
All groups were sacri ced on the third day after drowning and lung tissue samples were collected for evaluation.
2.11 Tissue collection, wet-to-dry ratio analysis and histological analysis Right lungs were collected for determined the lung wet-to-dry ratio, western blot and biochemical analysis. After the lung tissue were weighed, they were dried in an oven at 60 ℃ for 72 hours to constant weight, and then the wet-to-dry ratio was calculated to evaluate tissue edema. Left lung tissue samples were embedded in para n after xed in 4% paraformaldehyde. Tissue pieces were cut into 4 µm sections and stained with hematoxylin and eosin (HE) for lung injury score (25).

Micro-computed tomographic analysis
Mice were anesthetized with iso urane continuously delivered through a nose cone and scanned using a Quantum FX micro-CT Imaging System (PerkinElmer, USA). Each mouse was scanned for 4 minutes under the parameters of 70 kV, 88 µA, 36 mm FOV. The data acquired by the scan was analyzed and 3D reconstructed using the Analyze 12.0 software at the same level setting.

Immuno uorescence Staining
To observe Nrf2 localization of MLE-12 cells. Cells were then xed in 4% formaldehyde and permeabilized with 0.1%Triton X. The cells were probed with Nrf2 antibodies followed by Alexa Fluor 488-conjugated secondary antibodies (Thermo Fisher Scienti c, USA). To visualize the nuclei, cells were then treated with 1 µg/ml DAPI for 10 min and then washed by PBS. Finally, added the anti-fade mounting medium and collected images using Zeiss LSM 880 laser confocal uorescence microscope (Carl Zeiss, Oberkochen, Germany).
To observe the expression of Nrf2 in mouse lung tissue. Para n slides were rehydrated in alcohol with decreasing concentrations and then placed in 10 mM sodium citrate buffer heated to 95 ℃ for 30 min for antigen retrieval. Then, the slides were permeabilized with 0.5% Triton-X and blocked with 5% BSA for 2 hours. The slides were probed with Nrf2 antibodies followed by Alexa Fluor 555-conjugated secondary antibodies (Thermo Fisher Scienti c, USA). The slides were incubated in DAPI for 10 minutes and then washed. Finally, the slides were mounted with an anti-fade mounting medium and collected images using Zeiss Axio Imager 2 uorescence microscope (Carl Zeiss, Oberkochen, Germany).

Western blot
The samples were homogenized in ice-cold RIPA buffer with protease inhibitor mixture, and the supernatant was collected after centrifugation (12,000 rpm, 10 minutes and 4 ℃). The protein concentrations were measured using a BCA kit, and proteins were then denatured at 100 ℃ for 5 min. Proteins were loaded onto a 10% SDS-PAGE gel and transferred to a nitrocellulose lter membrane. Blots were blocked at room temperature for 1 h and then incubated with primary antibodies at 4 °C overnight.
After blocking and washing, the blot was incubated with the secondary antibody for 2 hours. Protein bands were visualized and analyzed by the ChemiDocTM XRS Plus luminescence image analyzer (Bio-Rad, USA) using the ECL system (Millipore, USA).

Reverse transcription-quantitative polymerase chain reaction
Total RNA isolation and quantitative real-time PCR were performed using the procedure described previously (26). The primers used in this study were synthesized by GENEWIZ Biotechnology Co., Ltd.

Protein-Protein interaction Analysis
Search Tool for the Retrieval of Reciprocity Genes (STRING) online database (http://string-db.org/) was used to evaluate protein-protein interaction network of Nrf2, Ptgs2, GPX4 and FTH1. Organism was selected "Mus musculus". The online database provided assessment and integration of protein interactions, including direct (physical) and indirect (functional) correlations (27).

Statistical analysis
All experiments were performed at least three times. Measurement data were expressed as means ± standard deviation (SD). Comparisons between groups were carried out by analysis of variance (ANOVA) with GraphPad Prism. P < 0.05 was considered statistically signi cant.

seawater exposure induced ferroptosis in MLE-12 cells
The cell morphology of MLE-12 cells exposed to seawater (0-12 h) was observed by bright eld microscopy. The results showed that seawater exposure caused a decrease in the number and changes in cell morphology of MLE-12 cells (Fig. 1A). CCK8 assay showed a time-dependent decrease in MLE-12 cell viability after seawater exposure (Fig. 1B). The results of ow cytometry detection of death also showed an increase in the proportion of cell death as the seawater exposure time prolonged ( Fig. 1C and D) (20).
These results indicate that seawater exposure causes MLE-12 cell damage and death. Based on the above experiments, this study selected seawater stimulation for 6 hours as the follow-up test conditions. As shown in Fig. 1E, seawater treatment induced a signi cant increase in Ptgs2 mRNA in MLE-12 cells, a putative ferroptosis molecular marker (28,29). Nrf2 mRNA and protein expression in MLE-12 cells also increased after 6 hours of seawater stimulation ( Fig. 1F and G).
To investigate whether ferroptosis participated in MLE-12 cell death induced by seawater drowning, we pretreated cells with the ferroptosis speci c inhibitor Fer-1. MLE-12 cells were treated with Fer-1 at a nal concentration of 0 µM to 40 µM for 24 hours ( Fig. 2A). It was found that the concentration of 10 µM did not signi cantly inhibit cell viability, and 10 µM was used as the concentration for subsequent experiments of Fer-1 (19). As shown in Figures. 2B-D, the percentage of cell death after Fer-1 treatment was reduced and cell viability was increased relative to the SW group. The uorescent indicator (DCFH-DA) and dihydroethidium (DHE) uorescent probes were used to monitor the generation of intracellular ROS in seawater-treated MLE-12 cells. The two detection methods consistently showed that seawater exposure caused a signi cant increase of intracellular ROS in MLE-12 cells, which was decreased by Fer-1 treatment (Fig. 2E and F). Lipid ROS accumulation is a typical feature of ferroptosis (30). The results of ow cytometry after BODIPY® 581/591 C11 staining showed that lipid ROS levels were signi cantly reduced after Fer-1 treatment compared with SW group (Fig. 2G and H). Glutathione depletion and lipid peroxide accumulation are important features of ferroptosis (31). MDA is the most common by-product of lipid peroxidation (32). Fer-1 treatment reversed the decrease of GSH level and SOD activity caused by seawater drowning (Fig. 2I and K). Fer-1 also reduced the content of MDA (Fig. 2J). These results suggest that ferroptosis is present in MLE-12 cell damage induced by seawater stimulation.

Nrf2 activation attenuates seawater-induced MLE-12 cell damage
To investigate the effect of Nrf2 on MLE-12 cell damage induced by seawater drowning, we treated cells with DMF (20 µM) and ML385 (20 µM). As shown in Figure.3A and B, DMF did signi cantly increase the expression of Nrf2 whereas ML385 treatment reduced the expression level of Nrf2. As a transcription factor, translocation into the nucleus is a key step for Nrf2 to play a regulatory role. Immuno uorescence images showed that DMF signi cantly promoted the translocation of Nrf2 into the nucleus, while ML385 suppressed this phenomenon (Fig. 3C). Flow cytometry results showed that DMF reduced the percentage of cell death caused by seawater stimulation, whereas ML385 reversed this effect ( Fig. 3D and E). The results of CCK8 also showed that compared with the SW group, the cell viability of the SW + DMF group increased, while the cell viability of the SW + ML385 group and the SW + DMF + ML385 group decreased (Fig. 3F). These results indicated that Nrf2 activation could attenuate cell death induced by seawater stimulation in MLE-12 cells.

Nrf2 acivation inhibited seawater-induced ferroptosis in MLE-12 cells
We further explored the relationship between Nrf2 activities and seawater-induced ferroptosis in MLE-12 cells. As shown in Figures.4A-C, DMF increased the GSH content and SOD activity, and decreased the content of MDA, whereas ML385 reversed these effects. Furthermore, the detection of intracellular ROS by the uorescent dye DCFH-DA showed that seawater-induced ROS levels were reduced after DMF treatment, while ML385 increased ROS levels (Fig. 4D). The same results were obtained for the uorescent images of DHE staining (Fig. 4E). The results of ow cytometry for detecting lipid ROS also indicated that ML385 abolished the effect of DMF on reducing lipid ROS accumulation induced by seawater stimulation (Fig. 4F and G).
Mitochondria are important organelles of oxidative metabolism and play a crucial role in ferroptosis (33). Mitochondrial membrane potential (MMP) was detected by JC-1 staining uorescence image and rhodamine 123. As shown in Figures. 5A and C, DMF has a protective effect on seawater-induced mitochondrial membrane potential reduction (mitochondrial depolarization), while ML385 reverses this effect. MitoSox Red is a mitochondria-targeted ROS dye. Fluorescence images and intensity results indicate that ML385 reverses the effect of DMF on decreasing mitochondrial ROS levels. (Fig. 5B and D). GPXs can use reduced GSH to eliminate peroxides, of which glutathione peroxidase 4 (GPX4) is the main neutralizer of lipid peroxides (34). We found that DMF treatment had an effect of improving seawaterinduced decrease in GPX4 mRNA levels in MLE-12 cells, whereas ML385 caused a decrease in GPX4 mRNA (Fig. 5E). FTH1 is an important gene for storing iron and maintaining iron homeostasis (35,36). As shown in Fig. 5F, DMF promoted the expression of FTH1 mRNA, whereas ML385 reversed this effect. As a potential marker of ferroptosis, Ptgs2 also showed a signi cant decrease in mRNA level relative to SW group after DMF treatment, while the levels of SW + ML385 group and SW + DMF + ML385 group were further increased (Fig. 5G). A network of Ptgs2, GPX4 and FTH1 that signi cantly interacted with Nrf2 was constructed using the String database (protein-protein interaction). The network graphic showed that ferroptosis-related genes FTH1, GPX4 and Ptgs2 were clearly associated with Nrf2 (Fig. 5H). The results of the above various tests indicated that DMF promoted Nrf2 expression inhibited ferroptosis in the MLE-12 cell drowning model, and this bene cial effect was abolished by the Nrf2 inhibitor ML385.

Inhibition of ferroptosis ameliorated lung injury in mice induced by seawater drowning
To further validate the above ndings, we performed in vivo experiments by using a mouse drowning lung injury model. As shown in Fig. 6A, on the third day after drowning, obvious edema and hemorrhage occurred in the gross anatomy of the lung, and the ferroptosis inhibitor Fer-1 treatment improved this condition. Compared with the SW group, the SW + Fer-1 group showed a signi cant decrease in lung wetto-dry ratio, which indicated a reduction in pulmonary edema (Fig. 6C). H&E-stained sections showed that Fer-1 improved lung injury caused by seawater drowning (Fig. 6B and D). We further tested mouse lung injury by Micro CT. As shown in Fig. 6F, seawater drowning caused obvious damage and deformation of the mouse lung, but Fer-1 treatment ameliorates this pathological change. The lung volume of the mice was calculated by the Analyze 12.0 software based on the Micro CT data. Compared with the SW group, the lung volume of the SW + Fer-1 group also recovered signi cantly (Fig. 6E). We further tested the content of GSH and MDA and SOD activity in lung tissue, and the results showed that Fer-1 treatment improved the reduction of GSH content and SOD activity and decreased MDA levels ( Fig. 6G-I). These results indicated that inhibition of ferroptosis could improve lung injury in mice caused by seawater drowning.

Nrf2 agonist DMF inhibited ferroptosis and improved lung injury in mice induced by seawater drowning
As shown in Fig. 7A and B, DMF promotes the expression of Nrf2 in mice lung tissue. Immuno uorescence images also showed the same conditions as Western blots (Fig. 7C). Compared with the SW group, the SW + DMF group had a lower lung wet-to-dry ratio, indicating an improvement in pulmonary edema (Fig. 7D). Gross anatomy images show that DMF treatment improves lung tissue hemorrhage and edema caused by seawater drowning (Fig. 7F). H&E slice staining images also showed that DMF treatment improved lung pathological damage in mice ( Fig. 7E and G). As shown in Figures.7H and I, Micro CT results showed that lung injury and deformation were improved in the SW + DMF group compared with the SW group, and the lung volume was also restored in the SW + DMF group. The results of GSH, MDA in lung tissue showed that compared with SW group, the content of GSH in SW + DMF group increased, and the content of MDA decreased ( Fig. 7J and K). qPCR results showed that seawater caused an increase of Ptgs2 mRNA expression in mouse lung, and DMF treatment reduced Ptgs2 mRNA levels (Fig. 7L). These data indicate that Nrf2 agonist DMF treatment inhibited ferroptosis and improved lung injury in mice caused by seawater drowning.

Nrf2 de ciency aggravated lung injury and ferroptosis in mice induced by seawater drowning
To con rm the protective effect of Nrf2 on drowning lung injury, we used Nrf2 −/− mice for further validation. As shown in Fig. 8A and 8B, we veri ed the knockout effect of Nrf2 in mouse lung tissue by Western blotting. The same result was obtained with the immuno uorescence image (Fig. 8C). The results of lung wet-to-dry ratio showed that knocking out Nrf2 promoted pulmonary edema (Fig. 8D). Gross anatomy and H&E staining images showed that seawater drowning caused more severe bleeding and pathological damage in Nrf2 knockout mice compared to wild-type mice (Fig. 8E-G). Similar results were obtained by Micro CT. The Nrf2 KO + SW group had more severe lung injury than the SW group, and the lung volume decreased more severely (Fig. 8H and I). Biochemical indicators showed that the lower levels of GSH and the higher MDA in Nrf2 −/− mice compared with those in wild-type mice (Fig. 8J and K). In addition, the expression of Ptgs2 mRNA in the Nrf2 KO + SW group was also higher than that in the SW group. (Fig. 8L). These results indicated that Nrf2 de ciency aggravated lung injury in mice induced by seawater drowning.

Discussion
Drowning is one of the three major causes of unintentional injury death in the world (37). Approximately 50,000 people annually die mainly due to ALI or ARDS while no speci c and effective treatments are currently available (5,37). Thus, it is very important to understand the key mechanisms and look for strategies to treatment seawater-induced ALI. It is generally recognized that many factors such as oxidation, in ammation, apoptosis and delayed cell proliferation be involved in the pathogenesis of seawater drowning-ALI (4), but which is the key factor is still unknown. In this study, we demonstrated that seawater stimuli directly induced the levels of intracellular ROS and lipid ROS, and reduced GSH content and led to severe mitochondrial damage in MLE-12 cells and mice. These results demonstrated that severe oxidative stress injury is the key factor in seawater drowning-induced ALI.
Ferroptosis is an iron-dependent, lipid peroxide-driven form of cell death. The new cellular death phenotype is mechanistically and phenotypically distinct from other cell death processes, e.g. apoptosis, autophagy, pyroptosis (14,38). However, whether and how ferroptosis plays a role in lung injury caused by seawater drowning has never been explored. In this study, we rstly found that seawater exposure induced MLE-12 cell damage and death and the ferroptosis inhibitor Fer-1 treatment improved cell viability and cell death. Furthermore, Fer-1 treatment reduced the levels of intracellular ROS and lipid ROS and MDA, and increased the levels of GSH and SOD. This indicated that ferroptosis participated in MLE-12 cell damage caused by seawater exposure. The mouse drowning model also veri ed that ferroptosis is present in lung injury caused by seawater drowning. Gross anatomical morphology, HE slices staining and micro CT results consistently showed that ferroptosis inhibition attenuated ALI in mice caused by seawater drowning.
Mitochondria are important organelles of oxidative metabolism and play a crucial role in ferroptosis (19).
Abnormal mitochondrial metabolism signi cantly contributes to rapid glutathione depletion and subsequent lipid ROS generation and ferroptosis (33). In this study, we monitored mitochondria function using C11 BODIPY 581/591 and mitochondrial superoxide images by MitoSox Red uorescent probe and DHE uorescent probe and Mitochondrial membrane potential (MMP) assay. We found that seawater stimulation caused an increase in intracellular ROS, lipid ROS accumulation in MLE-12 cells. Furthermore, Fer-1 treatment reduced the levels of intracellular ROS and lipid ROS and MDA, and increased the levels of GSH and SOD. Here, we presented evidences to demonstrate that the mitochondrion is indeed a crucial player in ferroptosis induced by seawater simulation.
The mechanisms about the negative regulation of ferroptosis are being hot topic in recent years as therapeutic means for multiple pathologies (20,21). Elucidating how ferroptosis provokes ALI will expose new therapeutic opportunities to treat these diseases. Nrf2 is a core player in the regulation of antioxidant molecules in cells, regulating a variety of genes (10,39). Nrf2 activation has a bene cial effect on ischemia-reperfusion (I/R)-induced lung injury(40), lipopolysaccharide-induced ALI (41) and ventilatorinduced lung injury (42). Nrf2 inhibition promoted erastin or artesunate-induced ferroptosis through regulating redox homeostasis (39,43). In this study, we found that seawater exposure induced an increase in Nrf2 expression in MLE-12 cells (Fig. 1F and G). In addition, we used DMF to induce Nrf2 expression and translocation into the nucleus, which improved MLE-12 cell damage caused by seawater exposure and ML385 reversed this effect. Furthermore, induction of Nrf2 expression by DMF signi cantly attenuated GSH depletion and accumulation of MDA, ROS and lipid ROS induced by seawater exposure in MLE-12 cells. In addition, detection of mitochondrial membrane potential and mitochondrial ROS content also indicated that induction of Nrf2 expression improved mitochondrial function in MLE-12 cells. Ptgs2 mRNA results also showed that Nrf2 inhibited ferroptosis caused by seawater exposure in MLE-12 cells (Fig. 5G). DMF treatment of wild-type mice also signi cantly ameliorated lung injury induced by seawater drowning. The results of GSH, MDA and Ptgs2 mRNA also indicate that DMF-induced Nrf2 inhibited ferroptosis in the lungs of mice caused by seawater drowning (Fig. 7J-L). We further used Nrf2 knockout mice to verify the role of Nrf2 on ferroptosis in seawater drowned mice. Nrf2 knockout mice had more severe lung injury than wild-type mice. Seawater drowning caused more serious ferroptosis in Nrf2 knockout mice. These results demonstrated in vivo levels that Nrf2 inhibited ferroptosis and alleviated lung damage caused by seawater drowning.
There many important genes regulating ferroptosis process, such as GPX4, Ptgs2 and FTH1. Ferroptosis process often led GSH depletion which involves in abnormal GSH synthesis cysteine supply controled by system xc-, glutathione reductase and GPX4 (31). GPXs synthesis GSH to eliminate lipid peroxides and prevent GSH depletion (34). In this study, we found Nrf2 agonist DMF up-regulated GPX4 mRNA expression. Meanwhile, we used the String database (protein-protein interaction) and found that Nrf2 and GPX4 did has positive relations. The p62-Keap1-Nrf2 antioxidant system may be responsible for the promoted function of Nrf2 on GPX4 expression (21). Nrf2-mediated upregulation of the iron storage protein ferritin promoted cellular proliferation (36). In this study, we found that Nrf2 activation increased FTH1 mRNA expression. Ptgs2, also known as cyclooxygenase 2 (Cox-2), is considered to be a typical indicator of ferroptosis (38). The network graphic showed that ferroptosis-related genes FTH1, GPX4 and Ptgs2 were associated with Nrf2, which molecularly explain the bene cent role in seawater drowninginduced oxidative stress damage.
Although the pathogenetic role of ferroptosis in lung injury induced by seawater drowning is still elusive, its involvement in multiple human diseases has been established. Ferroptosis is a key mechanism for cell death associated with ischemic organ injury, neurodegeneration and cancer (14,20,34). GPX4 inhibition induced ferroptosis could prevent cancer resistant to chemotherapy (28,44). Noteworthily, the latest research ferroptosis inhibitor attenuated cigarette smoke-induced chronic obstructive pulmonary disease (45) and radiation-induced lung brosis through the activation of Nrf2 pathway (46). These ndings suggest that modulating ferroptosis might be potential therapeutic approache in treating lung diseases. Our study supported the notion that Nrf2 plays an important role in attenuating seawater drowninginduced lung injury (Fig. 9).

Conclusions
In this study, we rstly demonstrated in vivo and in vitro that Nrf2 can inhibit ferroptosis and alleviate ALI induced by drowning. These results elucidate a new mechanism underlying drowning-induced oxidative stress damage and identify Nrf2 as a potential therapeutic target for the treatment of lung injury.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval
The study all experiments were conducted in accordance with established guidelines and approved by the Animal Care and Use Committee of Jiangnan University (JN.No20180615b0841230).

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.

Footnotes
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