(C) PLOS One [1]. This unaltered content originally appeared in journals.plosone.org. Licensed under Creative Commons Attribution (CC BY) license. url:https://journals.plos.org/plosone/s/licenses-and-copyright ------------ Exosomal miR-224 contributes to hemolymph microbiota homeostasis during bacterial infection in crustacean ['Yi Gong', 'Guangdong Provincial Key Laboratory Of Marine Biology', 'Shantou University', 'Shantou', 'Southern Marine Science', 'Engineering Guangdong Laboratory', 'Guangzhou', 'Institute Of Marine Sciences', 'Xiaoyuan Wei', 'Wanwei Sun'] Date: 2022-02 It is well known that exosomes could serve as anti-microbial immune factors in animals. However, despite growing evidences have shown that the homeostasis of the hemolymph microbiota was vital for immune regulation in crustaceans, the relationship between exosomes and hemolymph microbiota homeostasis during pathogenic bacteria infection has not been addressed. Here, we reported that exosomes released from Vibrio parahaemolyticus-infected mud crabs (Scylla paramamosain) could help to maintain the homeostasis of hemolymph microbiota and have a protective effect on the mortality of the host during the infection process. We further confirmed that miR-224 was densely packaged in these exosomes, resulting in the suppression of HSP70 and disruption of the HSP70-TRAF6 complex, then the released TRAF6 further interacted with Ecsit to regulate the production of mitochondrial ROS (mROS) and the expression of Anti-lipopolysaccharide factors (ALFs) in recipient hemocytes, which eventually affected hemolymph microbiota homeostasis in response to the pathogenic bacteria infection in mud crab. To the best of our knowledge, this is the first document that reports the role of exosome in the hemolymph microbiota homeostasis modulation during pathogen infection, which reveals the crosstalk between exosomal miRNAs and innate immune response in crustaceans. Exosomes are small membrane vesicles of endocytic origin which are widely involved in the regulation of a variety of pathological processes in mammals. Yet, although the antibacterial function of exosomes has been discovered for many years, the relationship between exosomes and hemolymph microbiota homeostasis remains unknown. In the present study, we identified the miRNAs packaged by exosomes that were possibly involved in Vibrio parahaemolyticus infection by modulating hemolymph microbiota homeostasis in crustacean mud crab Scylla paramamosain. Moreover, it was found that miR-224 was densely packaged in exosomes after Vibrio parahaemolyticus challenge, resulting in the suppression of HSP70 and disruption of the HSP70-TRAF6 complex in recipient hemocytes, then the released TRAF6 was further interacted with Ecsit to regulate ROS and ALFs levels, which eventually affected hemolymph microbiota homeostasis to cope with pathogenic bacteria infection. Our finding is the first to reveal the relationship between exosomes and hemolymph microbiota homeostasis in animals, which shows a novel molecular mechanism of invertebrate resistance to pathogenic microbial infection. Funding: This study was financially supported by 2020 Li Ka Shing Foundation Cross-Disciplinary Research Grant (2020LKSFG01E) ( https://lksf.org/ ) (Received by YLZ), Key Special Project for Introduced Talents Team of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) (GML2019ZD0606) ( http://www.gmlab.ac.cn/ ) (Received by SKL), National Natural Science Foundation of China (31802341, 42076125, 41876152) ( http://www.nsfc.gov.cn/ ) (31802341 is received by YG, 42076125 and 41876152 are received by SKL), Natural Science Foundation of Guangdong Province, China (2018A030307044) ( http://gdstc.gd.gov.cn ) (Received by YG) and Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Teams (2019KJ141) ( http://dara.gd.gov.cn/ ) (Received by SKL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The open circulatory system of crustaceans makes it an ideal carrier for exosomes to perform immune-related functions. However, the role of exosomes in maintaining hemolymph microbiota homeostasis remains unclear. In the light of this, the current study explored the relationship between exosomes and hemolymph microbiota in mud crab. Exosomes released from V. parahaemolyticus-infected mud crabs could reduce crab mortality due to V. parahaemolyticus infection by maintaining the homeostasis of hemolymph microbiota. Moreover, miR-224 was found to accumulate in exosomes after V. parahaemolyticus infection, which resulted in suppression of heat shock protein 70 (HSP70) and disruption of the HSP70-TNF receptor associated factor 6 (TRAF6) complex. The released TRAF6 then interacted with evolutionarily conserved signaling intermediate in Toll pathways (Ecsit) to regulate mitochondrial ROS (mROS) production and the expression of anti-lipopolysaccharide factors (ALFs) in the recipient hemocytes, which eventually affected hemolymph microbiota homeostasis in response to the infection. Crustaceans have an open circulatory system, where hemocytes, oxygen, hormones and nutrients circulate together in the hemolymph [ 17 ]. Symbiotic microorganisms are indispensable inhabitants in the host [ 18 ], with growing evidence showing the presence of diverse microorganisms in the hemolymph of aquatic invertebrates including shrimp [ 19 ], scallop [ 20 ] and crab [ 21 ]. Generally, the proliferation of microbiota in the nutrient rich hemolymph environment is tightly controlled by host immune factors such agglutination, phagocytosis, production of antimicrobial peptides and reactive oxygen species (ROS) [ 19 , 22 ]. For instance, it has been shown that a shrimp C-type lectin, MjHeCL, maintains hemolymph microbiota homeostasis by modulating the expression of antimicrobial peptides [ 23 ]. Hemolymph symbiotic microbiota in hemolymph is believed to be engaged in multiple functions in the host, including competing with invading pathogens or stimulating the host to mount an immune response during pathogens infection [ 24 , 25 ]. Unfortunately, uncontrolled proliferation of hemolymph microbiota could result in host diseases such as “Milky Disease” or “Early Mortality” [ 26 , 27 ], which highlights the importance of hemolymph microbiota in host immune system and disease prevention. MicroRNAs (miRNAs), a class of small non-coding RNAs with 18–25 nucleotides in length, can interact with the complementary sequences on the 3’ untranslated region (3’UTR) of target mRNA to either arrest translation or degrade the mRNA of the target genes [ 10 , 11 ]. Apart from their endogenous functions, miRNAs can be packaged into exosomes to modulate the expression of specific target genes in recipient cells [ 12 , 13 ]. Furthermore, recent studies have revealed that loading of miRNAs into exosomes is a selective process and can reflect the dysregulated miRNA composition in donor cells [ 14 ]. It has been demonstrated that alteration of exosomal miRNA composition has great influence on the biological activities of exosomes that have been taken-up during pathogens invasion [ 15 , 16 ]. It is thought that exosome-mediated intercellular transfer of miRNAs can regulate pathogens spread and immune defense in recipient cells, which suggest that exosomal miRNAs could play potential role as novel tools for intercellular communication. Exosomes are nanosized (30–150 nm in diameter) extracellular vesicles formed in multivesicular bodies and released into the extracellular environment under physiological and pathological conditions [ 1 , 2 ]. Specific proteins highly enriched in exosomes such as TSG101, CD63, CD81 and flotillin 1, usually serve as indicators for the identification of exosomes [ 3 ]. Exosomes can be secreted by various donor cells and transferred to target cells by fusing with cytomembranes, which serve as mediators during intercellular communications via transporting bio-cargoes, such as nucleic acids, proteins and lipids [ 4 , 5 ]. Given their role as a form of intercellular vesicular transport, numerous studies have pointed out the importance of exosomes during pathogen infection and immune response [ 6 , 7 ]. It is believed that pathogen-infected cells are capable of secreting exosomes that contain pathogens or host genetic elements to neighboring cells to help modulate host immune response, which has huge impact on the fate of the infection process [ 8 , 9 ]. However, little is known about how exosomes regulate host immune response and impact on pathogen infection, especially in crustaceans. Results The involvement of exosomes in anti-bacterial response in mud crab To explore the involvement of exosomes from mud crab in bacterial infection, exosomes were isolated from the hemolymph of V. parahaemolyticus-challenged mud crabs (i.e., exosome-Vp) and Phosphate buffer saline (PBS)-injected control crabs (i.e., exosome-PBS). After that, a series of tests required [28] were performed to evaluate the quality of the isolated exosomes. The typical cup-shaped structures of isolated exosomes were observed under an electron microscope (Fig 1A) and their sizes were measured by Nanosight particle tracking analysis (Fig 1B). The isolated particles were further ascertained as exosomes by determining the exosomal protein markers Flotillin-1, TSG101 and the cytoplasmic marker (Negative control) Calnexin using Western blot analysis (Fig 1C). These results indicate successful isolation of exosomes from mud crabs challenged with V. parahaemolyticus and PBS, and there is no major difference between them. PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 1. Exosomes secreted from Vibrio parahaemolyticus-infected mud crab participate in anti-bacterial regulation. (A-B) Exosomes isolated from mud crabs injected with PBS and V. parahaemolyticus were detected by electron microscopy (A) and Nanosight particle tracking analysis (B). Scale bar, 200 nm. (C) Western blot analysis of exosomal protein markers (Flotillin-1 and TSG101) and cytoplasmic marker (Negative control) Calnexin in cell lysate and exosomes. (D) The delivery of exosomes to mud crab hemocytes. The indicated exosomes (Dio-labeled, green) were injected into mud crabs for 6 h, after which hemocytes (DiI-labeled, red) were isolated and analyzed by confocal microscopy. Scale bar, 20 μm. (E) Effects of exosomes on mud crab mortality. The specific treatments are shown on the top and the mortality was examined daily. (F) Effects of exosomes on bacteria number in mud crab hemolymph. Hemolymph bacteria number for the different treatments were counted using a fluorescence microscope at 100× magnification. (Vp means V. parahaemolyticus, exosome-Vp or exosome-PBS means exosomes isolated from the hemolymph of crabs challenged with V. parahaemolyticus or PBS). Significant statistical differences between treatments are indicated with asterisks (**, p<0.01). https://doi.org/10.1371/journal.ppat.1009837.g001 Next, the ability of the isolated exosomes to be internalized by mud crab hemocytes was analyzed by labeling the isolated exosomes with DiO (green) before being injected into mud crabs. When hemocytes from the injected crabs were collected and labeled with DiI (red) before being examined with a confocal laser scanning microscope, the results showed that the isolated exosomes could be internalized in hemocytes (Fig 1D). Moreover, through flow cytometry analysis, we found that exosome-Vp and exosome-PBS possess similar binding activity to the recipient hemocytes (S1 Fig). To explore the involvement of exosomes in mud crab during pathogenic bacteria infection, the isolated exosomes (exosome-Vp and exosome-PBS) were mixed with V. parahaemolyticus before being injected into mud crabs to determine the mortality rate, Wild Type (WT) or V. parahaemolyticus-treated mud crabs serve as control groups. As shown in Fig 1E, there was significant reduction in the mortality rate of mud crabs injected with exosome-Vp mixed with V. parahaemolyticus compared with mud crabs injected with exosome-PBS mixed with V. parahaemolyticus, which suggested that exosomes isolated from V. parahaemolyticus-challenged mud crabs have a protective effect on pathogenic bacteria infection. Moreover, when the relative abundance of hemolymph bacteria in mud crabs was determined, the results revealed that exosome-Vp was able to inhibit the rapid increase in hemolymph bacteria during infection (Fig 1F). These data of bacteria abundance and mortality rate showed that exosome-Vp possesses a protective effect on the mortality and proliferation of bacteria in the hemolymph, which may suggest a role in microbiota homeostasis and immune response. Taken together, these results suggest that exosomes secreted by V. parahaemolyticus-challenged mud crabs play a role in anti-bacterial response in mud crabs, probably by helping to maintain homeostasis of hemolymph microbiota. Exosomes modulate hemolymph microbiota homeostasis To ascertain the regulatory function of exosomes in modulating the mud crab hemolymph microbiota homeostasis, we determined the expression of antimicrobial peptides (AMPs) and ROS level, which are essential in regulating hemolymph microbiota homeostasis [19,22]. The results revealed that exosome-Vp treatment could significantly increase ROS levels in mud crabs during pathogenic bacteria infection compared with the exosome-PBS (Fig 2A and 2B). Similarly, transcript levels of antimicrobial peptides (ALF1, ALF4 and ALF5) were significantly increased in mud crabs treated with exosome-Vp (Fig 2C). Next, the bacteria species and composition of hemolymph microbiota were analyzed using 16S rDNA sequencing. As shown in Fig 2D, the diversity of hemolymph microbiota in mud crabs decreased significantly during V. parahaemolyticus infection. However, hemolymph microbiota diversity was maintained during the infection following treatment of mud crabs with exosome-Vp as compared with exosome-PBS. When the composition of hemolymph microbiota was analyzed at the phylum level, the proportion of Proteobacteria, Tenericutes and Firmicutes increased during V. parahaemolyticus infection, while the proportion of Acidobacteria, Actinobacteria and Chloroflexi decreased. On the contrary, when mud crabs were treated with exosome-Vp, microbiota homeostasis was maintained in mud crabs during the infection (Fig 2E). A similar trend was observed when the top 35 genera of hemolymph microbiota was analyzed (Fig 2F). Meanwhile, it was found that some bacteria highly presented during V. parahaemolyticus (Lactobacillus, Moheibacter, etc) were decreased in the presence of both exosome-Vp and exosome-PBS, which might be mediated by the common bio-molecules packaged in them (Fig 2F). All these results suggest that during pathogenic bacteria infections in mud crabs, exosomes could maintain the homeostasis of hemolymph microbiota probably by regulating the levels of mROS and ALFs. PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 2. Exosomes regulate hemolymph microbiota homeostasis through activation of ROS and ALFs. (A-B) The effects of the indicated exosomes on ROS production during V. parahaemolyticus infection in mud crabs. The level of ROS was measured by fluorescence microscopy. Scale bar, 50 μm (A) and microplate reader (B). (C) The effect of exosomes on the mRNA levels of ALF1-ALF6, and β-actin was used as internal reference, each treatment contains 5 crabs and three independent experiments were performed. (D) The effects of the indicated exosomes on hemolymph microbiota diversity. Mud crabs were co-injected with exosomes and V. parahaemolyticus for 48 h, after which hemolymph was collected and subjected to 16S rDNA sequencing. (E-F) The effects of the indicated exosomes on the composition of hemolymph microbiota at phylum (Top 10) (E) and genera (Top 35) (F) levels. Data represent mean ± s.d. of triplicate assays (*, p<0.05; **, p<0.01). https://doi.org/10.1371/journal.ppat.1009837.g002 Functional miRNA screening in exosomes To determine the functional exosomal miRNAs that are crucial in modulating hemolymph microbiota homeostasis, miRNA sequencing was carried out on exosome-Vp and exosome-PBS. Among the differentially expressed exosomal miRNAs, the top 6 miRNAs (Fig 3A) which include miR-291, miR-343, miR-224, miR-189, miR-60 and miR-156 were selected to investigate their role in V. parahaemolyticus infection in mud crabs. To screen for the potential functional miRNAs in exosome-Vp, ALF1 was used as the indicator. The miRNA mimics and anti-miRNA oligonucleotides (AMOs) of these miRNAs were synthesized and co-injected with V. parahaemolyticus into mud crabs followed by qPCR analysis of ALF1 expression. The results revealed that injection of mud crabs with miR-224 mimics increased the expression of ALF1, while injection with AMO-miR-224 decreased the ALF1 expression (Fig 3B and 3C). PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 3. Exosomal miR-224 modulates hemolymph microbiota homeostasis in mud crabs. (A) miRNA sequencing analysis for exosome-V.p and exosome-PBS is presented as a heatmap. The top three up- and downregulated miRNAs in the indicated exosomes are listed. (B-C) The effects of the indicated miRNAs on ALF1 expression in mud crabs. Mimics (B) or AMOs (C) of the indicated miRNAs were co-injected with V. parahaemolyticus into mud crabs for 48 h, followed by the analysis of ALF1 expression using qPCR. Data were presented relative to the value of Vp+ mimic-NC or Vp+ AMO-NC group, which were treated as standard “1”. mimic-NC and AMO-NC stand for disordered nucleic acids which serve as the negative controls. (D) The expression levels of miR-224 in mud crabs challenged with different exosomes, U6 was used as an internal reference. (E-F) The participation of miR-224 in exosome-mediated ROS production. The indicated exosomes, AMO-miR-224 and V. parahaemolyticus were co-injected into mud crabs, followed by the detection of ROS using fluorescence microscopy, Scale bar, 50 μm (E) and microplate reader (F). (G) The effect of miR-224 silencing on exosome-mediated ALFs regulation. (H-I) The involvement of miR-224 in exosome-mediated hemolymph microbiota homeostasis. Hemolymph was collected from mud crabs with the indicated treatments, following by determining the bacterial cell count (H) and species (I) analysis. The data of Vp+Exosome-PBS and Vp+Exosome-Vp groups were from Fig 2D. Each experiment was performed in triplicate and data are presented as mean ± s.d. (*, p<0.05; **, p<0.01). https://doi.org/10.1371/journal.ppat.1009837.g003 To ascertain whether exosome-Vp was involved in regulating hemolymph microbiota homeostasis via exosomal miR-224, the relative expression level of miR-224 was determined in exosome-Vp and exosome-PBS injected mud crabs. The results revealed significant upregulation in the expression of miR-224 in the exosome-Vp injected mud crabs compared with exosome-PBS (Fig 3D), indicating that exosome-Vp treatment could led to miR-224 accumulation in the recipient cells. Next, the involvement of miR-224 in the exosome-mediated regulatory process was examined by co-injecting V. parahaemolyticus with exosome-PBS, exosome-Vp or exosome-Vp and AMO-miR-224 into the mud crabs. The level of ROS in the exosome-Vp and AMO-miR-224 co-injected mud crabs was significantly lower compared with the other mud crabs (Fig 3E and 3F). A similar trend was observed in the expression levels of ALF1, ALF4 and ALF5 for these mud crab samples (Fig 3G). In addition, 16S rDNA sequencing analysis revealed a disruption in the exosome-Vp-mediated hemolymph microbiota homeostasis upon miR-224 silencing (Fig 3H and 3I). These results suggest that V. parahaemolyticus-derived exosomes could help to maintain hemolymph microbiota homeostasis of mud crab by miR-224, a miRNA densely packaged in exosomes after V. parahaemolyticus challenge. Interactions between miR-224 and its target gene To explore the pathways mediated by miR-224 in mud crab, the genes targeted by miR-224 were predicted by Targetscan and miRanda software. The prediction revealed that HSP70 was the only target gene predicted by both software (Fig 4A), moreover, the sequence alignment results indicated that the seed sequence of miR-224 could be completely complementary with the 3’UTR of HSP70 mRNA (Fig 4A). Therefore, HSP70 was preferred as the potential target gene regulated by miR-224. To ascertain this prediction, synthetic miR-224 and EGFP-HSP70-3’UTR or the mutant EGFP-ΔHSP70-3’UTR were co-transfected into Drosophila S2 cells [29], EGFP-HSP70-3’UTR only was used as control group (Fig 4B). When the EGFP fluorescence activity of these transfectants was observed under a fluorescence microscopy and a microplate reader, a significant decrease in fluorescence intensity was observed in cells co-transfected with EGFP-HSP70-3’UTR compared with control (Fig 4C and 4D), which indicates that miR-224 potentially interacts with HSP70 to modulate its expression. PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 4. HSP70 is the direct downstream target for miR-224 in mud crab. (A) Target gene prediction of miR-224 using Targetscan and miRanda softwares. (B) Cloning of wild-type and mutated 3’UTRs of HSP70 into the pIZ-V5-EGFP plasmid. The sequences targeted by miR-224 are underlined. (C-D) The direct interactions between miR-224 and HSP70 in insect cells. Drosophila S2 cells were co-transfected with miR-224 and/or the indicated plasmids for 48 h, followed by analysis of the relative fluorescence intensities. Data were presented relative to the value of miR-224+ EGFP-HSP70-3’UTR group, which was treated as standard “1”. (E-F) The effect of miR-224 silencing on the expression level of HSP70 in mud crab post-injection with AMO-miR-224. The mRNA (E) and protein (F) levels were examined at 48 h post-injection. Gray-scale value quantification was conducted using Image J software. (G-H) The effect of miR-224 overexpression on the mRNA and protein levels of HSP70 in mud crabs. (I) The co-localization of miR-224 and HSP70 mRNA in mud crab hemocytes. miR-224 and HSP70 mRNA were determined with FAM-labeled miR-224 probe (red) and Cy3-labeled HSP70 mRNA probe (green), Cy3-labeled Tubulin mRNA probe (green) was used as a negative control, the arrows indicated co-localization. Experiments were performed in triplicates, with the data shown representing the mean ± s.d. (**, p<0.01). https://doi.org/10.1371/journal.ppat.1009837.g004 To investigate the interaction between miR-224 and HSP70 in mud crabs, miR-224 was silenced or overexpressed followed by HSP70 detection. The results revealed significant increase in both mRNA and protein levels of HSP70 after AMO-miR-224 treatment (Fig 4E and 4F). On the contrary, the mRNA and protein levels of HSP70 decreased upon miR-224 overexpression (Fig 4G and 4H). Furthermore, fluorescence in situ hybridization (FISH) analysis was carried out to determine the subcellular location of miR-224 and HSP70 in mud crabs hemocytes. When miR-224 and HSP70 mRNA were labeled with fluorescent probes before being observed under a confocal microscopy, miR-224 and HSP70 mRNA were found to co-localize in hemocytes of the mud crabs (Fig 4I), and Tubulin mRNA were used as a negative control (Fig 4I). All these results suggest that HSP70 is the direct target gene of miR-224 in the mud crabs. Effect of HSP70 on the modulation of hemolymph microbiota homeostasis To ascertain whether HSP70 is involved in the modulation of miR-224-mediated hemolymph microbiota homeostasis, miR-224-depleted mud crabs were injected with HSP70-siRNA before being infected with V. parahaemolyticus and the levels of ALFs and ROS were detected in hemocytes. The results revealed significant increase in the expression of ALF1, ALF4 and ALF5 in the HSP70-siRNA treated group compared with controls (Fig 5A). Similar results were obtained for ROS levels (Fig 5B and 5C). Moreover, the expression of HSP70 was significantly decreased in exosome-Vp treated mud crabs as compared with control (Fig 5D), which indicates that HSP70 participates in exosome-mediated regulatory process. Besides, in HSP70-depleted mud crabs co-injected with V. parahaemolyticus and exosome-PBS, there were lower hemolymph bacteria numbers but higher hemolymph bacteria diversity (Fig 5E and 5F), which suggest that exosome-PBS could also maintain hemolymph microbiota homeostasis when the expression of HSP70 is suppressed. These results suggest that exosomal miR-224 contributes to hemolymph microbiota homeostasis by targeting HSP70 in mud crabs. PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 5. Role of HSP70 in exosomal miR-224-mediated hemolymph microbiota homeostasis. (A) The participation of HSP70 in miR-224-mediated ALFs regulation in mud crabs. AMO-miR-224 was co-injected with HSP70-siRNA into V. parahaemolyticus-challenged mud crabs, followed by analysis of the expression levels of ALFs using qPCR. Data were presented relative to the value of Vp group, which was treated as standard “1”. (B-C) The involvement of HSP70 in miR-224-mediated ROS production. The level of ROS in mud crab hemocytes was determined using fluorescence microscopy, Scale bar, 50 μm (B) and microplate reader (Data were presented relative to the value of Vp group, which was treated as standard “1”) (C). (D) The effect of the indicated exosomes on HSP70 expression. Isolated exosomes from mud crabs treated with PBS and V. parahaemolyticus were injected into mud crabs for 48 h, followed by determination of HSP70 protein level using Western blot analysis, tubulin was used as an internal reference. (E-F) The effect of HSP70 silencing on exosome-mediated hemolymph microbiota homeostasis. Hemolymph was collected from mud crabs with the indicated treatments and subjected to 16S rDNA sequencing, then the bacterial cell count (E) and species (F) were analyzed. The data of Vp+Exosome-PBS and Vp+Exosome-Vp groups were from Fig 2D. All the data are the average from at least three independent experiments, mean ± s.d. (*, p<0.05; **, p<0.01). https://doi.org/10.1371/journal.ppat.1009837.g005 [END] [1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1009837 (C) Plos One. "Accelerating the publication of peer-reviewed science." Licensed under Creative Commons Attribution (CC BY 4.0) URL: https://creativecommons.org/licenses/by/4.0/ via Magical.Fish Gopher News Feeds: gopher://magical.fish/1/feeds/news/plosone/