In mammals, calsequestrin is encoded by two genes, CASQ1 and CASQ2, which are expressed almost exclusively in skeletal and cardiac muscles, respectively. Phylogenetic analysis indicates that calsequestrin is an ancient gene in metazoans, and that the duplication of the ancestral calsequestrin gene took place after the emergence of the lancelet. CASQ2 gene variants associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) in humans are positively correlated with a high degree of evolutionary conservation across all calsequestrin homologues. The mutations are distributed in diverse locations of the calsequestrin protein and impart functional diversity but remarkably manifest in a similar phenotype in humans.Calsequestrin is among the most abundant proteins in muscle sarcoplasmic reticulum and displays a high capacity but a low affinity for Ca Consequently, the SR controls excitation–contraction coupling3 without compromising Ca2+ requiring cellular processes that are normally associated with the endoplasmic reticulum4. There are two well defined structural and functional regions of the SR in cardiac muscle: the longitudinal SR that runs parallel to the myofibrils and the junctional SR that forms multiple membrane contacts with T-tubule membrane contact sites7. SR-T-tubules membrane contact sites play an important role in initiation of the excitation–contraction coupling7. The longitudinal SR is enriched with Ca2+-ATPase (SERCA) which is responsible for the rapid removal of Ca2+ from the cytoplasm to initiate muscle relaxation6. The junctional SR is the primary site of Ca2+ release via the Ryanodine Receptor (RyR)/Ca+2 channel which triggers muscle contraction. The SR luminal Ca2+ binding protein calsequestrin is a high capacity, low affinity Ca2+ binding protein that forms oligomeric structures that regulate RyR activity via interactions with RyR, triadin and junctin14. There are two isoforms of calsequestrin, which are encoded by two different genes: cardiac calsequestrin (CASQ2) and skeletal muscle calsequestrin (CASQ1)15. The crystal structure of calsequestrin indicates that the protein contains three thioredoxin-like domains reminiscent of ER luminal oxidoreductases17.The sarcoplasmic reticulum (SR) is a high specialized membrane network that supports mechanical muscle functions requiring large fluxes of Ca19. CPVT results from defects in intracellular Ca2+ handling by cardiomyocytes. Two major variants have been associated with the CPVT disorder. The autosomal dominant form is associated with mutations in the cardiac ryanodine receptor (RyR2) gene and accounts for ~ 50% cases, while a recessive form with mutations in the cardiac isoform of calsequestrin (CASQ2) accounts for 2–5% cases. Other mutations also found in the CALM1, CALM2, CALM3 , and TRDN (encodes triadin) gene account for < 2% of CPVT cases24. Thirteen mutations in the CASQ2 gene have been identified in CPVT patients, in sudden death syndrome26, and three of them are non-synonymous polymorphisms (cSNP)27. Several biochemical and cell biological studies of R33Q, L167H, and D307H calsequestrin mutants indicate that these mutations lead to impaired Ca2+ storage and Ca2+ release from the SR41. Recently new calsequestrin mutations have been identified including K180R, D351G, G332R, and P329S46.Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by ventricular arrhythmias leading to sudden deathCasq1, Casq2 and pre-duplicate calsequestrin in the phylogenetic analysis. We showed that calsequestrin is an ancient protein in the metazoan, and that the duplication of the calsequestrin gene took place after the divergence of the lancelet but before divergence of Chondrichthyes. We noted that calsequestrin mutations, associated with CPVT, positively correlated with an increase in the degree of evolutionary conservation of the mutated sites. Furthermore, we carried out biochemical and biophysical analysis of seven CPVT related mutants , and whole exome sequencing variants  selected based on their linked to the human CPVT phenotype and located within highly conserved thioredoxin-like domain. The mutations are distributed in diverse locations of the calsequestrin protein but remarkably manifest in a similar phenotype in humans.In this study, we examined the evolutionary constraints of the CPVT related calsequestrin mutations, and included casq1 and casq2) was carried out with the aim of clarifying the distribution and conservation of each paralogue across animals, and thus deduce the timing of the gene duplication event and relate this information to calsequestrin mutants responsible for CPVT. Homology searching was undertaken in 23 metazoan genomes and three outgroup lineages to identify calsequestrin homologues. We identified unambiguous calsequestrin homologues in most of the vertebrate and invertebrate lineages . We were unable to identify any calsequestrin sequences in the insect lineage. This likely represents a bona fide loss in this line given the positive identification of a homologue in the spider Parasteatoda tepidariorum.Each of these lineages, as well the invertebrates and hemichordates, possess only a single calsequestrin gene, leaving the timing of when casq1 independently. We identified casq1 paralogues in the Chondrichthyes despite robustly classifying casq2 being present and the duplication having taken place prior to this point  to investigate Ca2+ binding, and this was monitored by circular dichroism (CD) analysis52. Upon adding Ca2+, wild-type Casq2 lost 18.3% α-helix and gained 16.79% β-sheet conformation  cross-linker  revealed that calsequestrin is an ancient protein within the metazoan, and duplication of the calsequestrin gene took place after the divergence of the lancelet but before divergence of Chondrichthyes. Duplication of the calsequestrin gene allowed for the differentiation of a muscle-specific form of the protein, namely cardiac calsequestrin (casq2) expressed in cardiomyocytes. In the heart Ca2+ release from the calsequestrin (Casq2) rich junctional SR is initiated by the Ca2+-induced Ca2+ release mechanism. In skeletal muscle, where Casq1 is expressed, Ca2+ release from the junctional SR is initiated by the depolarization-induced Ca2+ release mechanism. Notably in mammalian species Casq1 is almost exclusively expressed in skeletal muscle  even including pre-duplication Casq. Because of a specific disease phenotype of Casq2 mutants, one a priori prediction is that the mutations associated with CPVT would be in sites conserved in Casq2 but divergent in Casq1 and preduplicates, providing gain-of function amino acid changes in Casq2 which were then disrupted by the disease phenotype-associated mutations. However, this was not what we observed. Instead we found strong conservation at these positions between the paralogues or indeed across all calsequestrin homologues. This suggests that the residues at these positions are critical for calsequestrin function. Specificity of cardiac disease seen with Casq2 mutants is likely due to the tissue-specific expression patterns of the paralogues. In humans there is little or no Casq1 paralogue expressed in cardiac tissue to compensate for Casq2 malfunction in CPVT.Phylogenetic analysis of CASQ2 variants from whole-exome sequencing clinical testing, P329S, G332R, and D351G63. In agreement with previous reports64, our studies showed that Casq2R33Q, Casq2L167H, and Casq2D307H differed in their biochemical properties. Casq2D307H was reported to have significantly reduced Ca2+ binding capacity17. In this study, in label-free MST Ca2+ binding assay the D307H mutant showed no measurable Ca2+ binding and altered protein tertiary structure. R33Q and L167H mutants retained Ca2+ binding capacity but had increased sensitivity to tryptic cleavage and lost Ca2+-dependent polymerization.For our biochemical studies, we have selected mutants linked to the human CPVT phenotype and located within highly conserved positions, namely, R33Q, L167H, K180R, D307H and three R33Q and Casq2L167H mutants form large oligomers insensitive to Ca2+, indicating that they lost Ca2+ depend polymerization, and are not able to depolymerize in response to Ca2+ depletion, a critical function that affects the RyR2 channel gating response to depletion of Ca2+ during muscle contraction54. Amazingly, Casq2D307H substitution from aspartic acid to histidine, in the highly conserved hydrophobic core of third thioredoxin-like domain of Casq2, results in the loss of low affinity Ca2+ binding to Casq2. This is likely due to disruption of the third thioredoxin-like domain, a highly conserved region in Casq2. Surprisingly, Casq2D307H polymerized in a Ca2+-dependent manner, and, just like the wild-type Casq2, it exhibited Ca2+-dependent confirmation changes31. Nevertheless, the loss of low affinity and high capacity Ca2+ binding sites, due to severe Casq2D307H misfolding, results in reduced Ca2+ storage at the junctional SR, and impaired Casq2/Ca2+-dependent regulation of RyR2 activity65. Animal studies revealed that in Casq2 D307H /D307H knock-in mice there is a reduction in the abundance Casq2 D307H protein66. Others reported no effect of D307H mutation of protein stability in either animal model or in cultured cardiomyocytes65. Expression of Casq2 L167H mutant did not affect the level of the endogenous wild-type Casq234. However, in R33Q knock-in mice there was a significant reduction in the abundance of Casq2R33Q/R33Q protein30. This likely contributes to the CPVT phenotype.The Casq227. Knollman’s group recently reported a CPVT-like phenotype in a K180R heterozygous knock-in mouse model46. Here we discovered that the Casq2K180R protein has indistinguishable biochemical properties from the wild-type Casq2, including Ca2+ binding affinity, secondary structure and conformation change in response to increased Ca2+ concentration, protein flexibility and conformation stability upon trypsin proteolysis, protein folding, and Ca2+ dependent polymerization. Recently, crystal structure studies of the Casq2K180R maps the mutation to the filament-forming interface67, and it was proposed that disrupted Casq2 polymer formation may be responsible for Casq2 mutant-associated CPVT. Casq2 binds directly to the luminal domain of ER stress sensor IRE1α at the junctional SR to prevent the activation of IRE1α55. Interestingly, of all mutants tested in this study, only Casq2K180R showed altered binding to the luminal domain of IRE1α. Whether this is associated with CPVT remains to be established.K180R, P329S, G332R, and D351G mutants have not been previously studied with respect to their biochemical properties. K180R is a newly identified Casq2 mutant, and the first autosomal dominant mutant found of Casq2D351G42, Casq2P329S39 and Casq2G332R39 are three novel Casq2 variants42 located in a highly conserved third thioredoxin-like domain. Casq2P329S and Casq2G332R have been identified as heterozygous carrier39. They are localized in the hydrophobic core of the highly conserved beta-sheet of the third thioredoxin-like domain and are highly conserved throughout the metazoan including Casq1 and pre-duplicate Casq. To our knowledge, there have been no reports on the biochemical and biophysical analysis of these mutants. Casq2D351G showed protein folding and function similar to wild-type protein, whereas Casq2P329S and Casq2G332R showed similar properties to Casq2D307H, including severe disruption in protein folding and impaired Ca2+ binding, indicative of an important structural and functional role for the highly conserved beta-sheet in the third thioredoxin-like domain of Casq2.Casq22+ binding, mutations in this region including D307H, P329S, and G332R, and all result in severely misfolded protein with reduced or lost Ca2+ binding. R33Q, L167H and K180R are located at the Casq2 front-to-front and back-to-back polymerization interface, causing dysfunction in the protein’s Ca2+ depend polymerization/depolymerization that would affect filament formation, as proposed by Titus et al.67. This may be the unifying feature of Casq2 mutants association with CPVT67.Overall, the three CPVT disease causing Casq2 mutants  and two heterozygous variants (P329S and G332R) may lead to CPVT via different mechanisms. The third thioredoxin-like fold domain contains one highly conserved beta-sheet consist of four beta-strands, which are essential for correct folding and CaHomo sapiens, Canis lupus familiaris, Bos taurus, Oryctolagus cuniculus, Sus scrofa, Rattus norvegicus, Mus musculus, Xenopus tropicalis, Gallus gallus, Taeniopygia guttata, Crocodylus porosus, Latimeria chalumnae, Danio rerio, Callorhinchus milii, Leucoraja erinacea, Branchiostoma floridae, Ciona intestinalis, Helobdella robusta, Drosophila melanogaster, Apis mellifera, Manduca sexta, Daphnia pulex, Parhyale hawaiensis, Parasteatoda tepidariorum, Caenorhabditis elegans, Nematostella vectensis, Mnemiopsis leidyi, Trichoplax adhaerans, Amphimedon queenslandica, Monosiga brevicollis, Salpingoeca rosetta, Capsaspora owczarzaki. Ensembl: Helobdella robusta. Skatebase.org: Leucoraja erinacea. hymenopteragenome.org: Apis mellifera. wfleabase.org: Daphnia pulex.The genomes used in the comparative genomics and phylogenetics analyses are publicly available and include the following from NCBI: H. sapiens CASQ1 and CASQ2 sequences as queries, BLASTp  searches were performed on the genomes of 28 metazoan organisms  was used for site-directed mutagenesis PCR with primers (Supp. Table A pET22b casq2 and casq2 mutants was cloned into pET22b expression vector. Proteins were expressed in E. coli BL21(DE3) and purified. Cells were grown in LB medium until the A600 reaches 0.6 at 37 °C, then induced with 1 mM IPTG at 37 °C for 3 h. Cells were crashed into a buffer containing 50 mM Tris–HCl, pH 8.0, 300 mM NaCl, and 10% glycerol, then purified by using a HisTrap HP purification column  and AKTA pure chromatography system (GE lifesciences 29018224). Purification was performed using binding buffer containing 50 mM Tris–HCl, pH 8.0, 300 mM NaCl, and protein eluted with buffer containing 50 mM Tris–HCl, pH 8.0, 300 mM NaCl, and 250 mM imidazole. The 6xHis tagged ER luminal domain of IRE1α (IRE1-NLD) was expressed in COS-1 cells and purified by Ni-NTA agarose chromatography55. Protein concentration was determined using Bio-Rad DC protein assay  as recommended by the manufacturer. Protein concentration was also determined by measuring A280 absorbance using Nanovue plus spectrophotometer .cDNA encoding wild-type canine cardiac 2PO4, pH 7.4, and 5 mM KCl, as NaCl interferes with CD analysis. CD scans were recorded using a quartz cell with a path length of 1 mm, response time of 2 s, scan speed of 10 nm/min, and band width of 1.0 nm. Ca2+-induced changes in CD spectra were monitored in the presence of 1 mM of EGTA and 6 mM of CaCl2. CD spectra analysis was carried out at 24 °C. The final spectra was an average of 5 measurements, after baseline subtraction. Analysis of the spectra was performed using K2D368. The following calculations were performed for analysis:CD spectra were recorded on a Jasco model J-810 spectropolarimeter. Far UV CD spectra were collected with 4.82 μM protein in buffer containing 10 mM NaH2dmol−1, θobs is the observed ellipticity in degrees, Mean residue ellipticity was calculated with formula:2, 250 μM EGTA, 0.05% Tween-20, and 2.5% glycerol. CaCl2 and EGTA concentrations were adjusted to obtain the desired free Ca2+ concentration: no Ca2+ , 5 mM . Free Ca2+ concentration was calculated using the Ca2+-EGTA Calculator TS v1.3 web tool69.MST analyses were carried out using a Monolith NT.115 instrument  or Monolith NT.LabelFree instrument . The ER-luminal domain of IRE1α (IRE1-NLD) was labelled using the Monolith NT Protein Labeling Kit RED-NHS  following the manufacture’s protocol. All experiments were carried out at room temperature in standard capillaries with 20% LED power (fluorescence lamp intensity) and 40% MST power (IR-laser intensity). The assay buffer contained 50 mM HEPES, pH 7.4, 150 mM KCl, 500 μM CaCl2+ binding to wild-type Casq2 or Casq2 mutants were carried out using a Monolith NT.LabelFree instrument in standard capillaries with 20% LED power and 60% MST power. The proteins were incubated for 10 min in a buffer containing 50 mM HEPES, pH 7.4, 150 mM KCl, 0.1% pluronic F-127, and 50 μM EGTA. An increasing concentration of CaCl2  was used. All MST data was analyzed by Monolith Affinity Analysis v2.2.6 software.Ca2, 250 μM EGTA, 0.05% Tween-20, and 2.5% glycerol.Thermal denaturation analysis of wild-type Casq2 or Casq2 mutants was carried out using Tycho NT.6. This label-free system is based on measurement of a protein’s intrinsic tryptophan fluorescence and records a protein’s unfolding profile in real-time as temperature is increased from 35 to 95 °C. Ten µl of 0.25 mg/ml protein was used in buffer containing 50 mM HEPES, pH 7.4, 150 mM KCl, 500 μM CaCl2+ concentration (167 µM), a discontinuous Tris–glycine polyacrylamide gel system consisting of a 5% stacking gel and a 10% separation gel was used under non-denaturing conditions. Proteins were diluted 3 × with non-denaturing loading dye . Proteins were separated in a Mini-PROTEAN electrophoresis chamber (BioRad) in a running buffer containing 25 mM Tris, pH 8.8, and 192 mM glycine, at 100 V, for 2 h at 4 °C. The proteins were stained with Stains-all solution70, Coomassie-blue, or transferred to nitrocellulose membrane for immunoblotting analysis.To determining the oligomerization state of Casq2 or Casq2 mutants in the presence of the same free Ca2, 250 μM EGTA, 0.05% Tween-20, and 2.5% glycerol. CaCl2 and EGTA concentrations were adjusted to the desired free Ca2+ concentration: no Ca2+ , 5 mM . Free Ca2+ concentration was calculated using the Ca-EGTA Calculator TS v1.3 web tool69. Proteins (150 μg of protein in 200 μl) were incubated in a reaction buffer with the desired free Ca2+ concentration for 20 min at 25 °C before addition of trypsin at the trypsin/protein ration of 1:50 (w/w), and samples were taken for SDS-PAGE analysis at 1, 2, 5, 10, 20, 30, 60, and 120 min. The samples were mixed with 4 × SDS-PAGE sample buffer (Bio-Rad) containing PMSF, and boiled at 100 °C for 2 min before SDS-PAGE. All experiments were repeated 3 times with protein from 2 separate purifications. The gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad).Cardiac calsequestrin (Casq2) and mutant proteins were subjected to proteolysis in a buffer containing 50 mM HEPES, pH 7.4, 150 mM KCl, 500 μM CaCl55. Protein concentration of wild-type and Casq2 mutant proteins was determined using Bio-Rad DC protein assay and the proteins were diluted to a final concentration of 10 µM in a reaction buffer containing 50 mM HEPES, pH 7.4, 150 mM NaCl, 250 μM EGTA, 500 μM CaCl2, and 0.05% Tween-20. Proteins were incubated with 20-fold molar excess of DSS for 1 h at 22.5 °C. The reaction was then quenched for 15 min with 100 mM Tris pH 7.4 followed by SDS-PAGE (10% acrylamide). Proteins were transferred to nitrocellulose membrane follow by immunoblotting with mouse anti-6xHis antibodies  or anti-calsequestrin antibodies .The homobiofunctional protein cross linker disuccinimidyl suberate (DSS)  was dissolved in DMSO at a final concentration of 10 mMp-value determined to be significant if less than 0.05.Statistical analysis was performed using GraphPad Prism version 7.0. The Student’s t-test was used to compare the mean of two independent groups, and one-way Anova was used to compare the mean of three or more independent groups, with a Supplementary Information 1.Supplementary Table S1.


We identified a bloom of Alteromonas (34.9%) at the primary nitrite maximum occurring in close proximity to the chlorophyll a peak in the blue hole. Genomic potential for nitrate reduction of Alteromonas might contribute to this maximum under oxygen decrease. Genes that would allow for aerobic ammonium oxidation, complete denitrification, and sulfur-oxidization were enriched at nitrate/nitrite-sulfide transition zone (90 and 100 m) of the blue hole, but not anammox pathways. Moreover, γ-Proteobacterial clade SUP05, ε-Proteobacterial genera Sulfurimonas and Arcobacter, and Chlorobi harbored genes for sulfur-driven denitrification process that mediated nitrogen loss and sulfide removal. In the anoxic bottom seawater (100-300 m), high levels of sulfate reducers and dissimilatory sulfite reductase gene (dsrA) potentially created a sulfidic zone of ~200 m thickness. Our findings suggest that in the oligotrophic Sansha Yongle Blue Hole, O2 deficiency promotes nitrogen- and sulfur-cycling processes mediated by metabolically versatile microbials.The Sansha Yongle Blue Hole is the world’s deepest (301 m) underwater cave and has a sharp redox gradient, with oligotrophic, anoxic, and sulfidic bottom seawater. In order to discover the microbial communities and their special biogeochemical pathways in the blue hole, we analyzed the 16S ribosomal RNA amplicons and metagenomes of microbials from seawater depths with prominent physical, chemical, and biological features. Redundancy analysis showed that dissolved oxygen was the most important factor affecting the microbial assemblages of the blue hole and surrounding open sea waters, and significantly explained 44.7% of the total variation, followed by silicate, temperature, sulfide, ammonium, methane, nitrous oxide, nitrate, dissolved organic carbon, salinity, particulate organic carbon, and chlorophyll  Intermediate layers of the ocean develop O2-deficient water masses, referred to as oxygen minimum zones (OMZs), due to limitation in photosynthetic O2 production and high-level aerobic respiration during the degradation of surface-derived organics2. In these OMZs, such as the Eastern Tropical South Pacific (ETSP) and the Arabian Sea, O2 concentrations fall below sensor-specific detection limits5. In contrast, the Peru Upwelling Region, the Namibian Shelf, and the Indian Continental Shelf experience episodic plumes of hydrogen sulfide (H2S)8. These sulfidic environments are also found in enclosed or semi-enclosed basins, including the Black Sea Basin12, the Baltic Sea Basin15, the Cariaco Basin17, and submarine caves, such as the Bahamian blue holes18, the Belize Blue Hole19, and the Sansha Yongle Blue Hole20.O2-deficient regions, microbial reactions control key steps in carbon, nitrogen, and sulfur transformation under successional redox gradients extending throughout the water column21. NO3− is the most energetically favorable terminal electron acceptor for anaerobic respiration, prompting the development of a dynamic nitrogen cycle22. Much of the nitrogen loss in the ocean (30–50%) occurs in OMZs23. Heterotrophic denitrification and autotrophic anaerobic ammonium oxidation (anammox) are generally responsible for fixed nitrogen loss31. Dissimilatory NO3− reduction to NH4+ (DNRA) takes place under suboxic or anoxic conditions and has the potential to moderate fixed nitrogen loss and to regenerate redox couples (NO2− and NH4+) for anammox5. In OMZs, heterotrophic NO3− reducers supply significant amounts of both NH4+ (via organic matter decomposition) and NO2− (via NO3− reduction) to the anammox process, suggesting a possible link between anammox and denitrification29. The relative contributions of denitrification and anammox to nitrogen loss from OMZs might depend on organic matter input, as well as on the availability of fixed nitrogen34. Sulfur cycling also plays an essential role in O2-deficient waters, coupling the production and consumption of H2S7. The γ-Proteobacterial clade SUP05 couple water column H2S oxidation to NO3− reduction; these bacteria are widespread in the sulfidic waters at the bases of H2S/NO3− transition zones in OMZs, including the Arabian Sea35, the ETSP37, the Peru Upwelling Region6, the Eastern Tropical North Pacific (ETNP)38, the Bay of Bengal39, the Cariaco Basin40, as well as the Baltic and Black Seas41. Chemolithoautotrophic sulfur-oxidizing and denitrifying ε-Proteobacteria, such as the Sulfurimonas subgroup, are most abundant under higher sulfidic water conditions, such as the Namibian Shelf7, the Cariaco Basin40, and the Baltic and Black Seas41. Denitrification and H2S oxidation might create an upper limit on the escape of H2S from anoxic waters, as well as provide autotrophic organic carbon resources, namely dark primary production. Thus, in O2-deficient waters, metabolically versatile microorganisms create complex networks of carbon-, nitrogen-, and sulfur-transforming reactions, which remain to be determined.In O42. The physiochemical characteristics are presented in detail in our parallel hydrochemical study20. Briefly, the blue hole has a sharp chemocline and sulfidic bottom waters. The O2 concentrations at surface layer in the blue hole were nearly equivalent to the maximum O2 concentrations in the euphotic layer of the surrounding open sea, as well as to the maximum O2 concentrations in the surface layers of global OMZs43. The O2 concentration declined below the detection limit (<1 μmol l−1) at 100 m using the Winkler method. The primary NO2− maximum  was identified at 30 m, and the secondary NO2− maximum  was identified at 90 m. The NH4+ concentration began to increase at 90 m, increasing to ~100 µmol l−1 at 150 m. This concentration was maintained throughout the bottom layer waters. Similar to NH4+, H2S concentration increased noticeably from ~10 µmol l−1 at 100 m, up to ~48 µmol l−1 in the deeper, euxinic waters (≥150 m) that was probably due to the much reduced ventilation. In the blue hole, a suboxic zone was identified at ~90 m, where NO3− (0.8 µmol l−1), NO2− (0.2 µmol l−1), O2 (13.4 µmol l−1), H2S (0.03 µmol l−1), and NH4+ (3.9 µmol l−1) co-existed. O2-free condition and trace amounts of NO3− and NO2− were observed between 100 and 300 m. These properties notably differed from other OMZs, including the ETNP, the OMZ off Chilean in the South Pacific Ocean, and the Arabian Sea, which are typically O2-free but NO2−-rich44. The H2S and NH4+ within the blue hole anoxic zones were several times higher than levels observed below the oxycline in the Cariaco Basin40, the Baltic and Black Seas41, and the OMZ off Peru in the South Pacific Ocean6, where trace amounts of NO3− were also detected. Particulate organic carbon (POC) concentration was low in the blue hole when compared to the Baltic sea, indicating that the blue hole had a poor nutrient input45. The blue hole is ~7 km and 70 km from Jinqing Island and Yongxing Island, respectively, and ~400 km south of Sanya42. Therefore, the anthropogenic activity has minimal influence. Together, the special geographical location and hydrochemical dynamics—in terms of the high levels of H2S and NH4+, as well as low levels of NO3−, NO2− and POC—of the blue hole might allow for distinct microbial community with diverse metabolic function to be sustained.The Sansha Yongle Blue Hole is located in the Yongle Atoll of the Xisha Islands, South China Sea. The cave entrance is shaped like a comma and has an average width of 130 m , two ocean sites (C3 and C4), and three lagoon sites , temperature (38.9%), H2S (34.8%), NH4+ (31.2%), CH4 (28.2%), nitrous oxide , NO3− (12.8%), dissolved organic carbon , salinity(11.7%), POC (9.2%), and chlorophyll a (7.3%) (P < 0.05). On the basis of the cluster analysis, all samples fell into three groups at the phylum level  and were characterized by high levels of H2S, NH4+, and CH4, suggesting highly reductive. The oxic-to-suboxic zone (30–90 m) in the blue hole displayed a similar microbial composition with deep waters of the surrounding open sea when compared with the anoxic bottom layer. This result is consistent with the observation that the functional capability of microbial communities at the shallow Landsort Deep of the Baltic Sea was similar to those of two deep communities: the 6 km-depth of a trench off Puerto Rico and the 1 km-depth of the Marmara Sea47. The similarities were both likely due to the stagnant conditions and hypoxia that shifted towards the surface of the water column47. Therefore, biochemical processes in deep waters might occur in shallow waters under O2 deficiency.Based on redundancy analysis  were clustered with the deep-water open sea samples Fig. . This su48. Consistently, metagenomic sequences of Cyanobacteria (25.4%), α-Proteobacteria (26.1%), γ-Proteobacteria (28.5%), and Bacteroidetes (5.4%) were dominant at 10 m in the blue hole  and 10 m (33.3%). This is consistent with the abundance of Bacteroidetes in other coastal areas (10–30%)52. Flavobacteriales are often associated with marine snow and marine phytoplankton blooms54. These bacteria attach to phytoplankton aggregates and efficiently degrade and preferentially consume high-molecular-mass organic matter as primary carbon and energy sources51.Based on the 16S rRNA amplicons, Cyanobacteria (10.9%), α-Proteobacteria (28.4%), γ-Proteobacteria (24.3%), Bacteroidetes (28.1%), and Actinobacteria (4.2%) were dominant at 0 m and 10 m in the blue hole Fig. . These pole Fig. . The relAlteromonas 16S rRNA sequences were abundant throughout the water column, especially at 30 m, 80 m, and 90 m (23.3–34.9%). Alteromonas species are widespread in shallow and deep waters of global oceans, including the ETNP OMZ57. Alteromonas species are particle-associated microaerophilic bacteria. In addition to relying on phytoplankton-derived organic matter for survival, Alteromonas species can also use NO3− as a nitrogen source58. SAR406 might participate in sulfur cycling via dissimilatory polysulfide reduction or sulfide oxidation59. The abundance of 16S rRNA sequences affiliated with SAR406 was 5.4–6.1% at 70–90 m in the blue hole, equivalent to SAR406 abundances at 150 m and 300 m at C3 and C4 (4.9–10.1%). SAR406 sequences were also highly abundant in the global OMZs59. In addition, 16S rRNA sequences affiliated with the methane-oxidizing archaean Marine Group II  were highly abundant in the blue hole at 70 m and 90 m . These levels were comparable to Marine Group II abundance at 150–300 m at C3 (5.2–12.7%). The nitrite-oxidizing autotrophic Nitrospina (phylum Nitrospirae) was abundant between 50 m and 90 m in the blue hole (3.3–7.2%). The greatest Nitrospina abundance was at 90 m in the blue hole (7.2%), at 300 m at C3 (8.6%), and at 300 m at C4 (4.2%), suggesting that this genus occupied a wide range of niches.Between 30 m and 90 m, the blue hole exhibited a sharp oxycline: from oxic (30–70 m), to hypoxic (80 m), and then to suboxic (90 m). The prevalent 16S rRNA amplicons across this transition included those affiliated with the γ-Proteobacteria (24.4–49.9%), Actinobacteria (11.3–22.6%), α-Proteobacteria (7.3–16.3%), Planctomycetes (3.5–9.6%), Euryarchaeota (0.2–10.9%), SAR406 (1.3–6.1%), and Cyanobacteria (0.8–6.2%) Fig. . Metagen2 was <1.0 µmol l−1, concentrations of H2S, NH4+, SiO32−, PO43−, and CH4 increased with depth, and only trace amounts of NO2− and NO3− were detected20. The microbial composition in this water layer was distinct, with the most abundant 16S rRNA amplicon sequences affiliated with the γ-Proteobacteria (11.9–42.2%), δ-Proteobacteria (12.0–18.7%), Candidatus OP3 (6.3–13.1%), Planctomycetes (2.0–9.3%), and Candidatus Parcubacteria (3.0–7.8%)  and Alteromonas , ε-Proteobacterial sulfur oxidizer Arcobacter (24.1% at 100 m), and phototrophic Prosthecochloris .In the anoxic deeper waters of the blue hole (100–300 m), O8%) Fig. . Also, m8%) Fig. . The 16S2-deficient environments often display ecologically specialized microbial populations, potentially mediating organic carbon turnover and syntrophic interactions. In the bottom layer waters of the blue hole, the clades of syntrophic taxa identified could potentially degrade lignocellulosic plant material or algal-derived complex organic polymers in order to produce hydrogen (H2), including Fibrobacter succinogenes (phylum Fibrobacteres)60, Latescibacteria61, and Firmicutes. The syntrophic bacteria also included taxa that convert small molecular compounds, such as glucose, pyruvic acid, short chain fatty acids, and glycerol to acetate and H2 for CH4 production—e.g., Thermotogae, Spirochaetae, Sebaldella termitidis (Fusobacteria), Elusimicrobium minutum (Elusimicrobia), Cloacimonetes, Atribacteria, Candidatus Acetothermus autotrophicum (Acetothermia), and Candidatus Hydrogenedentes62. Therefore, the blue hole represented a great amount phylogenetic and functional diversity of microbial communities that could drive matter and energy transformation throughout the water column.The O4+ is a central component of the marine nitrogen cycle. Sources of marine NH4+ include the degradation of organic nitrogen compounds, ammonification, N2 fixation, hydrolysis of urea, and DNRA63. We identified genes encoding molybdenum-iron nitrogenase  in the blue hole, affiliated with Cyanobacteria, Chlorobi, Bacteroidetes, Proteobacteria, Firmicutes, and Verrucomicrobia. The gene of nifH increased with depth, indicating that the microbial fixation of N2 was more common in deep waters of the blue hole  was associated with the clades of Cyanobacteria and Alteromonas australica (γ-Proteobacteria) , and was primarily detected in γ-Proteobacteria and δ-Proteobacteria  Fig. . The genria Fig. .Figure 43− reduction to NO2−—can be catalyzed by nitrate reductases. The metagenomes in the blue hole were enriched in the narG gene, which encodes respiratory nitrate reductase, and the napA gene, which encodes periplasmic nitrate reductase at 90 m, accounting for 10.3% and 102.3% of prokaryotic community, respectively . The proportion of narG gene was much higher at 150 m and 300 m than at 90 m, Alteromonadales and unclassified bacteria contributed to the high abundance, however, the capacity for these populations to perform NO3− reduction under trace NO3− and NO2− conditions is unknown. The napA gene sequences primarily matched γ-Proteobacteria , ß-Proteobacteria (Sulfuricella denitrificans and Burkholderia xenovorans), and ε-Proteobacterial genus Arcobacter. In addition, napA gene from Alteromonas macleodii accounted for up to half of all napA gene sequences at 30 m, suggesting that these species might be responsible for the PMN formation , iron-containing nitrite reductase (nirS), nitric oxide reductase (norB), and nitrous oxide reductase (nosZ), but were not detected at high frequencies in comparison to narG and napA. NO2− reduction to NO is mediated by nirK/nirS, and the greatest number of nirK gene was detected at 90 m, where it was present in ∼16% of the prokaryotic community   Fig. . NO reduely Fig. . N2 prodnce Fig. . The Rho2− production via NH4+ oxidation. The amoB gene encoding ammonia monooxygenase was primarily associated with Nitrosopumilus (Thaumarchaeota), reaching a maximum of 10.9% of the prokaryotic community at 90 m in the blue hole  and in parallel with NO3− concentration, indicating that the NO2−-oxidizing chemoautotroph might produce the observed NO3−  could not be detected in the metagenomes from the blue hole water column, suggesting a low abundance of Nitrospina. The relative abundance of Nitrospina might be overestimated based on measured 16S rRNA amplicons.Ammonia monooxygenase catalyzes NOole Fig. . This ma3− Figs.  and 6d. Scalindua is the only genus of anammox bacteria found in marine environments68. Low abundance of 16S rRNA amplicons matching Scalindua was present at the water depth between 80 m and 100 m (0.01–0.02%) in the blue hole, where NH4+ and NO2− overlapped at 80 m and 90 m, and NO2− began to disappear at 100 m. However, Scalindua-related sequences were not recovered in the metagenomes from the blue hole. This suggested that denitrification could be the dominant pathway of N2 formation in the blue hole, considerably outpacing anammox. The NO2− depletion in the bottom waters could limit the anammox pathways, although high NH4+ concentration was detected. Moreover, H2S could also inhibit the anammox activity69. This phenomenon was also detected in the OMZ off Peru in association with a giant H2S plume6. In contrast, abundant anammox activity was detected in the suboxic zone of the Black Sea where high levels of NO3− and NO2− were present69.To date, a peak base (~50 m)   at 30 m in the blue hole  supplemented with 300 µmol l−1 NaNO3 for 3 d, and NO2− accumulation was evident . This suggested that Alteromonas macleodii could perform NO3− reduction in the stagnant water. Altogether, in the O2-limited blue hole, a PNM at shallow water depth was identified and the denitrification activity of Alteromonas species might play important role in generating the PNM. Additionally, denitrification by aggregate-associated bacteria may shift the PNM towards the chlorophyll a peak in an O2-deficient marine system, which may previously have been overlooked. In addition, a primary NH4+ maximum was detected between 20 m and 80 m in the blue hole20. Low abundance of amoB gene sequences coupled with NH4+ substrate at 30 m could also partly contribute to NO2− accumulation  Fig.  and the  m) Fig. , we firs m) Fig.  and C4 ( m) Fig. . Unexpecak Figs.  and 7e. ole Fig. . Of thesied Fig. . Moreove0 m Fig. . Therefoion Fig. .Figure 72 environments, a SNM is often detected below the PNM73. NO2− in the SNM is mainly produced by dissimilatory NO3− reduction, an alternative respiratory mechanism that becomes favorable when O2 is limited. We observed a SNM in the blue hole at 90 m in close proximity to the base of the NO3− maximum (where O2 concentration had decreased to 13.4 µmol l−1) , ε-Proteobacteria (Arcobacter and Sulfurimonas), and Chlorobiaceae. Therefore, the sulfur-driven chemolithotrophic denitrification could also be a crucial method for SNM formation. In addition, amoB gene reached a maximum of 10.9% of the community at 90 m, which might also be partly responsible for the NO2− accumulation  Figs.  and 7a. 1) Figs. . Both naole Fig. . Thus, aion Fig. .2 depletion, both episodic plumes of H2S in continental shelf regions and permanent H2S under sulfidic conditions are produced by SO42–-reducing bacteria from SO42– 7. Based on 16S rRNA amplicons, diverse SO42−-reducing populations were detected at 90 m, accounting for 0.4% of total prokaryotes in the blue hole, which increased rapidly between 100 m and 300 m (10.6–16.7%). These SO42−reducers included Desulfatiglans  and Desulfurivibrio . In addition, an unclassified genus in the Desulfobacteraceae (2.4–3.9%) and an unclassified genus in the Desulfobulbaceae (0.4–5.8%) were also identified. Among these taxa, Desulfococcus (0.04–0.21%) and Desulfovibrio (0.05–0.21%) were also detected in OMZ waters off the Chilean Coast4. The relative abundances of sequences associated with the Desulfovibrionaceae, Desulfarculaceae, Desulfobulbaceae, and Desulphobacteraceae were represented in Fig. dsrA) were present in high proportions between 90 m and 300 m (1.0–9.3% of the community)  Fig. . In contion Fig.  in the b2, NO3−, and metal oxides are available as electron acceptors76. At these interfaces, H2S can be oxidized using the sulfide: quinone oxidoreductase enzyme (Sqr) and flavocytochrome c/sulfide dehydrogenases (Fcc), forming SO3−. The SO3− can be further oxidized to SO42− by the adenylylsulfate reductase (Apr) and sulfate adenylyltransferase (Sat). Elemental sulfur and S2O32− are presumably oxidized to SO42− via the sulfur-oxidizing multienzyme complexes (Sox)77.Clades of sulfur-oxidizing bacteria are particularly enriched at the oxic–anoxic interfaces, where Osqr), flavocytochrome c/sulfide dehydrogenase (fccABC), sulfate adenylyltransferase (sat), adenylylsulfate reductase (apr) and sulfur-oxidizing multienzyme complexes (soxABCD and soxYZ) in the blue hole. These genes were detected in various combinations across diverse sulfur-utilizing taxa, primarily affiliated with γ-, ε-Proteobacteria, and Chlorobi. The greatest abundances of fccB and soxZ genes were present at the suboxic layer (90 m)    were dominant at 90 m, while genes of sqr and soxZ affiliated with ε-Proteobacterial genera Sulfurimonas and Arcobacter were enriched at 100 m  Fig. , while s m) Fig. , coincidty) Fig. . Genes e0 m Fig. . Depth-s78. The high abundance of fccB gene(11.4% at 90 m and 10.3% at 100 m) and 16S rRNA amplicons (7.2% at 100 m), within the narrow layer might indicate that Chlorobiaceae members could potentially couple H2S oxidation to phototrophy, even at extremely low-light intensities  were enriched in the suboxic and anoxic zones of the blue hole. Genes of sqr , fccB , soxZ (5.0% at 90 m) and napA  were recovered, implying that Candidatus Thioglobus may prefer to reside within suboxic zone. These results support finding from recent surveys indicate that γ-Proteobacterial SUP05 can oxidize sulfur by denitrification, and is most abundant in slight to moderate redoxclines, thereby linking sulfur cycling to N-loss pathways38.The metagenomic data suggested that sulfur oxidizing genes found in soxZ gene related to Sulfurimonas occupied 13.8% of the community at 100 m, although a minor component of the 16S rRNA amplicons (0.4%) affiliated with Sulfurimonas was detected. In addition, up to half of the norB sequences were related to Sulfurimonas species, further suggesting that the sulfur-oxidizing genus Sulfurimonas also supported reductive nitrogen metabolism. Sulfurimonas species are also widespread in the sulfidic anoxic waters of the Benguela system off Namibia7, as well as in the anoxic waters of the Baltic Sea, the Black Sea41, and the Cariaco Basin42. The sqr sequences were present in high abundance in Arcobacter (7.2% at 90 m and 25.0% at 100 m). Correspondingly, 16S rRNA amplicons affiliated with Arcobacter were also most abundant at 100 m (24.1%)  were 99% identical to gill epibionts of hydrothermal vent gastropods, and Arcobacter clones from the Saanich Inlet, from the near-shore anoxic basin, and from the costal oxycline, respectively. Another Arcobacter-affiliated sequence also had 96% identity to Arcobacter nitrofigilis and 95% identity to Arcobacter sulfidicus , as well as oxidizing HS–/S2– to S0  and SO32− to SO42− (soxACD and soxYZ) for energy generation. Additionally, Arcobacter-affiliated sequences contained genes encoding clades of proteases, peptidases, and oligopeptidases, as well as enzymes critical for the oxidative tricarboxylic acid (TCA) cycle (citrate synthase). However, no glycerases were identified. This indicated that Arcobacter species used proteins, amino acids, propionates, and TCA cycle intermediates, but not carbohydrates79. We also identified gene sequences encoding key enzymes of the rTCA cycle for chemoautotrophic CO2 fixation, including citrate lyase (aclB), pyruvate flavodoxin oxidoreductase (porA), and 2-oxoglutarate-acceptor oxidoreductase (oorA). Therefore, Arcobacter species had the genomic capacity to grow chemolithoautotrophically via H2S or S2O32− oxidation that is linked to diverse steps of denitrification, as well as heterotrophically on various organic compounds. The metabolic versatility of Arcobacter might provide a competitive advantage in the energy-limited blue hole.In contrast to SUP05-related sequences, ε-Proteobacteria preferentially colonized anoxic and highly sulfidic environments in the blue hole. The 1%) Fig. . Based ocus Fig. . Arcobacx coupling to sulfur oxidation pathways has been documented in diverse taxa from the H2S/NO3− transition zones in OMZs41. In the blue hole, sulfur-oxidizing denitrifiers—such as γ-, ε- Proteobacteria, and Chlorobiaceae—were enriched at 90 m and 100 m, supporting sulfur oxidation that is coupled to reductive nitrogen metabolism. It is obvious that sulfur-based denitrification occurs in this zone (90 m), where NO3−/NO2− and H2S overlapped. Meanwhile, amoB gene from Nitrosopumilus (Thaumarchaeota) was recovered at 100 m, indicating that NH4+ oxidation could provide the NO2− substrate necessary for denitrification, although this process is transient and cryptic, as trace NO3−/NO2− was detected at 100 m. This is in good agreement with a previous report on the anoxic water at Landsort Deep of the Baltic Sea47. We speculate that H2S produced by heterotrophic sulfur reducers could support sulfur-driven chemolithotrophic denitrification, which mediates both nitrogen loss and H2S removal from the blue hole.The microbial reduction of NO4 . Gene encoding methyl-coenzyme M reductase (mcrA), the best diagnostic enzyme for anaerobic methanogenesis, was not found in the metagenomic data. This could be explained by low levels of archaeal 16S rRNA amplicons. However, metagenomic and metatranscriptomic data in the 300 m surface sediment revealed a mcrA gene belonging to Methanosarcinales, suggesting active methanogenesis (unpublished data). Based on this study’s 16S rRNA amplicons and metagenomic sequences, coupled with recent published literatures, we propose three methanogenic pathways in the bottom waters of the blue hole. (1) Methanococcoides and Methermicoccus adopt methylotrophic pathways, including one-carbon compound pathways such as methanol conversion to CH480. Consistently, gene sequences for key enzymes were found, such as trimethylamine-corrinoid protein Co-methyltransferase and Methylated-thiol-coenzyme M methyltransferase. (2) Methanosaeta and Methanosarcina catalyze the acetoclastic pathway (acetate conversion to CH4)82. (3) The family Methanomicrobiaceae catalyzes the hydrogenotrophic pathway (H2 + CO2 → CH4)83. In terms of abundance, methylotrophic methanogenesis was the major pathway in the blue hole, in agreement with previous reports that some methanogens can survive in the presence of SO42- reducers by consuming noncompetitive methylated substrates84. In contrast, SO42− reduction processes could compete for these substrates, , potentially leading to a low proportion of sequences related to hydrogenotrophic and acetoclastic methanogenesis. Sequences affiliated with CH4-oxidizing archaeal Thermoplasmata displayed comparable abundance among the blue hole and the open sea waters, potentially explaining the low concentration of CH4 (<9 nmol l−1) in the oxic layers.In the blue hole, sequences associated with methanogens  were identified at 150–300 m, with a total abundance of 0.02–0.04% of total 16S rRNA amplicons Fig. . The tot2 deficiency is ongoing in global oceans, and understanding the biogeochemical responses to deogxygenation in various marine ecosystems will help our adaptation to such changes. The Sansha Yongle Blue Hole can act as an indicator of how O2 loss might influence microbially mediated biochemical processes in oligotrophic marine ecosystems.The O2 plays the most important role in affecting the microbial assemblages of the blue hole and surrounding open sea waters . The microbial composition occurring in oxic-to-suboxic zone has characteristic of that in the deep waters of surrounding open sea. That means, biochemical processes (e.g. NO2− oxidation by Nitrospina and CH4 oxidation by archaean Marine Group II) in deep waters could occur in shallow waters when O2 is deficient. Moreover, heterotrophic aggregate-associated Alteromonas blooms and might enhance the NO3− reduction process under O2 decrease, shifting the PNM towards the chlorophyll a peak. These all might influence carbon- and nitrogen-transforming reactions in the marine ecosystems.O3−/NO2−-H2S transition zone sunstains a diverse microbial community capable of sulfur oxidation by denitrification in the blue hole, such as γ-, ε-Proteobacteria, and Chlorobi. These are ubiquitous in diverse suboxic marine environments. The depth-specific patterns and metabolic versatilities enable to prevent the escape of H2S produced from the bottom layer waters. On the other hand, low level of NO2− and high level of H2S might limit anammox process, leading to NH4+ excessive.The NOChanghe Ocean, a cargo ship, and an anchored working platform as previously described20. We established six sites in the Sansha Yongle Blue Hole and the surrounding waters: SYBL, C1, C2, C3, C4, and C5  was attached to a Conductivity Temperature Depth profiler  to measure chlorophyll a. Chlorophyll a was calculated from in vivo uncalibrated fluorescence. Prokaryotes were counted using a FACSCalibur flow cytometer  following the protocols of Marie et al.84.Samples were collected in May 2017 aboard the R/V We collected 40 water samples for DNA extraction across all six sites: SYBL0m, SYBL10m, SYBL30m, SYBL50m, SYBL70m, SYBL80m, SYBL90m, SYBL100m, SYBL125m, SYBL150m, SYBL200m, and SYBL300m; C3–0m, C3-10m, C3-30m, C3-50m, C3-75m, C3-100m, C3-150m, C3-200m, C3-300m; C4-0m, C4-10m, C4-30m, C4-50m, C4-75m, C4-100m, C4-150m, C4-200m, C4-300m; C1-0m, C1-10m; C2-0m, C2-10m, C2-30m; C5-0m, C5-10m, C5-30m. We sampled C4-300m (C4–300m-1) and SYBL125m (SYBL125m-1) repeatedly.Total genomic DNA was extracted from each sample using a FastDNA Spin Kit for Soil  following the manufacturer’s instructions. The concentration and quality (A260/A280 ratio) of each DNA sample were measured using a NanoDrop 2000 spectrophotometer .85 and 806 R (GGACTACHVGGGTATCTAAT)86; an eight-base barcode unique to each sample was added to each sequence. PCRs were performed in triplicate. Each 50-µl PCR contained 5 μl of 10 × KOD Buffer, 5 μl of 2.5 mmol 1−1 dNTPs, 1.5 μl of each primer (5 μ mol 1−1), 1 μl of KOD polymerase, and 100 ng of template DNA. The amplification cycling program was an initial denaturation at 95 °C for 2 min, followed by 27 cycles of denaturation at 98 °C for 10 s, annealing at 62 °C for 30 s, and extension at 68 °C for 30 s, with a final extension at 68 °C for 10 min. PCR products were purified using the AxyPrep DNA Gel Extraction Kit  following the manufacturer’s instructions. Equimolar volumes of purified amplicons were pooled and paired-end sequenced with an Illumina HiSeq. 2500 PE250 , following standard protocols, by GeneDenovo Biotechnology Co., Ltd. .The V3–V4 region of the 16S ribosomal RNA gene was PCR amplified using primers 341 F (CCTACGGGNGGCWGCAG)Raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (accession numbers: SAMN1036346–SAMN10363471).87, with a minimum overlap of 10 bp and a mismatch error rate of 2%. We recovered 75044−109589 raw tags from each sample. Noisy sequences of raw tags were filtered using the QIIME (V1.9.1)88 pipeline with specific filtering conditions89 to obtain high-quality cleaned tags. All chimeric tags were removed. The remaining effective tags were clustered into OTUs with ≥ 97% similarity using the UPARSE pipeline90. The tag sequence with the highest abundance was selected as the representative sequence for each cluster. The representative sequences were assigned to organisms by a naive Bayesian model using the ribosomal database project classifier (Version 2.2)91, which is based on the SILVA database92. The abundances of major microbial divisions are shown as a percentage of total identifiable 16S rRNA gene sequences. Phylogenetic trees were constructed using the neighbor-joining algorithm implemented in MEGA493. Bootstrapping was performed by resampling 1000 times. Bootstrap values <50% are not shown. The scale bars represent estimated changes per nucleotide.Paired-end clean reads were merged as raw tags using FLSAH (v 1.2.11)94 . Overall, de novo assembly statistics were determined using BWA 95, which calculated the percentage of paired or singleton reads realigned to the assembly. The unmapped reads from each sample were pooled and re-assembled using MEGAHIT to generate mixed assemblies. For each sample, the sample-derived and mixed assemblies were combined to obtain a final assembled contigs. A total of 2.1 Gb data were recovered from each sample.We used 1 μg DNA per sample  as input material for DNA library preparations. Sequencing libraries were generated using the NEBNext Ultra DNA Library Prep Kit for Illumina , following the manufacturer’s instructions. Index codes were added to attribute each sequence to a sample. The index-coded samples were clustered using a cBot Cluster Generation System , following the manufacturer’s instructions. After cluster generation, library preparations were sequenced on an Illumina HiSeq. 2500 platform (Illumina), and paired-end reads were generated. We recovered 66312588~87965958 clean reads from each sample. The Illumina sequencing data were assembled individually and by sample using MEGAHIT96. All predicted ORFs ≥300 bp in length were pooled, and ORFs more than ≥95% identical present in ≥90% of all reads were combined with CD-HIT97 in order to reduce the number of redundant genes in the downstream assembly step. Reads were realigned to predicted genes, and read numbers were counted using BWA. The final gene catalogue included only non-redundant genes with gene read counts >2. All unique ORFs were annotated against the Kyoto Encyclopedia of Genes and Genomes using DIAMOND98. Reads were filtered, and taxonomic profiles were generated based on cleaned reads with MetaOthello99.We predicted the open reading frames (ORFs) of the final contigs (>500 bp) using MetaGeneMarkrpoB). Abundances per gene were normalized to gene length4.Abundances of metabolic function genes were calculated relative to the putative single copy per organism of RNA polymerase subunit B 100. Redundancy analysis (RDA) was performed using Canoco 4.5 to assess the relationships between the biophysiochemical variables and microbial composition101. The significance of the variable was tested using Monte Carlo permutation tests with 499 unrestricted permutations (P < 0.05). Chlorophyll a and 14 physiochemical variables at the SYBL, C3 and C4 were standardized to Z–score values . These 14 physiochemical variables and methods were based on our parallel study20, and were shown in Table 2−, NO3−, NH4+, SiO32−, H2S, N2O, suspended particulate matter (SPM), CH4, dissolved organic carbon (DOC), particulate organic carbon (POC), temperature, salinity, particulate nitrogen (PN), and dissolved oxygen (DO).The Hellinger distances among the relative abundances of phyla were calculated for all samples. Pearson’s correlation analyses were carried out with SPSS statistics 17.0 software to test relationships among relative abundances of different microbial groups and environmental variables.The similarity of the bacterial and archaeal composition across samples was analyzed by hierarchical clustering analysis in the “vegan” R package (R version 3.4.3)Supplementary information.


In this study, plasmonic Ag-decorated 2D MoS2 nanosheets were vertically grown on graphene substrates in a practical large-scale manner through metalorganic chemical vapor deposition of MoS2 and thermal evaporation of Ag. The plasmonic Ag-decorated MoS2 nanosheets on graphene yielded up to 10 times higher photo-to-dark current ratio than MoS2 nanosheets on indium tin oxide. The significantly enhanced PEC activity could be attributed to the synergetic effects of SPR and favorable graphene/2D MoS2 heterojunction. Plasmonic Ag nanoparticles not only increased visible-light and near-infrared absorption of 2D MoS2, but also induced highly amplified local electric field intensity in 2D MoS2. In addition, the vertically aligned 2D MoS2 on graphene acted as a desirable heterostructure for efficient separation and transportation of photo-generated carriers. This study provides a promising path for exploiting the full potential of 2D MoS2 for practical large-scale and efficient PEC water-splitting applications.A controllable approach that combines surface plasmon resonance and two-dimensional (2D) graphene/MoSThe online version of this article (10.1007/s40820-020-00512-3) contains supplementary material, which is available to authorized users. In addition, vertically packed 2D sheets offer higher volume than laid sheets for interacting with incoming photon flux on a unit substrate area. He et al. demonstrated that the edge-on structure of MoS2 flakes/TiO2 nanowires improves the photocatalytic hydrogen evolution of MoS2 [2 nanosheets vertically grown on supporting electrode substrates, such as indium-tin oxide (ITO) and ITO/TiO2 nanowires [2 nanosheets on graphene have not been reported despite its considerable potential.Photoelectrochemistry (PEC) and photocatalysis of semiconductors have been extensively studied as effective approaches for energy conversion, such as hydrogen gas production by water splitting, and for environmental applications, such as air/water purification, water disinfection, and hazardous waste remediation –7. Recenn nature . In partonolayer –10. The  hopping . Thus, t of MoS2 . Recentlanowires , 13. How2 to minimize Ohmic junction losses. Graphene has attracted huge research attention as a promising conducting layer not only because it displays remarkable electron mobility  but also due to its favorable electric contact with MoS2. Chang et al. reported the enhanced photocatalytic hydrogen evolution of MoS2/graphene as a result of the improved charge transport of graphene [2 nanoparticles/graphene for enhanced PEC hydrogen production [2 and graphene exhibited a ΔGH value that is close to zero, which is ideal for hydrogen evolution reactions [2/graphene heterostructures have been synthesized by wet-chemical and mechanical transfer approaches, which are unsuitable for controlled synthesis or vertical stacking of few-layer MoS2 nanosheets on graphene electrode substrates [2 layer thicknesses and produced the randomly assembled structures of 2D MoS2 and graphene [The support substrate should be made highly conductive and form an appropriate energy band alignment with MoSgraphene . Carraroeactions . Howeverbstrates –18. Wet-graphene , 15, 19.2 can be remarkably enhanced by employing plasmonic metal nanoparticles (NPs), such as Ag or Au. Plasmonic metals improve the optical absorption over the entire solar spectrum as well as broaden and tune the optical absorption behavior, depending on their composition, size, and shape [2 by overcoming the Schottky barrier (~ 0.8 eV) of Au/MoS2 [2, CdS, ZnO, and BiFeO3 [2 nanosheets on graphene can act as desirable heterostructures to synergistically exploit the SPR effects in terms of energy band diagram and physical nanostructure architecture.The optical absorption of 2D MoSnd shape , 21. Ag nd shape , 22. Pland shape , 23. Kan Au/MoS2 . Plasmond BiFeO3 –32. To m2 sheets that are vertically aligned on graphene. Few-layer MoS2 sheets were vertically grown on graphene in a controlled manner at relatively low temperatures (250 °C) through metalorganic chemical vapor deposition (MOCVD) to minimize damage to the graphene. For the SPR effect, Ag NPs were formed on 2D MoS2 sheets on graphene through simple thermal evaporation of Ag  SPR-enhanced optical absorption and photo-generation of charge carriers and (2) efficient separation and transportation of photo-generated e–h pairs through few-layer MoS SPR-enha4 and H2 gases at 950 °C for 5 min. The ICP power and growth pressure were fixed at 200 W and 1 Torr, respectively. The synthesized graphene on Cu was transferred on an ITO glass substrate 6 and H2S gas  as Mo and S precursors, respectively. Mo(CO)6 was vaporized at 20 °C and transferred into a quartz reaction tube with Ar gas of 25 standard cubic centimeters per minute (SCCM). The flow rate of H2S gas was 75 SCCM. The growth pressure and time were fixed at 1 Torr and 5 min, respectively. Ag NPs were formed on ITO/graphene/MoS2 through thermal evaporation of Ag at room temperature. The size and coverage of Ag NPs on few-layer MoS2 were controlled by various nominal Ag deposition thicknesses of 2, 4, and 8 nm. The Ag contents per electrode area were estimated to be 2.1, 4.2, and 8.4 μg cm−2 for 2, 4, and 8 nm of Ag, respectively.Graphene was synthesized on Cu foils (Alfa Aesar) by using inductively coupled plasma (ICP) CVD with CH2 F30 S-Twin). The structural properties of MoS2 were characterized by TEM and micro-Raman spectroscopy by using an excitation band of 532 nm and a charge-coupled device detector. The chemical states and composition of the samples were characterized by X-ray photoelectron spectroscopy . Optical properties were evaluated by UV–visible  and photoluminescence (PL) spectroscopy (excitation at 532 nm). Photoexcited carrier behavior was investigated by time-resolved PL (TRPL) measurements. The samples were excited using a 467 nm pulsed laser, and the transient signal was recorded using a time-correlated single-photon counting spectrometer (Horiba Fluorolog 3). The energy level of MoS2 was evaluated via UV photoelectron spectroscopy .The morphology of the samples was investigated via scanning electron microscopy  and transmission electron microscopy . A Pt plate and KCl-saturated calomel (Hg/Hg2Cl2) were used as counter and reference electrodes, respectively. The electrolyte solutions were prepared with 0.3 M KH2PO4 + 0.3 M KOH and 0.5 M Na2SO3 + 0.5 M Na2SO4. The light source was simulated AM 1.5G irradiation of 100 Mw cm−2 delivered by a 150 W Xe arc lamp. The current density–voltage characteristics were recorded using a source meter (Keithley 2400). Electrochemical impedance spectroscopy (EIS) measurement was performed under constant light illumination (100 mW cm−2) at a bias of 0.6 V with varying AC frequencies from 100 kHz to 100 mHz. The incident monochromatic photon-to-current conversion efficiency (IPCE) of the electrode structure was measured using a grating monochromator in the excitation wavelength range of 300–800 nm. The hydrogen gas products were analyzed using a YL 6500 gas chromatograph  equipped with a flame ionization detector and a thermal conductivity detector. A gas volume of 0.5 mL was injected into columns of 40/60 Carboxen-1000 for GC analysis.PEC cells were fabricated on 1 × 2 cm2 nanosheets with a height of ~ 200 nm and length of ~ 150–250 nm were vertically aligned and densely packed on the ITO/graphene substrate . The A1g modes can be attributed to the in-plane vibration of Mo and S atoms and the out-of-plane vibration of S atoms, respectively. The positions and relative frequency difference (RFD) of A1g peaks are strongly correlated with the number of MoS2 layers [2, the RFD value (22.3 cm−1) of −1) and A1g peaks (407.3 cm−1) corresponds to a few layers of MoS2. The MoS2 sheets grown on ITO (hereinafter referred to as ITO/MoS2) showed similar size and morphology as the counterpart sample, namely G/MoS2 . The RED value of A1g peaks of ITO/MoS2 was also similar to that of G/MoS2 .MoSmentclasspt{minima2 layers , 36. For2 with nominal Ag thicknesses of 2, 4, and 8 nm, respectively . The size of the Ag NPs on MoS2 was successfully manipulated by varying the nominal deposition thickness of Ag through thermal evaporation. For G/MoS2/Ag-2, Ag NPs with a size of ~ 3–5 nm were formed on the MoS2 nanosheet surface . The NP size increased to ~ 10–20 nm for G/MoS2/Ag-4. By increasing the nominal deposition thickness to 8 nm, the size of Ag NPs increased to ~ 20–40 nm. In addition, large Ag clusters of ~ 60–100 nm were partially formed. The high-resolution TEM lattice image revealed that the NPs were metallic Ag  and Ag NP-decorated MoS2 (G/MoS2/Ag-4) exhibited nearly identical XPS spectra at the Mo 3d core level, indicating that the single phase of semiconducting 2H MoS2 remained stable after Ag NP decoration. This structural stability is highly advantageous for 2D MoS2 in semiconducting photoelectrode applications. Figure p core level of MoS2. The spectra were deconvoluted into two peaks at 163.2 and 162.0 eV, corresponding to the S 2p1/2 and S 2p3/2 orbital of divalent sulfur, respectively [p1/2 and S 2p3/2 peaks of G/MoS2 and G/MoS2/Ag-4 were almost identical, suggesting a single phase of 2H MoS2 for both samples. The 2H phase of both samples was also confirmed by TEM . The reduced carrier lifetime can be attributed to the benefits of graphene/MoS2 heterojunction for efficient separation and transportation of photo-generated carriers to the semiconductor/liquid interface [The G/MoS Κ point . Notablyers Fig. a. The avnterface .Fig. 4a 2 exhibited smaller EIS semicircles than ITO/MoS2, whose radius mirrors the charge transfer resistance (Rct). The Nyquist plots were fitted using a simplified Randles circuit , constant phase element (Q), and diffusion of species in electrolyte solution represented by Warburg impedance (W). The Rct values are listed in Supporting Information (Table S1). G/MoS2 had Rct values of 3264 and 1959 Ω, whereas ITO/MoS2 exhibited Rct values of 4236 and 2766 Ω in the dark and under illumination, respectively. Moreover, the Rct (dark)-to-Rct (photo) ratio (1.67) of G/MoS2 was greater than that of ITO/MoS2 (1.53), suggesting that the photo-generated e–h pairs were efficiently separated and transported through the graphene/MoS2 heterojunction.EIS study was conducted to further understand the charge transport property. Figure 2 heterojunction, G/MoS2 exhibited significantly higher PEC activity through the measured potential range than ITO/MoS2  at 0.4 V (at which the photo-to-dark current ratio (Iph/Idark) was at maximum) than ITO/MoS2 (0.19 mA cm−2). The maximum Iph/Idark value of G/MoS2 was approximately 16 at 0.4 V, whereas that of ITO/MoS2 was approximately 4 at 0.8 V. The water oxidation onset potential (~ 0.13 V), which is generally defined by the potential at the intersection of the dark current and the tangent at the maximum slope of the photocurrent, of G/MoS2 had a cathodic shift of ~ 0.41 V with respect to that (~ 0.54 V) of ITO/MoS2. The rapidly increasing photocurrent density of the samples above 0.6 V resulted from the considerably high dark current, which can be attributed to the electrocatalysis and electro-corrosion of MoS2, in which active S atoms can react with redox species in the solution [Considering the benefits of graphene/MoSoS2 Fig. c. G/MoS2solution , 14.2. The characteristic A1g modes of the Ag-decorated samples were redshifted with respect to those of G/MoS2-250 because of the stiffening of A1g vibrations  of Ag-decorated MoS2 samples were redshifted with respect to that (~ 750 nm) of G/MoS2. The enhanced visible light and broadened absorption near the IR region can be attributed to the strong coupling between the excitons and surface plasmons of Ag NPs [2 showed a PL peak position of 676 nm, corresponding to the energy of the exciton A, representing the direct bandgap transitions at the Κ point of 2D MoS2. With increasing Ag NP sizes, the PL peak was gradually redshifted, reaching 688 nm for G/MoS2/Ag-8.Raman spectroscopy characterization was performed to investigate the interaction of Ag NPs with MoS in MoS2 . MoS2/Agand MoS2 . Figure mentclasspt{minimauin zone . In compf Ag NPs , 21. The2 exhibited the shortest carrier lifetime of 3.1 ns, whereas G/MoS2/Ag-4 and G/MoS2/Ag-8 yielded longer carrier lifetimes of ~ 3.8 ns  and under illumination (~ 1284 Ω), suggesting the effective assistance of the carrier transfer through the Ag NPs on the MoS2 surface. Moreover, G/MoS2/Ag-4 yielded the highest Rct (dark)/Rct (photo) of 2.00 /Rct (photo) can result from the SPR-enhanced photo-generation and transfer rate of the charge carriers. In addition, the heterojunction of MoS2/Ag can play a significant role in improving the charge separation and transfer rate [TRPL study was conducted to investigate the plasmonic effect of Ag NPs on the dynamic carrier behavior. G/MoS ns Fig. c. The calectrons . Ag-deco dark ~ 272 Ω and fer rate , 43.2 substrate was assumed including the following: (1) 3-nm-diameter NPs with a pitch of 20 nm (referred to as AgNP-3), (2) 15-nm-diameter NPs with a pitch of 30 nm (referred to as AgNP-15), and (3) 60-nm-diameter NPs with a pitch of 65 nm (referred to as AgNP-60). The electric field vector of the source oscillated along the x-axis, while the propagation vector is along the z-axis. Figure λSPR) for AgNP-3, AgNP-15, and AgNP-60. The simulated UV–Vis absorption spectrum of pristine MoS2 substrate was in good agreement with the corresponding experimental result , consistent with the experimentally observed broadened absorption near the IR region. AgNP-15 exhibited the strongest SPR effect, resulting in the highly amplified local electric field intensity in MoS2 /Rct (photo). Meanwhile, the SPR effect of G/MoS2/Ag-2 was relatively weak because of its low surface coverage of tiny Ag NPs of less than 10 nm  simulations of the interaction of materials with the incident electromagnetic radiation were performed based on Maxwell’s equations. Ag hemispheres in an open-air environment under the illumination of a plane-wave source were considered . A 2D periodic orientation of the NPs on a three-layer-thick MoSactivity , 45. In activity , 46. Thu2, its electronic structure was studied by UPS. The work function of MoS2 (4.82 ± 0.15 eV) was determined based on the difference between the photon energy of excited radiation (21.2 eV) and the spectrum width which is measured from the valence band and secondary edges  was 1.36 eV  [2, working as a photoanode. As shown in Fig. 2 for efficient extraction of electrons to the cathode.To gain insight into the carrier transport property across the heterojunction of graphene/MoS eV Fig. b. Consid 4.3 eV) . This el2, G/MoS2/Ag-2, G/MoS2/Ag-4, and G/MoS2/Ag-8 under illumination. The Ag-decorated MoS2 samples exhibited significantly higher PEC activities than G/MoS2, whereas the dark currents were almost identical . Subsequently, G/MoS2/Ag-4 yielded 2.5 times higher Iph/Idark value than G/MoS2. In addition, the Ag-decorated MoS2 samples showed a significant cathodic shift of onset potential up to 0.2 V with respect to that of G/MoS2. The Ag-decorated MoS2 samples also did not exhibit any anodic peaks before the onset potential, whereas G/MoS2 yielded minor but noticeable peaks. The anodic peaks can be originated from surface states of 2D MoS2 nanosheets, which can be filled and deactivated by plasmon-excited electrons [2 samples  was estimated using the following equation to further quantify PEC performance [J is the photocurrent density (mA cm−2) at the applied potential, Eo is the standard reversible potential (1.23 V), Vapp is applied potential, and Plight is the power density of illumination. The photoconversion efficiencies of Ag-decorated MoS2 samples were significantly higher than that (0.8% at − 0.45 V) of G/MoS2  [2 nanosheets on TiO2 nanorods (0.81%) [2/BiVO4 heterojunctions (2.67%) [2 nanosheets on polydopamine-modified TiO2 nanotubes (1.56%) [2 and G/MoS2/Ag-4 did not change significantly after 1 h of illumination, whereas the photocurrent of ITO/MoS2 decreased continuously . After PEC measurement for 1 h, the MoS2 nanosheets on ITO (ITO/MoS2) were significantly damaged, whereas G/MoS2 and G/MoS2/Ag-4 showed slight morphological changes . We recently reported that such morphological changes in MoS2 nanosheets were due to the decomposition of MoS2, mainly the loss of S elements [Figure lectrons . Passivalectrons  and prolformance :1\docume (1.27%) , MoS2 na 0.81%) , Ag-embe (2.67%) , and MoS (1.56%) . Moreove1% , Ag-electrons . The phomentclasspt{minima2 evolution studies were carried out to gain insights into the enhanced PEC performance by G/MoS2 heterojunction and SPR effects. Figure 2, G/MoS2, and G/MoS2/Ag-4). G/MoS2/Ag-4 exhibited significantly improved photoconversion efficiencies over the overall incident-light waveband, suggesting fortified carrier photo-generation and transfer dynamics. Furthermore, a significant IPCE enhancement was observed at the ~ 650–750 nm region, which was associated with the SPR effect of Ag NPs. Hydrogen evolution from dark cathode (Pt) was measured at 0.6 V versus Hg/Hg2Cl2 by using a three-electrode configuration during 15 min. The amount of H2 produced was significantly increased by G/MoS2 heterojunction and SPR effects, suggesting that the photocurrent was attributed to the water-splitting reaction. In addition, the Faradaic efficiency of G/MoS2/Ag-4 was estimated to be approximately 90.4%, implying that few side reactions occurred during water splitting [IPCE and Hplitting .Fig. 9a 2 nanosheets were prepared on graphene in a practical manner through MOCVD of MoS2 and thermal evaporation of Ag. G/MoS2 showed up to four times higher Iph/Idark than ITO/MoS2 because of the efficient separation and transportation of the photo-generated carriers by the graphene/2D MoS2 heterojunction. The PEC activity of G/MoS2 was further enhanced by plasmonic Ag NP decoration. G/MoS2/Ag-4 yielded 10 times higher Iph/Idark value than ITO/MoS2. The maximum photoconversion efficiency of G/MoS2/Ag-4 was 2.2% at − 0.58 V. The significantly improved PEC performance was attributed to the synergetic effects of SPR and graphene/2D MoS2 heterojunction. Plasmonic Ag NPs enhanced visible-light and near-IR absorption of 2D MoS2, resulting in significantly increased the photo-generation rate of e–h pairs. Subsequently, the e–h pairs were efficiently separated and transported to catalytic surfaces across the favorable graphene/2D MoS2 heterojunction and along the highly conductive edges of the vertically aligned 2D MoS2, thereby significantly enhancing the PEC activity. This study offers a practical large-scale approach that combines the potential of SPR and graphene/2D MoS2 heterojunction effects for efficient PEC applications.Plasmonic Ag-decorated vertically aligned few-layer MoSSupplementary material 1 (PDF 798 kb)Below is the link to the electronic supplementary material.


An investigation of metabolite formation, identified as Capridine β reduction product (IE1) by the fungus Candida albicans was performed. IE1 exhibited no activity against fungal cells due to an inability to enter the cells. Although no antifungal activity was observed, in contrast to Capridine β, biotransformation metabolite totally inhibited the yTOPOII-mediated relaxation at concentrations lower than detected for m-AMSA. The closely related Capridine β only slightly diminished the catalytic activity of yTOPOII.In the last few years, increasing importance is attached to problems caused by fungal pathogens. Current methods of preventing fungal infections remain unsatisfactory. There are several antifungal compounds which are highly effective in some cases, however, they have limitations in usage: Nephrotoxicity and other adverse effects. In addition, the frequent use of available fungistatic drugs promotes drug resistance. Therefore, there is an urgent need for the development of a novel antifungal drug with a different mechanism of action, blocking of the fungal DNA topoisomerases activity appear to be a promising idea. According to previous studies on the m-AMSA moderate inhibitory effect on fungal topoisomerase II, we have decided to study Capridine β  antifungal activity, as well as its inhibitory potential on yeast topoisomerase II (yTOPOII). Results indicated that Capridine β antifungal activity depends on the kind of strains analyzed (MICs range 0.5–64 μg mL Acridine derivatives are a class of compounds with a broad spectrum of biological activity and are very interesting for scientists. Many compounds containing the acridine chromophore were synthesized and tested, and the aminoacridines found wide use, both as antibacterial agents and as antimalarials . The pla1-Nitro-9-aminoacridine derivative Capridine β, 9[2′-hydroxyethylamino]-4-methyl-1-nitroacridine  , similarDue to the fact that m-AMSA, a 9-aminoacridine derivative, is a human topoisomerase II poison and has been shown to have a moderate inhibitory effect on fungal topoisomerase II  along wiAs Capridine β antifungal activity has been previously observed , we haveC. albicans ATCC 10231 growth kinetics in the absence and presence of Capridine β and Amphotericin B at concentrations corresponding to ½ × MIC, 1 × MIC and 2 × MIC indicates its high antifungal efficacy, slightly lower than that of Amphotericin B activity (−1) do not result in a 90% growth inhibition  analyses of the cell free extracts obtained after 20 h of compound (0.2 mM) incubations with C. albicans ATCC 10231 are presented in Due to the significant activity of Capridine β against fungal cells, we have decided to examine whether the original form of the compound or the product of biotransformation is responsible for its activity. Fungal degradation of acridine compounds under an aerobic condition is not well described. Biotransformation experiments were conducted by incubating The metabolic profile of the studied compound, obtained by HPLC, indicated the presence of three main peaks at retention times (r.t.) 12.5 min (M1), 18.3 and 19.1 min (M2) C. The reBased on the liquid chromatography mass spectrometry (LC-MS) data , two metC. albicans seems to be similar to that obtained for mammalian cells under hypoxia or reducing conditions  C16H15N3O3—298.2.9-(2′-hydroxyethylamino)-4-methyl-1-nitroacridine (Capridine β) 1H NMR (MeCapridine β (0.3 mmol) was hydrogenated in the presence of 10% Pd/C  in 5 mL methanol by passing gaseous hydrogen through them at room temperature for 24 h. After the time of hydrogenation, the catalyst was filtered off and the solvent evaporated. The product in the form of a base was dissolved in methanol (10 mL) and acidified by the HCl/diethyl ether. After diethyl ether, adding the desired product was obtained. 6+TFA) δ: 11.35 , 8.31 , 8.08 , 7.81 , 7.40 , 7.35 , 6.79 , 3.95–4.00 , 3.64–3.67 , 2.43 ; ESI-MS [M+H+] C16H17N3O—268.1. 9-(2′-Hydroxyethylamino)-1-amino-4-methylacridine (IE1). Yield 92%; m.p. 221–223 °C; 1H NMR  in a shaking incubator. For growth on solid media, 1.5% agar was added to the YPG medium.The following fungal strains were used: Candida albicans ATCC 10231 strains were grown in the YPG medium overnight (16–18 h) at 30 °C, washed twice with sterile water and resuspended in the fresh YPG medium at a cell density of 2 × 108 cells mL–1. In addition, 20 μL of the stock solution of Capridine β in DMSO was added to 980 μL of cell cultures to obtain 0.2 mM final concentrations. The control cells were treated with the same amount of solvent (DMSO). The cells were incubated at 30 °C for 20 h. After incubation, the cells were harvested by centrifugation  and washed with water. Then, the cells were resuspended in 0.5 mL of 60% MeOH at 4 °C and disrupted with the use of zirconium-glass beads (0.5 mm in diameter) by vigorous shaking in four 5-min cycles interrupted by 2-min ice-cooling. The samples were then centrifuged  and filtered  prior to the HPLC analysis.For studies on cellular metabolism, The LC-DAD-MS system consisted of a liquid chromatograph, a degasser, a binary pomp, an auto-sampler, and a column oven was combined with a diode array detector (DAD) and MS detector with an electrospray source (AJS ESI) and quadrupole analyzer . The ChemStation software was used to control the LC-MS system and for data processing. The column effluent passed a DAD before arriving in the MS interface.4 in water, pH 7.0) and mobile phase B (100% methanol) was used. The gradient profile was set as follows: 0 min—15% effluent B, 20 min—80% effluent B, 22 min—100% effluent B, 23 min—100% effluent B, 24 min—15% effluent B, 30 min—15% effluent B. The flow rate was 1 mL/min, the column temperature was 25 °C, and the injection volume was 20 μL.Chromatographic separations were performed on a Zorbax SB-C18 column . For the separation, a gradient of mobile phase A .The data were collected in a MS scanning mode (MS2 SCAN) with the range 150–700 (4 fungi colony-forming units (CFU)/mL. Plates were incubated for 24 h at 37 °C and growth was then quantified by measuring an optical density at 600 nm, using a microplate reader . The MIC was defined as the lowest drug concentration in which at least a 90% decrease in turbidity, in comparison to the drug-free control, was observed. The antifungal activity was determined in a RPMI-1640 medium buffered to a pH value of 7.0. The final concentration of the compound solvent (DMSO) did not exceed 2.5% volume of the final suspension in each well, and did not influence the growth of the microorganism.Antifungal in vitro activity was determined by the modified M27-A3 specified by the CLSI . Wells cCandida albicans ATCC 10231 strains were grown in the YPG medium overnight (16–18 h) at 30 °C, washed twice with a sterile phosphate buffered saline (PBS) and resuspended in RPMI 1640 at a cell density of 2 × 106 cells mL–1. The inoculum (500 μL) was added to 500 μL RPMI 1640 with 100 μM of the tested compounds. After 180 min, 200 μL of the cells suspension was washed three times with a sterile phosphate buffered saline (PBS), re-suspended in 25 μL 90% (v/v) glycerol/10% (v/v) 1× PBS, and transferred to a microscopic slide. The cells were examined with the Olympus BX-60 fluorescence microscope  equipped with the Olympus XC50 digital camera and cellSens Dimension imaging software.v/v) glycerol). The reaction was initiated by the addition of an enzyme, allowed to proceed at 30 °C for 30 min and terminated by the addition of 40% (w/v) sucrose, 100 mM Tris-HCl pH 8, 10 mM EDTA, 0.5 mg mL−1 Bromophenol Blue. A two-step extraction with chloroform:isoamyl alcohol (24:1) and butanol water were made and mixtures were analyzed on the 1% agarose gel in a 1× TAE buffer, 3 h, 4.5 V cm−1. The gel was stained in a GelRed 3× staining solution for 30 min and photographed with a Gel Doc XR+ Gel Documentation System . The relaxation inhibition effectivity (EC50) of the analyzed compounds was determined by densitometry quantification of the transition from supercoiled to relaxed forms and was expressed in relation to the control.The inhibition of yeast Topoisomerase II was analyzed according to the relaxation assay kit from Inspiralis . Briefly, 500 ng of supercoiled pBR322 DNA, 1 mM ATP, 1–200 μM of the analyzed compound were mixed with a reaction buffer (1 mM Tris.HCl (pH 7.9), 10 mM KCl, 0.5 mM MgCl 2, 0.2%  at room temperature with a 1 cm path cell. Compounds at concentrations of 0.1 mM were dissolved in a phosphate-buffered saline (PBS), pH 7.4. Fluorescence spectra were recorded with the use of the TECAN Spark 10M microplate mode. In addition, 100 μL of the analyzed compounds at 0.1 mM concentrations were mixed with 100 μL of MQ water or 0.1 mM DNA sequences: DNA-1 5′→3′: CGATATCG  and DNA-2 5′→3′: CCCTAGGG  dissolved in MQ water. The fluorescence spectra of Capridine β, IE1, and m-AMSA with or without DNA were recorded at the excitation wavelength of 410 nm, within the excitation range used for microscopic studies.DNA topoisomerases are enzymes that catalyze changes in the spatial structure of DNA and play an important role in replication, transcription, and recombination. Beyond their normal functions, those enzymes are significant molecular targets in antimicrobial and anticancer chemotherapy. Our results indicate that their fungal counterpart may also become a promising antifungal target. The evaluation of biological properties of Capridine β indicated that despite its high antifungal activity, the ability to enter the cell and biotransformation into a product that influences the effectiveness of fungal topoisomerase is crucial for its activity. Summing up, the search for antifungal drug candidates targeting topoisomerases among acridine derivatives is undoubtedly worth continuing.


Expression of chemokine receptor CX3CR1 is reportedly restricted to several cell types including natural killer cells, cytotoxic T cells, monocytes, and macrophages. However, its expression and function on exosomes, which are nanosized extracellular vesicles known to act as mediators of intercellular communications, remain unclear. Here, we investigated CX3CR1 expression on exosomes isolated from various cell types. Although we found that all the exosomes tested in our study highly expressed CX3CR1, this chemokine receptor was expressed only inside, but barely on, their source cells. Moreover, exosomal CX3CR1 was capable of binding soluble CX3CL1. Therefore, our study suggests that CX3CR1 is a novel and ligand-competent exosome receptor.  •CX3CR1 is highly expressed by exosomes.•Expression of CX3CR1 is restricted within, but not on, the cells.•Exosomal CX3CR1 is capable of binding soluble CX3CL1.•CX3CL1 binding of exosomes may deprive their source cells of the chance to bind this chemokine. Among t0 nm–5 μmApart from their functionality vis-à-vis integrins, chemokine receptors on exosomes have been shown to play a role in tissue-specific homing or chemotaxis in both cells and exosomes. The CXCR4 and CCR2 on bone-marrow stromal cell-derived exosomes exert a promoting effect on the migration of multiple myeloma cells . In addiWhen cognate chemokines are bound, the chemokine receptors present on the plasma membrane are generally internalized in early endosomes and are then trafficked to MVBs . These uThe chemokine receptor CX3CR1 mediates cell adhesion and trafficking through its interaction with chemokine CX3CL1  . CX3CR1 Here, we examined the exosomal expression of CX3CR1 by using many cell types, revealing its high expression by all the exosomes used, and further showing the exosomal binding to CX3CL1. Based on the compelling results obtained in this study, CX3CR1 represents a ligand-competent chemokine receptor highly expressed by exosomes.22.1in vitro culturing and activating primary T cells as previously described [2 supply. T cells activated for 48 h were moved onto antibody-uncoated dishes and further incubated for 72 h in the same culture media supplemented with interleukin-2 (1 ng/ml) . As mentioned above, to eliminate extracellular vesicles (EVs) including exosomes in FBS, FBS was centrifuged at 76,000 g for 18 h at 4 °C using polypropylene centrifuge tubes , a swing bucket rotor  and an L60 Ultracentrifuge (Beckman Coulter) and the supernatant solution was filtered through 0.22-μm filter units . This filtrated FBS was considered to be exosome-depleted and was used in this study for exosome isolation from the cells.All the study has been done with mouse cell lines and primary cells, unless otherwise specified. The spleen cells isolated were used for escribed . In brieSeveral mouse tumor cell lines including TK1, CT26.WT, EL4, LTPA, and B16F10 cells were obtained from ATCC . RAW264.7 (mouse macrophages) and THP-1 (human monocytes) were also from ATCC. MLO-Y4 (osteocyte) and MLO-A5 (osteoblast) cells were purchased from Kerafast . All cells were cultured, according to the manufacturers’ instructions, for 48 h in RPMI1640, DMEM , or MEMα (Thermo Fisher Scientific) media supplemented with 10% EV-depleted FBS and penicillin/streptomycin.2.2C57BL/6J mice (8–11 weeks old) were purchased from CLEA Japan  and maintained at the Experimental Animal Facility of Mie University. Experimental animal protocols were approved by the Ethics Review Committee for Animal Experimentation of Mie University . Spleens, bone marrows, and lungs were separated from the mice and used to isolate cells by using a mechanical dissociation with Falcon 40-μm cell strainers . The media collected in the cell isolation procedure were used to isolate exosomes (see section 2.3g for 10 min at 4 °C to remove cells. The supernatant was spun at 2000 g for 20 min at 4 °C to eliminate apoptotic bodies. The supernatant was centrifuged in an L60 Ultracentrifuge (Beckman Coulter) at 24,000 g for 20 min at 4 °C. The supernatant was then subjected to a second centrifugation at 110,000 g for 2 h at 4 °C. The pelleted exosomes were subsequently suspended in phosphate-buffered saline (PBS) buffer . In turn, this exosome solution was passed through a 0.22-μm filter unit and spun at 110,000 g for 2 h at 4 °C. The pellet (exosomes) was suspended in PBS buffer. The concentration was measured with a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). The particle size was characterized by using a dynamic light scattering (DLS) device .Exosomes were isolated as previously described with minor changes ,27,28. B2.4The exosomes were conjugated to 4-μm latex beads for efficient detection and then stained with fluorescently labeled monoclonal antibodies as previously shown . In brie2.5Antibodies to CD9 (HI9a), CD63 (NVG-2), CD63 (H5C6), CD81 (Eat-2), CCR9 (9B1), CXCR4 (L276F12), and CX3CR1 (SA011F11) were purchased from BioLegend . Isotype controls including Rat IgG2a, Rat IgG2b, Armenian Hamster IgG, Mouse IgG2a, and Mouse IgG1 were also from BioLegend. The antibody to CD9 (KMC8) was obtained from BD Biosciences. The antibodies to CCR7 (4B12), CCR10 (248918) were acquired from R&D Systems. The cells or microbead-conjugated exosomes were stained with the fluorescently labeled antibodies, washed twice with PBS containing 2% FBS and 2 mM ethylenediaminetetraacetic acid (EDTA) , and analyzed by using BD Accuri C6 flow cytometer and software (BD Biosciences). For this method of microbead conjugation of exosomes, only positive events can be detected and fluorescently quantified in the flow cytometry. In some experiments, total (intracellular plus surface) staining experiment was done by using FIX and PERM Kit (Thermo Fisher Scientific) according to manufacturer's instructions.2.6The exosomes conjugated to the microbeads were incubated with recombinant mouse fractalkine-His (R&D Systems) at 37 °C for 1 h, washed once with PBS containing 2% FBS and 2 mM EDTA, and labeled with phycoerythrin (PE)-anti-His Tag antibody (Biolegend). After wash, fluorescence intensity for fractalkine binding was analyzed by using BD Accuri C6 flow cytometer and software.33.1To characterize the exosomes, we examined expression of tetraspanin markers . All the exosomes isolated from different types of mouse cells used in this study were positive for indicated tetraspanins, although they exhibited the expression levels different depending on source cells A–D. We tWe sought to examine expression pattern of selected chemokine receptors on T-cell exosomes that were isolated from activated primary T cells . We disc3.2We next inquired whether CX3CR1 levels are intracellularly sustained, by using two T-cell lines (TK1 and EL4). Expressions of CCR9, CXCR4, and CX3CR1 were poor on the surface of TK1 and EL4 cells, except for CXCR4, which was moderately expressed by EL4 cells A. In theSome of macrophages have been known to express CX3CR1 ,31. UsinCX3CR1 was superior to other chemokine receptors in its exosomal acquisition, suggesting the possibility that the intracellular compartment of this chemokine receptor might largely be restricted to the membrane of MVBs . In this3.3To determine whether CX3CR1 expression by exosomes is limited to those isolated from the aforementioned cell types or is otherwise widespread, we used various cell lines to isolate their exosomes separately and examined their expression levels of CX3CR1, CCR9, and CXCR4. The exosomes from several cancer cells including B16F10 , CT26.WT , and LTPA  were chosen to test their chemokine-receptor expression levels. These cancer-cell exosomes were found to express typical tetraspanins with some variations depending on their parent cell types B. We fouNext, we isolated the exosomes from spleen, bone marrow (BM), and lung cells of mice and then confirmed for their CD9 and CD63 expressions D, in ordIn order to rule out a possibility that this marked CX3CR1 expression of the exosomes may be manifested due to any remaining EVs in their depleted FBS supplemented to culture media, we next decided to test this. PBS (1 ml), PBS (1 ml) plus EL4 EV sample (5 μg), EV-depleted FBS (1 ml), and FBS (1 ml) were first coupled to 10 μl latex beads as done with the same methods as exosomes. Then, all samples stained equally for CX3CR1 as well as CD9 and CD63 were analyzed using flow cytometry. On note, all three samples except for EL4 EV-containing conjugates did not show any positive signals for the expression of CD9, CD63, and CX3CR1 . TherefoFurthermore, because expression of CX3CR1 was remarkable on all the exosomes tested in current study, it may be open to question that the anti-CX3CR1 antibody is too blunt to discriminate the exosomes positive from negative for mouse CX3CR1. We thus tested this by using the exosomes isolated from THP-1 cells (human monocytes) as a negative control, despite CX3CR1 expression on human exosomes remains obscure and should be carefully determined in a separate experimental setting in near future. THP-1 exosomes positive for human CD9 and CD63 were negative for CX3CR1 , indicat3.4To examine the function of exosomal CX3CR1, we explored its binding to fractalkine (CX3CL1), a ligand unique to CX3CR1 . We chosBased on the results shown in this study, two possibilities regarding the regulatory function of CX3CR1-expressing exosomes can be raised. First, those exosomes expressing CX3CR1 are thought to bind both membrane-bound and soluble CX3CL1 produced by counterpart cells before do source cells. The exosomes isolated from the two selected cell types proved to be capable of binding recombinant CX3CL1 A, thus sOurs is the first study to report exosomal expression of chemokine receptor CX3CR1. In particular, CX3CR1 was prevalently and highly expressed by all of the exosomes isolated from the different cell types tested in this work. The various source cells contained high level of CX3CR1 in their intracellular compartments, presumably on the membrane of MVBs, which are the exosomal reservoirs inside cells. Moreover, those exosomes were capable of binding fractalkine. Our study concludes that CX3CR1 is a novel and ligand-competent molecule of exosomes. In future investigations, it would be worthwhile to elucidate: i) the underlying mechanisms by which CX3CR1 expression is maintained at such high levels on all of the exosomes; and ii) the exosomal impact on any functional changes undergone by the cells or tissues that express membrane-bound CX3CL1.Eun Jeong Park: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - original draft, review & editing, Visualization, Funding acquisition. Phyoe Kyawe Myint: Investigation, Methodology. Michael G. Appiah: Investigation, Methodology. Patsorn Worawattananutai: Investigation, Methodology. Janjira Inprasit: Investigation, Methodology. Onmanee Prajuabjinda: Investigation, Methodology. Zay Yar Soe: Investigation, Methodology. Arong Gaowa: Investigation, Methodology. Eiji Kawamoto: Investigation, Methodology. Motomu Shimaoka: Conceptualization, Data curation, Supervision, Project administration, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that might have influenced the work reported in this paper.