This study aimed to investigate the thermal behavior and composition of volatile compounds, fatty acids and polyphenols in paprika obtained from peppers of different countries. The thermal analysis revealed various transformations in the paprika composition, namely drying, water loss and decomposition of volatile compounds, fatty acids, amino acids, cellulose, hemicellulose and lignin. The main fatty acids found in all paprika oils were linoleic (20.3–64.8%), palmitic (10.6–16.0%) and oleic acid (10.4–18.1%). A notable amount of omega-3 was found in spicy paprika powder varieties. The volatile compounds were classified into six odor classes (citrus (29%), woody (28%), green (18%), fruity (11%), gasoline (10%) and floral (4%)). The total polyphenol content ranged between 5.11 and 10.9 g GA/kg. Capsicum annuum L.) followed by milling of the dried pepper to obtain a fine powder [Paprika is widely used within the food industry as a natural colorant  due to its ability to improve upon the flavor of food through its characteristic taste and pungency . Paprikae powder . Drying e powder . The inte powder . Most mae powder . In papre powder . Carotene powder . The type powder . In addie powder . Recentle powder .The formation of volatile compounds that generate the characteristic aroma is caused by chemical conversions, such as hydrolytic reactions, amino acid conversion, oxidative degradation reactions of lipids (fatty acids) and carotenoids (lipid-soluble pigments), Maillard reactions and caramelization browning . Some stPaprika oil contains saturated (SFAs), monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids. SFAs are undesirable in large quantities because they can cause cardiovascular problems. Moreover, they are stable at room temperature, and the unsaturated fatty acids start to increase the fluidity and oxidation process, leading to the formation of free radicals. Replacing SFAs with PUFAs has positive benefits for cholesterol in the blood. Among all PUFAs, linoleic C18:2) and α-linolenic acids (C18:3) are essential fatty acids for food :2 and α-. Zaki et2 extraction, ultrasound, enfleurage and microwave irradiation. Oil is the fluid separated from the plant in the presence of a solvent. The most common solvent used for the extraction of oil is n-hexane. Studies show that polar solvents can extract all fatty acids and are preferred for this purpose. In addition, extraction methods influence the amounts of fatty acids. Combining solvent extraction, microwave irradiation, ultrasonic extraction and supercritical carbon dioxide techniques could significantly improve oil performance [2 extraction of dry paprika and n-hexane, and the results showed that there is no difference between the two extraction methods. [The methods used for oil extraction from plants are solvent extraction, maceration, cold-press extraction, steam distillation, COformance . Kostrzemethods. . Ultrasomethods. .This study focused on the determination of the key volatile compounds, the comparative analysis of fatty acids, polyphenol and thermal behavior among wild-harvested and commercial paprika samples obtained from pepper varieties from different countries. Ultrasound-assisted extraction (UAE) was applied to extract oil from paprika powder varieties. The interest of this study consists in the following: (i) the literature is enriched with the obtained extensive characterization of different paprikas, (ii) the relationship between the thermal behavior and the content of volatile compounds, cellulose, hemicellulose, lignin and fatty acids, (iii) the aroma profile and classification of volatile compounds by classes of chemical compounds and types of aromas and (iv) the statistical analysis of the content of volatile substances and fatty acids of the eight varieties of paprika.3OH), chloroform (CHCl3), potassium chloride (KCl), sodium sulfate (Na2SO4), isooctane (C8H18), potassium hydroxide (KOH), sodium hydrogen sulphate monohydrate (NaHSO4·H2O), ethanol (C2H6O), diethyl ether (C2H5)2O, phenolphthalein (C20H14O4), sodium chlorite (NaClO2), sulphuric acid (H2SO4) and sodium hydroxide (NaOH), all of analytical grade, were purchased from Merck . The standard FAME mixture  was purchased from Sigma-Aldrich . Ultrapure water (18.2 MΩ·cm−1 at 20 °C) was obtained from a Direct-Q3 UV Water Purification System .The chemicals, including methanol (CHPaprika 1 (P1) was obtained from red Kapia peppers of Romanian origin, light reddish-brown in color, very pleasant in smell, sweet feeling with a well-defined pepper aroma.Paprika 2 (P2) was obtained from hot peppers, with Romania as the country of origin. The uniform powder had a specific smell of pepper with a spicy taste.Paprika 3 (P3) was obtained from golden pepper, with Romania as the country of origin—a yellow-brown powder with a specific and pleasant smell.Paprika 4 (P4) was obtained from hot peppers, with Morocco as the country of origin—light red-orange powder, with a specific smell of peppers and a sweet spicy taste.Paprika 5 (P5) was obtained from red Kapia peppers, with Turkey as the country of origin, and it was characterized by a light red-orange color, specific sweet pepper smell and a weaker taste compared to the others.Paprika 6 (P6) is a ground chili originating from India, with a light red-brown color and a very spicy, aromatic and pleasant taste.Paprika 7 (P7) is obtained from red Kapia peppers originating from China. The taste and smell of this red-orange colored paprika powder is pleasant and specific but weakly pronounced compared to the rest of the samples.Paprika 8 (P8) is obtained from Kapia peppers originating from Hungary. It is characterized by a red-brick color, with a taste and smell specific to this particular pepper assortment.Each variety of paprika (P1–P8) was prepared under the same working conditions. After washing the peppers, they were cut into rings and placed in the oven at a temperature of 80 °C and then left to dry until they reached a light red-brown color and acquired a crunchy texture. After cooling, they were chopped.All the samples were freeze-dried  at −40 °C and −25 psi for 24 h to uniformize their moisture content. The freeze-dried samples were ground using an agate mortar and pestle to obtain homogenized powders. The moisture of the samples was determined by drying the samples to a constant mass at 105 °C in a universal oven .v/v) and introduced into an ultrasonic bath  for 15 min (repeated for four times) at room temperature, according to Pohndorf et al. (2016) with modifications [2SO4 to obtain a clear solution. The solvent was evaporated using the rotary evaporator Laborota 4010 , and the oil obtained was dried at 60 °C in an oven. The yield and the lipid content were calculated using Equation (1).Lm is the extracted lipid weight, and pm is the mass of dried paprika powder. The dried samples (1 g) were extracted with 20 mL chloroform: methanol  were converted to FAME using transesterification methods according to [The oils obtained using ultrasound extraction with chloroform: methanol  (2 mol/L) and vigorously stirred for 30 s. Lastly, the mixture was treated with 1 g of sodium hydrogen sulphate to avoid saponification of methyl esters and neutralize excess alkali. Each oil sample was trimethylated and analyzed in three replicates. Fatty acid compositions were determined using a gas chromatography coupled with flame ionization detector technique, after lipid extraction and transesterification to fatty acid methyl esters. The obtained lipids were converted into FAMEs by transesterification with potassium hydroxide. The samples (0.06 g) were dissolved in isooctane, treated with 0.2 mL methanolic potassium hydroxide solution , using phenolphthalein (2%) as the indicator. The FFA content was calculated in accordance with Equation (2):V is the volume of KOH used for titration (mL), C is the concentration of KOH used for titration, and m is the biomass of oil used for analysis. The free fatty acids were determined by titrating the oil obtained with KOH (0.1 M in ethanol). An amount of −1. The injection volume was 1 µL in a 1:20 split mode. The GC oven temperature program consists of three stages: 60 °C for 1 min, 60 to 200 °C , 200 to 220 °C . The temperature of the injector and detector was set to 250 °C. FAs in samples were identified by comparing their retention time with that of the Supelco FAME standard mixture.The FAME content was determined using GC-FID  equipped with a Zebron ZB-WAX capillary column (30 m × 0.25 mm × 0.25 µm) and a flame ionization detector . The gas carrier was helium with a constant flow rate of 1 mL minThe thermal behavior of all samples was evaluated based on thermal analysis (TG-DTA) carried out with a Mettler-Toledo TGA/SDTA851. The measurements were performed at a heating rate of 20 °C/min in a controlled atmosphere using air or nitrogen with a flow rate of 60 mL/min.−1, increased to 230 °C at a rate of 15 °C·min−1 and then held at a plateau for 7 min. Mass spectra were recorded in electron impact (EI) ionization mode at 70 eV using a TSQ 9000 MS, Thermo Fischer Scientific mass spectrometer. The quadrupole mass detector, ion source and transfer line temperatures were set at 150, 230 and 280 °C, respectively. Mass spectra were scanned in the range m/z 50–450 amu. VOCs were identified by comparing the mass spectra with the NIST 14 database system library and linear retention index. The criteria for compound identification required a mass spectrum matching score of ≥80%. The results were expressed as a percentage of the relative peak area (% RPA) of a peak in each paprika sample that was calculated by dividing the peak area by the total peak area of all identified peaks in each chromatogram. The total ion chromatogram (TIC) of each sample was used for peak area integration.For the HS-SPME GC-MS analysis of volatile organic compounds, 3 g of ground paprika P1–P8 was transferred to a 20 mL headspace vial, and 3 mL of NaCl saturated solution was added to enhance the volatile organic compounds in the headspace and to inhibit any enzymatic reactions. The method was developed according to Martín et al., with improvement [All measurements were conducted in triplicate, and data are presented as the mean ± standard deviation.Polyphenols were measured using the Folin–Ciocalteu colorimetric method using a Perkin Elmer Lambda 25 spectrophotometer to measure the blue complex at 760 nm, and gallic acid was used as a reference standard . All mea2 in acetic acid (10%). The content of cellulose was determined by treatment of holocellulose with NaOH (17.5%). The lignin was determined as present residue after treatment of samples with 72% H2SO4 solution.The content of cellulose, hemicelluloses and lignin in paprika varieties was determined according to Senila et al. . The conFor the statistical processing of the data, OriginPro Data Analysis and Graphing Software  was used. Descriptive data analyses, including standard deviation and Pearson correlation, explained by very strong correlation (0.9–1), strong correlation (0.70–0.89), moderate correlation (0.40–0.7), weak correlation (0.10–0.39) and negligible correlation, were realized. Two different sets of variables, including fatty acids and volatiles and polyphenols, were evaluated in order to separate the geographical provenance and types of the paprika samples. The paprika samples were grouped according to sets of variable contents by Agglomerative Hierarchical Clustering (AHC) using the squared Euclidian distance and the Ward method for combining clusters, using XLStat software version 2019.3.2 .The decomposition stages of the paprika samples were investigated in both air  and nitrThe thermal behavior is different for decompositions in a nitrogen atmosphere  comparedIn our previous studies on coffee , the theCapsicum annum pepper species and reported the variation of lignin during the maturation period. It was concluded that the maturation process caused the lignin content to decrease. The process can be explained by the rearrangement of cell structure by plant maturation and the interaction between lignin-like substances derived from phenyl propanoid precursors [Lignin is a natural phenolic polymer found in higher plant tissues and the second most abundant polymer after organic cellulose . In all ecursors . Celluloecursors .The cellulose content varied in the following order (%): P2 (32.2 ± 2.1) > P4 (28.6 ± 1.8) > P6 (25.5 ± 2.0) > P1 (23.1 ± 1.9) > P5 (23.0 ± 1.3) > P7 (21.1 ± 1.6) > P8 (19.2 ± 1.2) > P3 (19.1 ± 1.0), whereas hemicellulose contents were (%) P6 (13.5 ± 0.8) > P4 (12.1 ± 1.0) > P2 (11.1 ± 0.9) > P8 (8.9 ± 0.5) > P1 (8.1 ± 0.5) > P5 (7.2 ± 0.6) > P7 (5.4 ± 0.3) > P3 (5.2 ± 0.4). The highest content of cellulose was found in spicy varieties. According to Mudrić et al., in Serbian paprika, the following sugars were identified: glucose, fructose, arabinose, xylose, mannose, disaccharides , trisaccharides and sugar alcohols . The resVolatile compounds of Romanian paprika were analyzed using SPME followed by GC-MS, as shown in Hydrocarbons had the highest proportion 50.6%, a in the Aldehydes 28.7%, a are forThe content of ketones 6.7%, a in paprAlcohol compounds 4.1%, a accountEsters 2.1%, a in paprAllium family, including garlic and shallot [Sulfur compounds are decomposition products of thiosulfinates and are derived from amino acid flavor precursors of the  shallot ,33,34. D shallot .The predominant aroma is citrus (29%), where the limonene and woody aroma (28%) of paprika is represented by furfural, 5-methyl furfural, α-pinene, β-pinene, 3-(bromomethyl)cyclohexane, benzaldehyde, β-myrcene, 1.3.8-p-menthathiene and cedrene . Green-aHowever, the cis (oleic acid) and trans (elaidic acid) isomers of C18:1 and cis (linoleic acid) and trans (linolaidic acid) isomers of C18:2 were not separated and were quantified together. The lipid content varied in the following order: P5 (16.0 ± 1.1%) > P2 (15.5 ± 1.0%) > P6 (13.7 ± 1.1%) > P7 (13.0 ± 1.0%) > P8 (12.4 ± 0.89%) > P4 (12.1 ± 0.90) > P1 (10.01 ± 0.90%) > P3 (4.2 ± 0.2%). The fatty acid methyl esters found in all oil from paprika powder varieties are presented in The PUFAs are divided into omega-6 (ω-6) and omega-3 (ω-3). The highest PUFA (ω-6) quantities found in all samples are reported for linoleic acid (C18:2(c + t)) and varied in the following order: P4 (64.7%) > P8(61.9%) > P6(61.4%) > P7 (56.8%) > P3(24.3%) > P1, P5 and P2 (approx.20%). The highest PUFA content was found in spicy samples. According to −1), and the lowest content was measured in P8 . The sample with the lowest total polyphenol content (sweet paprika spice) had only about 50% of the content of paprika delicate with the highest value; there is only a weak connection between the pungency of the spices and the polyphenolic amount [Polyphenols are often responsible for the antioxidant capacity of plant products, and they could be important constituents to explain the protective effects of plant-derived foods and beverages . The resc amount . The amoc amount .Hierarchical clustering (dendrogram), AHC of the paprika varieties presented in In the case of volatile compounds, four clusters can be observed. A very strong positive correlation was obtained for d-carvone with camphene and β-pinene (0.99). Four clusters of volatile compounds were identified b. The prThis study conducted a comparative analysis of volatile compounds, fatty acids, cellulose, hemicellulose, lignin and total polyphenols and evaluated the thermal behavior among paprika samples from different countries. The thermal analysis of paprika samples was in agreement with the moisture evaporation of adsorbed water and solvent (at 64–73 °C and a mass loss of 3.6–4.6%), decomposition of volatile compounds (206–214 °C and mass loss of 29.6–33.7%), decomposition of fatty acids and proteins (at 324–344 °C and mass loss of 22.1–24.5%) and degradation of cellulose, hemicellulose and lignin . In total, 32 volatile compounds divided into seven chemical classes were identified in the samples, including 16 hydrocarbons, 5 aldehydes, 4 ketones, 3 alcohols, 2 esters, 1 sulfur compound and 1 heterocyclic compound. The predominant aromas in paprika samples were citrus  and woody (28%), and other aromas identified in smaller quantities were green (18%), fruity (11%), gasoline (10%) and floral (4%). The limonene was the most abundant in the paprika samples, although its amount varied between samples as follows: P7 (100%) > P8 (42.8%) > P6 (41.6%) > P3 (30.2%). The highest PUFA (ω-6) quantities were found in hot pepper from Turkey, India, China and Hungary. Linoleic acid (C18:2(c + t)) is the major PUFA fatty acid. The highest SFA content was found in golden pepper. The highest content of polyphenols was measured in P3 (10.9 g GA/kg), and the lowest content was measured in P8 . This could be influenced by pepper variety and also be dependent on the time of harvesting and processing. The obtained results will help create new research perspectives regarding the use of paprika powders in the food, cosmetic and pharmaceutical industries, thus creating new natural ingredients and bioactive compounds.


Mitochondrial fission protein 1 (FIS1) is conserved in all eukaryotes, yet its function in metazoans is thought divergent. Structure-based sequence alignments of FIS1 revealed a conserved, but noncanonical, three-residue insert in its first tetratricopeptide repeat (TPR) suggesting a conserved function. In vertebrates, this insert is serine (S45), lysine (K46), and tyrosine (Y47). To determine the biological role of the “SKY insert,” three variants were tested in HCT116 cells for altered mitochondrial morphology and recruitment of fission mechanoenzyme DRP1 and mitophagic adaptor TBC1D15. Similar to ectopically expressed wildtype FIS1, substitution of the SKY insert with alanine (AAA) fragmented mitochondria into perinuclear clumps associated with increased mitochondrial DRP1. In contrast, deletion variants (either ∆SKY or ∆SKYD49G) elongated mitochondrial networks with reduced mitochondrial recruitment of DRP1, despite DRP1 coimmunoprecipitates being highly enriched with ΔSKY variants. Ectopic wildtype FIS1 drove co-expressed YFP-TBC1D15 entirely from the cytoplasm to mitochondria as punctate structures concomitant with enhanced mitochondrial DRP1 recruitment. YFP-TBC1D15 co-expressed with the AAA variant further enhanced mitochondrial DRP1 recruitment, indicating a gain of function. In contrast, YFP-TBC1D15 co-expressed with deletion variants impaired mitochondrial DRP1 and YFP-TBC1D15 recruitment; however, mitochondrial fragmentation was restored. These phenotypes were not due to misfolding or poor expression of FIS1 variants, although ∆SKYD49G induced conformational heterogeneity that is lost upon deletion of the regulatory Fis1 arm, indicating SKY–arm interactions. Collectively, these results support a unifying model whereby FIS1 activity is effectively governed by intramolecular interactions between its regulatory arm and a noncanonical TPR insert that is conserved across eukaryotes. MFF recruits DRP1 for midbody, housekeeping fission, whereas FIS1 recruits DRP1 to peripheral sites for mitophagic removal   proteins A 35, 36, 36A (35A) . To date regions . FIS1 is regions , 40, 41. regions . Consist regions .Figure 1In the current study, we searched for unifying mechanisms that could account for conservations of FIS1 functions and the observed differences between vertebrate and invertebrate species. Structure and phylogeny-based sequence alignments revealed a three-residue insert in the N-terminal TPR that is uniquely conserved as Ser-X-X in all species. Moreover, this insert is conserved as Ser-Lys-Tyr (SKY) in all vertebrates. Here, we report that the conserved SKY insert is not a stringent structural requirement for human FIS1 but is indispensable for its mitochondrial recruitment of TBC1D15 complexes that appear crucial to FIS1’s mitochondria division functions in vertebrates. Coimmunoprecipitation experiments suggests that ΔSKY variants drastically reduce TBC1D15 recruitment, although DRP1 recruitment is retained. Furthermore, we show that FIS1-induced fission of mitochondrial networks can be potently upregulated or downregulated by simply perturbing insert residues. Overall, our findings provide useful insights into elucidating unifying structural mechanisms that govern FIS1 activity and suggest differences between vertebrate and invertebrate FIS1 highlighted by insert residues.B). Strikingly, these alignments revealed a noncanonical TPR feature in the first, but not the second TPR of FIS1: instead of the canonical 34 amino acids that define a TPR, FIS1’s first TPR (N-TPR) contains an additional stretch of three amino acids—serine, lysine, and tyrosine—inserted within the turn region of the canonical helix-turn-helix of a TPR (B). Curiously, a three-residue insert is present in all known FIS1 sequences and occurs as an invariant SKY in vertebrates (C). As the “SKY insert” is not required to specify the TPR fold, we infer that it is not conserved for structural purposes, but rather for FIS1 activity.We used structure-based sequence alignments to compare human proteins containing TPRs with FIS1 B. Strikiof a TPR B. Curioutebrates C. As theα RMSD = 1.1 Å) with only a slightly longer turn for FIS1 (A). This suggested that replacing the SKY insert with the turn from the TPR consensus sequence would not perturb the FIS1 fold. TPRs have a characteristic three-residue turn with φ, ψ backbone torsional angles that, according to Effimov’s convention  substituting three Ala residues for SKY (AAA), (ii) deleting the SKY insert (ΔSKY) that retains the central Asp to give N-D-D, (iii) deleting the SKY insert that substitutes the central Asp (D49) with the canonical Gly (ΔSKYD49G) to give N-G-D, and (iv) a control that retained the SKY insert but replaced the succeeding Asp with Gly (D49G).To investigate the functional relevance of the SKY insert, we designed a FIS1 variant with a short canonical TPR turn lacking the insert. This was accomplished by analyzing the TPRs from a well-characterized consensus TPR sequence that adopts the canonical structure. This consensus TPR is an entirely non-native sequence designed from statistical thermodynamic analysis of TPR sequences and was shown to fold into the desired TPR structure, indicating the robustness of the design and TPR fold . Structufor FIS1 A. This snvention , correspm). The WT cytoplasmic domain is quite thermally stable with a Tm of 81.8 ± 0.1 °C and neither alanine substitutions (AAA) nor the control construct (D49G) impacted thermal stability compared to WT (B). Deletion of the SKY insert (ΔSKY) modestly decreased the Tm to 71.5 ± 0.2 °C consistent with the assumption that these residues are dispensable for the TPR fold. However, the ΔSKYD49G construct dramatically decreased the Tm to 59.9 ± 0.2 °C. To understand this, we turned to two-dimensional NMR spectroscopy of these proteins uniformly labeled with 15N that allows for individual residue contributions to the overall protein fold. All constructs showed similar chemical shift dispersion to WT indicating well-folded proteins (C). However, ΔSKYD49G NMR data showed an increased broadening of resonances throughout the spectrum consistent with a significant degree of conformational heterogeneity. Moreover, cross peaks for N-terminal residues 1 to 8 corresponding to the “Fis1 arm” were not detected. To test the role of the FIS1 arm in this conformational heterogeneity, we created a ΔSKYD49G variant lacking the N-terminal arm (ΔNΔSKYD49G) and assessed its structure by thermal melt and NMR. Deletion of the FIS1 arm restored the Tm to a value similar to ΔSKY (73.6 ± 0.5 °C) and showed resonances largely similar to WT with little indication of conformational heterogeneity . We observed that protein expression levels between transfected FIS1 variants were not similar (A). Therefore, to rule out the possibility that morphological and/or functional changes between variants were simply a result of poor or inconsistent protein expression levels across the variants, we included only transfected cells that expressed moderate FIS1 levels for subsequent analyses (B). The changes in mitochondrial morphology were quantified by using MitoGraph (https://rafelski.com/susanne/MitoGraph) to determine the mitochondrial area, which showed a statistically significant decrease for WT compared to vector alone . As a control for the TPR domain, a commonly used FIS1 variant (5LA) that replaces five conserved TPR Leu residues with Ala was expressed  cells. Ectopic overexpression of WT FIS1 induces uniform fragmentation and collapse of mitochondrial networks around the nucleus collectively resulting in perinuclear clumps confirming the findings by others  cells. E, 47, 48 Fig. 3 similar A. Therefanalyses B. The chxpressed , 30. As C). Mitochondrial recruitment of DRP1 is potently induced upon ectopic FIS1 overexpression consistent with earlier findings . Therefore, we asked if mitochondrial DRP1 recruitment was significantly perturbed between WT and variant FIS1 overexpression in HCT116 cells. Consistent with an elongated mitochondrial network, mitochondrial DRP1 colocalization decreased by nearly 2-fold for both ΔSKY overexpressing cells. A similar decrease in mitochondrial DRP1 was observed for the known loss of function variant 5LA although expression of this variant induced mitochondrial clumping. In contrast, both AAA and D49G variants recruited DRP1 to mitochondria similar to WT FIS1. The reduction in mitochondrial area induced by FIS1 variants correlated reasonably well (R2 = 0.65) with their ability to recruit DRP1 with the notable exception of 5LA . We also noted less Drp1 signal in both the ΔSKY and 5LA expressing cells. To investigate if the observed differences in mitochondrial phenotypes were due to alterations in FIS1-DRP1 complex formation, we expressed variants in HCT116 cells, then harvested endogenous Drp1 complexes under cross-linking conditions was scored as the ratio of immunoprecipitated to input FIS1. Expectedly, we observed a nearly 4-fold increase in FIS1–DRP1 interaction when WT FIS1 (0.7 ± 0.1) was expressed compared to vector (0.2 ± 0.1) alone. Compared to WT FIS1, AAA had slightly higher ratios (0.9 ± 0.1), while D49G had the least ratio of all variants (0.7 ± 0.3). Unexpectedly, we observed high ratios for loss of function variants ΔSKY, ΔSKYD49G, and 5LA , suggesting that compared to WT FIS1 expression, there is an increase in FIS1–DRP1 interaction . This unexpected trend suggests that loss of function variants  can still recruit DRP1 in the absence of fission. The abnormally high ratios observed in ΔSKY and 5LA (1.31 ± 0.9 and 3.92 ± 1.9) are consistent with the modest expression of these variants that was restricted to the mitochondria and is in contrast to ectopic WT FIS1 that was less restricted and also highly expressed in the cytosol . Taken together, our results indicate that deleting the SKY insert leads to large perturbations in FIS1–DRP1 complex formation that appear to impair mitochondrial fission.The striking morphological changes induced by ectopic FIS1 involves mitochondrial recruitment of nonresident factors such as the highly conserved dynamin family GTPase, DRP1 , 16, 41.Fig. 3findings , 16, 41 A). Mitochondrial recruitment of YFP-TBC1D15 was evaluated by measuring colocalization between YFP and immunostained TOM20 (B). Without FIS1 overexpression, the TBC1D15 signal is predominantly cytoplasmic and does not concentrate on mitochondrial networks, consistent with endogenous FIS1 levels in HCT116 cells being quite low (B). By contrast, WT FIS1 expression triggers a robust transition of cytosolic TBC1D15 pools onto mitochondrial sites as discrete foci or puncta, which was concomitant with FIS1-induced mitochondrial fragmentation and perinuclear clumping. For FIS1 variants, coexpression of YFP-TBC1D15 impaired the YFP-TBC1D15 cytoplasm-to-puncta transition. To quantify this transition, the mean and mode values of cellular YFP-TBC1D15 signal were measured and reported as mean:mode ratios (C). For vector alone, the mean and mode are essentially equivalent, reflecting the even distribution. For WT FIS1 expression, the mean:mode ratio decreases by 40%, reflecting a decrease of uniform, cytoplasmic YFP-TBC1D15, and the formation of TBC1D15 punctate structures that reside on mitochondrial surfaces (C). As expected, the 5LA variant impaired both mitochondrial recruitment and formation of TBC1D15 puncta with similar mean:mode ratios to vector alone (B and C). In the case of AAA, puncta formation appeared to be dysregulated because compared to WT, YFP-TBC1D15 mitochondrial recruitment was reduced by half (B), and puncta formation appeared unperturbed (C). Although, punctate structures in AAA were noticeably larger but fewer than WT as indicated by the increase in mean:mode values. Both ΔSKY constructs similarly impaired mitochondrial recruitment, and puncta formation was almost completely abolished by ΔSKY (B and C). ΔSKYD49G caused the formation of fewer punctate structures, a phenotype that is between ΔSKY and D49G, the latter of which appeared to be more effective at driving puncta formation than WT (C). To determine if these observed changes were indeed due to disruptions in Fis1–TBC1D15 interactions, we isolated TBC1D15 complexes from cells coexpressing FIS1 and YFP-TBC1D15 by coimmunoprecipitation and then probed for FIS1. In line with previous reports, we observed that TBC1D15–Fis1 complex formation is almost nonexistent at endogenous FIS1 levels (D). In contrast, complex formation is robust when WT FIS1 or D49G is ectopically expressed with YFP-TBC1D15. In line with previous reports, FIS1–TBC1D15 complexes were significantly disrupted by 5LA. Similarly, complex formation was impaired by AAA and even more so for both ΔSKY constructs (D). We, therefore, conclude that the FIS1 SKY insert is required for proper TBC1D15 recruitment.The FIS1 TPR domain is exposed to the cytoplasm, where it also recruits other binding partners to help govern mitochondrial network morphology. One such class of proteins are the cytoplasmic TBC1 effectors important to many cellular functions, including serving as GTPase-activating proteins for Rab family proteins. One TBC1 protein recruited by FIS1 is TBC1D15, and we next explored if the SKY variants impacted TBC1D15 recruitment. For this, the FIS1 constructs were cotransfected with YFP-TBC1D15, and mitochondrial networks were visualized by immunofluorescence of the mitochondrial outer membrane marker TOM20 A. Mitoched TOM20 B. Withouuite low B. By cone ratios C. For vesurfaces C. As expor alone , B and C by half B, and puerturbed C. Althou by ΔSKY , B and C than WT C. To det1 levels D. In connstructs D. We, thA and B). In the absence of exogenous TBC1D15, the AAA variant drove a similar clumped morphology to WT , which was quantified again by using MitoGraph to measure the mitochondrial area . Surprisingly, compared to WT, coexpression of AAA with YFP-TBC1D15 caused mitochondrial clumps to resolve into much smaller clumps, indicated by a 10% decrease in mitochondria . Coexpression of either ΔSKY constructs with YFP-TBC1D15 reversed the elongated mitochondrial morphology of these variants as indicated by similar mitochondrial areas to WT (A and B). Coexpression of D49G with YFP-TBC1D15 led to decreased mitochondrial area consistent with its increased TBC1 recruitment. By contrast, coexpression of the 5LA variant did not show increased mitochondrial fragmentation with YFP-TBC1D15, consistent with 5LA’s defective ability to support TBC1 recruitment onto mitochondrial sites. These results indicate that insert perturbations modulate FIS1 activity through TBC1D15 recruitment, since ΔSKY loss of function is rescued by TBC1D15 expression, supporting an integral role for TBC1D15 in FIS1-driven changes in mitochondrial morphology.We next asked if coexpression of TBC1D15 with FIS1 variants impacted mitochondrial morphology and DRP1 recruitment. To ensure that functional analyses were not biased by differential protein expression levels, we again gated for only cells that moderately expressed FIS1 , A and BA and C). Across all conditions, expression of TBC1D15 increased mitochondrial localization of DRP1. This is most notable for vector-transformed cells with endogenous FIS1 expression, as they showed a bimodal distribution of DRP1 recruitment in the violin plot and a reversal of the DRP1 poor recruitment phenotype upon TBC1D15 expression (C). In the presence of TBC1D15, the AAA variant increased DRP1 recruitment by over 20% compared to WT consistent with its more pronounced effect on mitochondrial morphology with decreased mitochondrial area. D49G showed a similar effect, although not statistically significant with respect to DRP1 localization. For the ΔSKY variants, we observed a bimodal distribution of DRP1 localization in the absence of TBC1D15 coexpression, which was similar to vector alone. This bimodal distribution was also eliminated upon TBC1D15 coexpression, although these ΔSKY variants still had impaired DRP1 localization compared to WT (A and C). Similar results were found for the 5LA variant. These data indicate that the expression of TBC1D15 potentiates mitochondrial DRP1 recruitment and partially rescues the fission defect in FIS1 ΔSKY variants. For example, ΔSKY variants reduced mitochondrial area while concomitantly increasing DRP1 colocalization (D). Interestingly, these correlational analyses revealed that ectopic TBC1D15 unmasks significant functional differences between WT FIS1 and the AAA variants that are otherwise obscured, suggesting that FIS1’s fission activity is dependent on TBC1D15 (D).To determine whether this rescue depended on DRP1, we immunostained for endogenous DRP1 in these experiments and assessed mitochondrial colocalization , A and Cpression C. In theed to WT , A and Clization D. Intere TBC1D15 D.AAA and D49G variants in the presence of ectopic TBC1D15. Interestingly, ectopic TBC1D15 also resolved mitochondrial clumps, presumably by potentiating mitochondrial DRP1 recruitment and fission. Thus, our results indicate that impairments to the mitochondrial fission machinery can have dominant effects—phenotypically in the form of mitochondrial clumping—that are mitigated by augmenting mitochondrial fission.Here, we report that mitochondrial fragmentation and perinuclear clumping typical of WT FIS1 overexpression were abolished upon deletion of the SKY insert, which we show is a noncanonical yet highly conserved insert into the N-terminal TPR of FIS1 . In thisAAA and D49G, but not ΔSKY variants. NMR chemical shift changes in arm residues upon deletion of SKY also support the possibility of arm–SKY intramolecular interactions (m to 73.6 ± 0.5 (not shown). Thus, multiple lines of evidence support that FIS1 activity requires intramolecular arm–SKY interactions that might govern the recruitment and assembly of effector proteins like TBC1D15 and DRP1 , and thus links TBC1D15 to both Fis1 activity and turnover (A and S3E). Interestingly, in the presence of YFP-TBC1D15, the AAA FIS1 variant shows a significant gain of function phenotype compared to WT  as a template  cells as previously described (The soluble domains of FIS1 and variants were recombinantly expressed as SUMO protease cleavable 6xHis-smt3 fusion constructs in escribed . Postcleescribed . SubsequF + mF∗T) + (SU + mU∗T)∗exp(ΔH/R∗(1/Tm-1/T)))/(1+exp(ΔH/R∗(1/Tm-1/T))) to determine the midpoint of the unfolding transition, Tm, and rendered as box and whisker plots using R (https://www.r-project.org/).Thermal unfolding was monitored by light scattering and intrinsic fluorescence at 330 nm and 350 nm using a NanoTemper Prometheus instrument. Briefly, FIS1 or variants were prepared at a final concentration of 20 μM in 100 mM Hepes, pH 7.4, 200 mM NaCl, 1 mM DTT, 0.02% sodium azide. High-sensitivity capillaries (MO-K022) were then filled with each sample in four replicates for thermal scans. A melting scan was performed using an excitation power of 100%, a temperature range of 25 °C to 95 °C, and a temperature ramp of 0.5 °C/min. The resulting light scattering data were fit to a two-state model using the method of Santoro–Bolen equation  with the1H,15 N heteronuclear single quantum coherence data were collected in 3 mm NMR tubes (Bruker) on a 14.1 T Bruker Avance II spectrometer equipped with a 5 mm TCI cryoprobe with a z-axis gradient. Data were collected on 100 μM 15N-FIS1 in 100 mM Hepes pH 7.4, 200 mM NaCl, 1 mM DTT, 0.02% (w/v) sodium azide, 10% 2H2O, 25 °C with eight transients, and 1024 (t2) × 300 (t1) complex points with acquisition times of 51.2 ms (1H) and 75 ms (15N). Spectra were processed with NMRPipe and analyzed with NMRAnalysis 2.5.2     and NMRAassign/)  using NMassign/) . Chemicaassign/)  and for HCT116 cells (American Type Culture Collection) were cultured in Mcoy5A supplemented with 10 mM glutamine, 10% fetal bovine serum (FBS), and 1% non-essential amino acid. See table of reagents in Eighteen to twenty-four hours posttransfection, the medium was aspirated and replaced with 4% paraformaldehyde (prewarmed to 37 °C) and incubated with gentle shaking at RT for 25 to 30 min (see table of reagents in https://imagej.net/software/fiji/). All immunofluorescence-based recruitment experiments were repeated three times and at least 30 cells or more  per experimental condition were manually cropped for statistical analyses. Prior to statistical analyses of morphology and DRP1 recruitment, FIS1-gating was done to exclude cells expressing higher than 1600 AU. For colocalization analysis, the FIJI coloc2 plugin was used to calculate Pearson’s correlation between endogenous DRP1 and mitoYFP, DRP1 and Tom20, or YFP-TBC1D15 and endogenous Tom20 as described . For morphology counts, cells were visualized using a 100× oil objective at 0.2-micron z-slices and 0.07-micron resolution and assessed by eye for the indicated morphology. Representative confocal images were acquired and processed using FIJI (escribed . We noteescribed . Maximumescribed . For anahttps://imagej.net/ij/) macro and screened for accurate mitochondrial segmentation as previously described . Once the run is complete, we use the Compass software to determine the signal area for each antibody. For area calculations, we use the dropped lines option. We additionally performed a total protein assay for loading level normalization using the Total Protein Detection Module (DM-TP01). The total protein area for FIS1 was normalized to overexpressed wildtype FIS1 and plotted for comparison. Conventional Western blots were developed and imaged by enhanced chemiluminescence (Bio-Rad) and quantified by densitometry using the gel analyses tool on FIJI (ImageJ).Transfected HCT116 cells were harvested using a radioimmunoprecipitation assay lysis kit (ProteinSimple CBS401), and cleared supernatants were saved at −20 °C until analyses. Capillary electrophoresis experiments were carried out using a JESS system (ProteinSimple) with the 25 capillary 12 to 230 kDa Separation module (ProteinSimple SM-W004), FIS1 antibody (Proteintech 10955-1-AP), and the Anti-Rabbit Detection Module (ProteinSimple DM-001). Setup and analysis were performed according to the manufacturer’s instructions. Briefly, samples were diluted to a final concentration of 0.2 mg/ml in 0.1× sample buffer and 5× fluorescent master mix. The biotinylated ladder and the samples are then heated at 95 °C for 5 min. Once all reagents were dispensed, the plate was covered, and centrifuged for 5 min at 1000 rpm. Runs were performed using the instrument default settings in the Compass software , and transferred to prechilled tubes. Washed cell pellets were cross-linked by resuspending in cross-linking buffer  and incubating either on ice or at RT for 8 min. The reaction was subsequently quenched by adding an equal volume of quenching buffer (1.25 M glycine in 1× HBSS). The recovered cell pellets were then resuspended in 300 μL of lysis buffer . Resuspended whole-cell lysates were then pulse sonicated on ice and incubated for at least 1 h at 4 °C to completely recover cross-linked complexes. At 4 °C, whole-cell lysates were centrifuged at 14,000 RPM for 10 min, and the supernatants were carefully collected in fresh prechilled tubes. Lysates were precleared with 10 μl of protein A/G beads, 30 μl was collected for input fractions, and the remaining supernatant was used for coimmunoprecipitation. Endogenous DRP1 was immunoprecipitated using a mouse monoclonal DRP1 antibody (sc-271583) pre-conjugated to A/G beads. Ectopic YFP-TBC1D15 was immunoprecipitated using a GFP-nanobody . Coimmunoprecipitates were recovered for SDS-PAGE and Western blot by boiling beads in 2.5× Laemmli buffer.https://github.com/Hill-Lab/. Raw data is available upon request.All R scripts used for data analysis and visualization are available upon request and/or for download at This article contains R. B. H. and R. T. have financial interest in Cytegen, a company developing therapies to improve mitochondrial function. A portion of the salary for R. T. is paid for by the company. Cytegen had no role in study design; in the data collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. The other authors declare that they have no conflicts of interest with the contents of this article.


Ru0.5Ir0.5O2 catalysts can form more Ru active sites with high oxidation states at lower applied voltages after Ir incorporation, which is confirmed by the pulse voltage induced current method. Also, The X-ray absorption spectroscopy data shows that the Ru-O-Ir local structure in two-dimensional Ru0.5Ir0.5O2 solid solution improved the stability of these Ru centers.The oxygen evolution reactions in acid play an important role in multiple energy storage devices. The practical promising Ru-Ir based catalysts need both the stable high oxidation state of the Ru centers and the high stability of these Ru species. Here, we report stable and oxidative charged Ru in two-dimensional ruthenium-iridium oxide enhances the activity. The Ru Stabilizing high oxidation state of Ru centers is important to achieve stable performance for acidic oxygen evolution reaction. Here the authors report two-dimensional ruthenium-iridium oxide for enhanced stability and activity for acidic water oxidation in proton exchange membrane electrolyzer. Currently, only Ir-based oxides8, Ru-based oxides12 and their derivatives have sufficient corrosion resistance to withstand the harsh acid corrosion and oxidation environments of OER13. The scarcity and relatively low OER activity of Ir are insufficient to meet industrial requirements14, while, Ru-based catalysts generally suffer from poor stability on account of the formation of soluble Ru oxides (such as RuO4) during OER process16. Up to date, the OER catalysts of RuIr bimetallic oxides have been extended from their component-dependence21, optimization of bimetallic oxide nanostructures  to modification of electronic properties27. Notably, the redox of Ru in RuIr bimetallic oxides could be affected by Ir species in RuIr bimetallic oxides system19, in which Ru exhibits a strong oxidation state28. Two sides of requirements must be met by a prospective RuIr oxide-based OER catalyst. One is the stable high oxidation state of the Ru active center, and the other one is to prevent the dissolution and inactivation of Ru species by excessive oxidation. Designing a RuIr oxide based OER catalyst that can be qualified for both requirements is quite challenging.Anodic oxygen evolution reaction (OER) is the bottleneck in the hydrogen production process of water electrolysis0.5Ir0.5O2 used as an OER catalyst, it shows excellent OER performance in acidic media, providing an anodic current density of 10 mA cm−2 at an overpotential of only 151 mV, and together with an activity retention time over a 618.3 h stability test at 10 mA cm−2. In addition, Ru0.5Ir0.5O2 also achieves a high mass activity of 730.4 A gIr + Ru−1 at 1.44 V vs. RHE and a high turnover frequency (TOF) of 6.84 s−1 at 1.44 V vs. RHE. The results of pulse voltage induced current (PVC), cyclic voltammetry (CV), density functional theory studies and transient light-induced voltage (TPV) tests showed that the origin of high activity in the Ru0.5Ir0.5O2 catalyst is more Ru active sites with high oxidation states at low applied voltage were formed after Ir incorporation, while increasing the oxidative charge concentration on the surface of the catalyst during the OER process. The X-ray absorption spectroscopy (XAS) measurements of Ru0.5Ir0.5O2 show that the stability was originated from the interaction within the local structure of Ru-O-Ir, and the characteristics of two-dimensional material structure.Here, a two-dimensional substitutional solid solution material, phase ruthenium-iridium oxide was successfully synthesized via a two-step molten-alkali process. We show that the stable and oxidative charged Ru two-dimensional RuIr oxides enhance the OER activity significantly. When the optimal Ru2 and IrO2, Ru/Ir atoms have the same valence state, the same crystal structure type and similar chemical properties, which are conducive to synthesis substitutional solid solution. In a typical experiment, IrCl3 (0.5 M) and KOH were mixed and thoroughly ground and the mixture was heated again by a mechano-thermal method29. The cooled solid was thoroughly ground again with addition of RuCl3 (0.5 M) and KOH and heated again by a mechano-thermal method to obtain Ru0.5Ir0.5O2 product29. Both heating processes are heated to 800 °C for 2 h. One thing should be noted that when further annealing Ru0.5Ir0.5O2 at 900 °C for 2 h, it obtains the mixture of thermally stable rutile phase IrO2 and RuO2  in the second synthetic step was further increased to more than 0.5 M , a large amount of soluble Ru/Ir complexes were generated in the obtained product  the catalytic active components (Ru/Ir atoms) in the catalysts. In RuO0.5Ir0.5O2 exhibits a typical two-dimensional (2D) sheet-like shape structure with a large diameter of about 3–5 μm. Atomic force microscopy (AFM) image further confirms the two-dimensional structure where the thickness of the Ru0.5Ir0.5O2 nanosheet is approximately 1.9 nm   and commercial RuO2 (C-RuO2) were characterized by X-ray diffraction (XRD), SEM, TEM, and HAADF-STEM. Both the structure characterizations showed the stable rutile phase C-IrO2 and rutile phase C-RuO2  image Fig.  and tran0.5Ir0.5O2. The sharp Bragg diffraction peaks in the XRD pattern illustrate the high crystallinity and ordered stacking of the 2D layers in Ru0.5Ir0.5O2  image . As depicted in the XPS spectra, Ru0.5Ir0.5O2 exhibits a meaningful negative-shift of Ru 3p3/2 and Ru 3p1/2 peaks (461.6 and 483.6 eV) in comparison with those for C-RuO2 (462.0 and 484.0 eV) 24. Ir 4 f XPS spectra , Ru0.5Ir0.5O2 shows a slight positive-shift. Which indicates that Ru0.5Ir0.5O2 has the higher Ir valence state than C-IrO230. The fitting parameters used of all peaks can be found in Supplementary Table The composition and chemical states of RueV) Fig. , which ctra Fig.  show the0.5Ir0.5O2, and the intensity analysis of which can give the clearer valence electronic state information31. As depicted in Fig. 0.5Ir0.5O2 shows the white-line adsorption energy of which is between that of C-RuO2 and Ru foil, indicating that the valence state of Ru in Ru0.5Ir0.5O2 is between 0 and +433. Meanwhile, by characterizing the intensity of white-line intensity of Ir L3-edge XANES spectra, the Ir valence state can be more easily observed. Figure 0.5Ir0.5O2 exhibits significantly higher than that of C-IrO2 and Ir foil, demonstrating that the valence states of Ir show an order of Ru0.5Ir0.5O2 > C-IrO2 > Ir foil34. The valance state results of Ru and Ir species were consistent with those discussed earlier in the XPS results. The Fourier transforms of the extended X-ray absorption fine structure (EXAFS) spectra 35. Compared with the reference samples, Ru-Ir, Ir-Ru scattering signals appeared in Ru K-edge  was used to explore the electron transition behavior and electronic structure of Rutra Fig.  at the Rdge Fig.  and Ir Ldge Fig.  WT-EXAFS0.5Ir0.5O2 was evaluated in O2-saturated 0.5 M H2SO4 electrolyte and compared with the advanced C-IrO2 and C-RuO2 electrocatalysts. The saturated calomel electrode (SCE) was used as a reference electrode and was calibrated prior to electrocatalytic testing  with iR-correction  of 1.30 V vs. reversible hydrogen electrode (RHE) at 0.3 mA cm−2. And to deliver a current density of 10 mA cm−2, Ru0.5Ir0.5O2 only requires a minimum overpotential (η10) of 151 mV, while the overpotentials (η10) of C-IrO2 and C-RuO2 were 321 and 297 mV, respectively. Besides, the lowest Tafel slope for Ru0.5Ir0.5O2 (45 mV dec−1) indicates that the fastest kinetic velocity compared to C-IrO2 (126 mV dec−1) and C-RuO2 (108 mV dec−1)  Fig. . In addiη10) at 10 mA cm−2 and the current density at 1.440 V vs. RHE for different electrocatalysts. It is noteworthy that the current density of Ru0.5Ir0.5O2 reaches 169.7 mA cm−2 at 1.440 V vs. RHE, which is 106.1 times and 141.4 times of C-IrO2 (1.6 mA cm−2) and C-RuO2 (1.2 mA cm−2), respectively. The gas chromatographic diagram analysis was further used to confirm the formation of O2 gas and evaluate the Faraday efficiency of O2. The data of the produced oxygen by Ru0.5Ir0.5O2 at 20 mA cm−2, 40 mA cm−2, 100 mA cm−2 in 0.5 M H2SO4 electrolyte were collected  and C-RuO2 (5.6 A gIr + Ru−1 at 1.440 V vs. RHE). The turnover frequency (TOF) of noble metal site was also calculated. Figure 0.5Ir0.5O2 (6.84 s−1 at 1.440 V vs. RHE) was nearly 136.8 and 171 times higher than that of C-IrO2 (0.05 s−1 at 1.440 V vs. RHE) and C-RuO2 (0.04 s−1 at 1.440 V vs. RHE).In order to compare the intrinsic activity of the prepared electrocatalysts, the mass activity and the turnover frequency (TOF) of the prepared electrocatalysts were calculated based on the total loaded mass of the noble metal (Ir + Ru). Figure 0.5Ir0.5O2, we explored the electrochemical double-layer capacitance (Cdl) test and the electrochemically active surface area (ECSA) was calculated by Cdl for activity normalization  sheet-like shape structure exhibited higher Cdl and ECSA than those of C-IrO2 and C-RuO2 , and C-RuO2 was inactivated after 23.5 h test (15.83 mV h−1). The amount of dissolved Ru and Ir ions in the electrolyte at different times during the stability test of Ru0.5Ir0.5O2 was determined by ICP-AES analysis  and Ru 3p1/2 (484.2 eV) peaks of Ru0.5Ir0.5O2 after the stability test were positive-shifted compared with before the stability test  and Ir 4f5/2 (64.6 eV) peaks showed that there was is a slight negative-shift compared with before stability tests . Supplementary Fig. 0.5Ir0.5O2 for 1000 cycles with a scan rate of 100 mV s−1. Supplementary Fig. 0.5Ir0.5O2 before and after 1000 cycles. After the ADT test, the overpotential (η @10 mA cm−2) of Ru0.5Ir0.5O2 was increased by 11 mV  was used as an anode catalyst in acidic PEM electrolyte (0.5 M H2SO4) at room temperature , CV results indicate that Ru species in Ru0.5Ir0.5O2 material are more easily oxidized than Ir species under the same conditions. It suggests that Ru0.5Ir0.5O2 is more likely to generate more high valence Ru active sites than C-IrO2 and C-RuO2 at low applied voltage, which may mainly be responsible for the good acidic oxygen evolution reaction (OER) activity over Ru0.5Ir0.5O239.The electrochemical cyclic voltammetry (CV) tests of Ru41. Therefore, in order to determine the instantaneous oxidation state of the Ru active site during the OER process, we used the pulse voltage induced current (PVC) test method to compare potential, charge and quantify the capacitance of the catalysts under different biases. The bias applied in the PVC test preferentially acted on the oxidation process of the metal, and the resulting oxidative charge on the metal surface is then involved in the OER reaction process, and the change in the oxidation state of the metal causes the capacitance of the catalyst to change43. Thus, the instantaneous oxidation state of the active site during OER can be determined, and the sensitivity can be adjusted according to the size of the applied bias interval. According to XANES data and CV data, Ru is easier to be oxidized in the Ru0.5Ir0.5O2 solid solution, so the changes in solid solution capacitance can be regarded as the change of Ru pseudo-capacitance, and the existence of pseudo-capacitance in Ir/Ru based materials is well known44. Figure iR correction potential. As shown in Fig. 0.5Ir0.5O2. By integrating the current (OER current is deducted as background current) response of the anodic voltage pulses, the charge  stored in the catalyst at a given potential can be quantified. As highlighted in Fig. 0.5Ir0.5O2 begins to drop from about 2.45 mF at 1.325 V vs. RHE. The capacitance of C-IrO2 and C-RuO2 begin to change at 1.485 V, 1.505 V vs. RHE, respectively. And the charge-log (current densities) profile , thus improving the OER activity of the catalyst.In addition, we carried out in situ Ru K-edge XAS to further observe the trend of Ru oxidation state change with applied voltage increasing. As depicted in Supplementary Fig. 46. The schematic diagram of the test device is shown in Supplementary Fig. 0.5Ir0.5O2, C-IrO2 and C-RuO2. The decay rate is usually described by fitting the time decay constant (τ) value 48. The τ values of Ru0.5Ir0.5O2, C-IrO2 and C-RuO2 were calculated to be 0.32254 ms, 0.18312 ms and 0.11633 ms, respectively. Because the Ru pseudo-capacitance in Ru0.5Ir0.5O2 is easy to change and the oxidation state of Ru is easy to increase. And before and after laser the irradiation of Ru0.5Ir0.5O2 can be regarded as the charge-discharge process of pseudo-capacitance in the material. Simultaneously, there are strong interactions in the local structure of Ru-O-Ir. These results cause that the electron transfer rate of Ru0.5Ir0.5O2 solid solution decreases significantly.The presence of the local structure of Ru-O-Ir and large amount of capacitance in Ir/Ru based materials may impede electron transmission. Based on this, the transient photo-induced voltage (TPV) test was measured as an effective means to explore the electron transfer behavior of different catalysts46. Supplementary Fig. 0.5Ir0.5O2, C-IrO2 and C-RuO2. None of these curves has obvious peak values, which means that there are no obvious static frequency and periodic frequency components in the TPV attenuation curve48. FFT can only identify the frequency component of TPV signal, but not the relationship between frequency and time. In order to study the non-static characteristics of TPV curves, Continuous Wavelet Transform (CWT) was applied to TPV data. 2D and 3D CWT spectra of Ru0.5Ir0.5O2, C-IrO2 and C-RuO2 are shown in Supplementary Fig. The fast Fourier transform (FFT) technique based on TPV data was used to further analyze the electron transfer behavior of catalysts45. In order to further analyze the dynamics of the electron transport process, the intensities of peak positions at different frequencies (10–80 Hz) were compared with the relationship between time47. It can be seen from Fig. 0.5Ir0.5O22 moves backwards at 10 Hz on the time scale, indicating that the charge transfer rate is slowing down. Similarly, comparisons at other higher different frequencies confirm this same conclusion , C-RuO2 , and Ru0.5Ir0.5O2 was calculated at different frequencies, respectively, using the time of the Ru0.5Ir0.5O2 (t1) as the peak reference value. As shown in Fig. 0.5Ir0.5O2 has the slowest transfer rate during the charge transfer process46.Low frequency represents slow electron transport, and high frequency means fast electron transport0.5Ir0.5O2 exhibits record OER activity in sulfuric acid electrolyte may mainly be due to more Ru active sites with high oxidation states generated at low applied voltage. And the local structure of Ru-O-Ir in Ru0.5Ir0.5O2 has strong interaction and high stability, which prevents excessive oxidation and dissolution of the active site.In summary, Ru0.5Ir0.5O2 catalyst. Consistent with the XRD pattern  simulations to rationalize the observed OER activity and understand the effect of Ru on the OER performance in Ruern Fig. , Ru0.5Ir5 Å Fig. . Both C-res Fig. . The latels Fig. , the cal0.5Ir0.5O2. As shown in Supplementary Fig. p binding energy exhibits a decrease from C-RuO2 to Ru0.5Ir0.5O2, indicating a lower Ru oxidation state in Ru0.5Ir0.5O2, consistent with experiment. Meanwhile, as shown in Supplementary Fig. f binding energy exhibits an increase from C-RuO2 to Ru0.5Ir0.5O2, indicating a higher Ir oxidation state in Ru0.5Ir0.5O2, consistent with experiment. These trends are also supported by the Bader charge analysis, as shown in Supplementary Table 0.5Ir0.5O2 increased compared to that of C-RuO2 and the length of Ir-O bonds also increases compared to that of C-IrO2, which is consistent with experimental observations. For solid solutions, there are two factors that affect the bond length of metals-oxygen. One is the crystal structure, and the other is the valence state. In the 1 T phase structure, the metal-oxygen bond length is longer than that of the rutile phase;29 For valence states, the higher valence state corresponds to the shorter bond length, while the lower valence state corresponds to the longer bond length. In this work, the crystal structure has larger impact on to the bond length of Ir-O, so the bond length of the Ir-O in Ru0.5Ir0.5O2 is longer than that of the corresponding rutile phase.We carried out additional XPS predictions and Bader charge analysis to verify the oxidation states of Ru and Ir species in Ru0.5Ir0.5O2 (01-10) edge-site50, C-IrO2 (110) surface and C-RuO2 (110) surface52 in C-IrO2, and C-RuO2, and the corresponding limiting overpotential is 1.00 V and 0.81 V, respectively. For Ru0.5Ir0.5O2, generation of O2 is the PDS with an overpotential of 0.36 V. Thus, our DFT simulations showed that the Ru0.5Ir0.5O2 has the lowest OER overpotential (ƞOER = 0.36 V) compared to C-IrO2 (ƞOER = 1.00 V) and C-RuO2 (ƞOER = 0.81 V), coincident with our experimental observation that OER stability and catalytic activity of Ru0.5Ir0.5O2 catalyst were significantly improved.We chose to study the catalytic performance of Ruce52Fig. . We calc0.5Ir0.5O2 as active sites. These simulations yielded a considerably lower overpotential of 360 mV, which aligns much more closely with the experimentally observed overpotential of 151 mV. We believe that this now adequately explained the difference between the predicted overpotential of DFT calculation and the experiment results. Remaining differences can be attributed to various sources of uncertainty inherent in both theoretical and experimental methods. Moreover, most of the existing DFT calculations exhibit a systematic overestimation of the OER overpotentials for IrO2 and RuO2 .In general, Ru0.5Ir0.5O2 substitutional solid solution prepared by a 2-step mechano-thermal method is highly OER active and acid-stable material. Ru0.5Ir0.5O2 exhibits a low overpotential of only 151 mV at 10 mA cm−2 and attributes its enhanced catalytic activity to the more Ru active sites with high oxidation states generated at low applied voltage, and the surface oxidative charge concentrations are increased. Ru0.5Ir0.5O2 also shows good stability after 618.3 h operation at 10 mA cm−2. In addition to the 2D structural characteristics of Ru0.5Ir0.5O2, a strong interaction in the local structure of Ru-O-Ir, namely, dominated its high stability, preventing the excessive oxidative dissolution of the active site.In conclusion, we demonstrate that the well-defined 2D Ru3, 99.9%) and Iridium chloride  were purchased from Alfa Aesar Co. Commercial ruthenium (IV) oxide  and commercial iridium (IV) oxide  were purchased from Aladdin Chemical Regent Co. Potassium hydroxide  was purchased from Sinopharm Chemical Reagent Co. Nafion solution (5 wt%) was obtained from Sigma-Alddrich Co. Isopropanol was purchased from Sinopharm Chemical Reagent Co. The continuous ultrathin carbon layer on a holey carbon/formvar support film were purchased from Zhongjingkeyi (Beijing) Film Technology Co., LTD. All other experimental reagents were analytical reagent grade, and all the aqueous solutions used in the experiment were prepared by double distilled water.Ruthenium chloride  IrCl3 and 2 g KOH) were mixed and heated by a mechano-thermal method29. Then the furnace was cooled down to room temperature. At the second step, the gotten solid production and other raw materials (104 mg (0.5 M) RuCl3 and 2 g KOH) were mixed and thoroughly ground and heated again by a mechano-thermal method29. Both heating processes are heated to 800 °C for 2 h. The as-prepared solid production was washed by double distilled water and dried by lyophilization to obtain Ru0.5Ir0.5O2.The RuTM K-AlphaTM+ spectrometer equipped with a monochromatic Al Kα X-ray source (1486.6 eV) operating at 100 W. Samples were analyzed under vacuum (P < 10−8 mbar) with a pass energy of 150 eV (survey scans) or 25 eV (high-resolution scans). The experimental peaks were fitted with Avantage software. The topographic height of surface was measured by Atomic force microscopic . The BET specific surface areas were characterized by American Micromeritics ASAP−2020 porosimeter. Inductively coupled plasma atomic emission spectra  was used to measure the dissolved Ru in electrolyte (0.5 M H2SO4) after the OER stability testing.X-ray powder diffraction  with Cu Kα radiation source (λ = 0.15406 nm) was used to characterize the phase and crystallization of the samples. Transmission electron microscopy (TEM) image, high resolution transmission electron microscopy (HRTEM) image and high-angle annular dark-field scanning TEM (HAADF-STEM) images were recorded via a Talos F200X transmission electron microscope (USA) under an accelerating voltage of 200 kV. Scanning transmission electron microscopy (STEM) results were collected on a fifth order aberration-corrected transmission electron microscope (JEOL ARM200CF) at 80 kV. Scanning electron microscopy (SEM) was conducted by using a Zeiss G500. X-ray photoelectron spectroscopy (XPS) was conducted on a Thermo Scientific−2). A saturated calomel electrode (SCE) and a platinum wire were used as the reference electrode and counter electrode. The electrochemical pulse voltammetry tests were measured in O2-saturated 0.5 M H2SO4. Keep the potential at low potential (Elow = 1.195 V vs. RHE) for 5 s, then switch to high potential (Ehigh) for 5 s and back to low potential for 5 s. The anodic current is a convolution of capacitive charge, catalyst oxidation and OER currents. The Ehigh was increased from 1.245 to 1.645 V vs. RHE (Ru0.5Ir0.5O2) or 1.345 to 1.745 V vs. RHE (C-IrO2 and C-RuO2) at a speed of 20 mV/step, and the Elow remained unchanged. The charge integration scheme applied in the main text is to integrate the current after deducting the background current (OER current is deducted as background current) in the PVC test40.PVC was performed by using a RRDE-3A Rotating Ring Disk Electrode Apparatus  when the current was tracked over a period of time (1600 rpm RDE). An GC RDE (3 mm in diameter) loaded with catalysts was employed as the working electrode .The electrolyte was prepared from 0.4 g of tetrabutylammonium perchlorate dispersed in 10 mL anhydrous acetonitrile and thoroughly deoxygenated with pure NThe TPV test is performed with a platinum network covered by the operating electrode of the power sample (1 × 1 cm) and a platinum wire as the counter electrode. The TPV was excited with a nanosecond laser radiation pulse (wavelength of 355 nm and the repetition rate was 5 Hz) from a third harmonic Nd:YAG laser . The TPV signals were amplified by an amplifier and were recorded by an oscilloscope. All measurements were performed at room temperature and under ambient pressure.0.5Ir0.5O2 and commercial catalyst (C-IrO2 and C-RuO2) were dispersed 5 mg in 0.9 mL isopropanol solution and 0.1 mL Nafion solution (0.5 wt%), and the ink was formed by ultrasonically ultrasonic action for 1 h to form a homogeneous ink. Then 4 μL of suspension was loaded on glass carbon electrode  to prepared the working electrode (mass loading ∼283 μg cm−2). Finally, the prepared working electrode was dried in room temperature. And the working electrode for stability test was prepared by 28.3 μL of suspension was loaded onto the carbon paper 0.5 × 1.0 cm (283 μg cm−2). A platinum wire was used as the counter electrode and SCE as the reference electrode. The SCE electrode were also calibrated in highly pure H2-saturated 0.5 M H2SO4 electrolytes bubbled with pure hydrogen gas with a scan rate of 5 mV s−1 . All linear sweep voltammetry (LSV) polarization curves were obtained with 95% iR-compensation at a scan rate of 5 mV s−1. The solution resistances were measured by electrochemical impedance spectra (EIS). The EIS test was performed at a frequency range of 100 kHz to 10 mHz with an amplitude of 5 mV. And measured on GCE (3 mm in diameter) with a mass loading of 283 μg cm−2 at 1.38 V vs. RHE.All electrochemical measurements of OER were carried out in a conventional three-electrode system performed at room temperature with a CHI 760E electrochemical workstation. The synthetic Ruisopropanol: Vwater = 1: 3,) and 100 μL of 5 wt% Nafion solution. The suspension was then ultrasonicated for 1 h until a well-dispersed catalyst ink was formed. The ink of Ru0.5Ir0.5O2 and Pt/C catalysts were sprayed onto Ti wafers (1 × 1 cm2), respectively. And pretreated Ti wafers (1 × 1 cm2) were used as cathode and anode gas diffusion layers (GDLs), respectively. The membrane electrode assembly (MEA) was constructed by placing Nafion 117 membrane in the middle of the Pt/C cathode catalyst-supported Ti wafer and Ru0.5Ir0.5O2 anode catalyst-supported Ti foam GDLs, followed by hot pressing at 130 °C for 5 min under a pressure of 2 MPa. The constructed MEAs were finally applied in the PEM electrolyzer. The PEM electrolyzer was performed by CHI 660E with a high-current amplifier (CHI 680 C) using 0.5 M H2SO4 as the electrolyte with the flowing rate of 10 mL min−1 at room temperature. The performance of the PEM electrolyzer was evaluated by measuring polarization curves from 10 to 2000 mA cm−2 at room temperature and ambient pressure. The stability of the PEM electrolyzer using Ru0.5Ir0.5O2 as the anode catalyst was evaluated by measuring the chronoamperometry test at the cell voltages of 1.43 V.For PEM electrolyzer test, a self-made cell was used as the PEM device and a cation exchange membrane (Nafion 117) as the membrane electrolyte. Ru0.5Ir0.5O2 catalyst (1.0 mg) was used as the anode catalyst and Pt/C  was used as the cathode catalyst. To prepare the cathode or anode catalyst ink, the catalyst was suspended into a 0.9 mL mixture of isopropanol and water .The conversion between the measured potential and the reversible hydrogen electrode (RHE) was calculated by Eq. :1\documeiR-compensation of the polarization curves of Ru0.5Ir0.5O2, C-IrO2 and C-RuO2.i is the tested current, and R is the solution resistance.Equation  determin0.5Ir0.5O2, C-IrO2 and C-RuO229.Equations –6 determ0.5Ir0.5O2 is calculated as follows Eq.  at non-Faradaic potential range was obtained by measuring the capacitance of double layer at solid-liquid interface employing cyclic voltammetry (CV) with different scan rates  in a range from 0.92 and 1.18 V vs. RHE. The Cs values were 0.035 mF cm−2.The electrochemical double-layer capacitance  at 1.05 V vs. RHE and ν is the scan rate.The C the Eq. :8\docume55 version 5.4.4, was used to simulate the density function theory (DFT). To account for the exchange-correlation interaction, a generalized gradient approximation (GGA) in the form of Perdew–Burke–Ernzerhof (PBE) functional was applied56. The Projector augmented-wave (PAW) method and the plane wave basis set were used to complete the calculations. In the settings of the computational simulation parameters, the cut-off energy of the plane wave of the PAW is set to 500 eV, and a vacuum layer of 15 Å thickness is introduced for all surfaces in the vertical direction. The Brillouin grid points are used as 3 × 3 × 1 Monkhorst-Pack K points. The convergence condition for the force is 0.03 eV/Å, and the convergence accuracy for the energy is 10-5 eV. Note that we constructed a single-layer Ru0.5Ir0.5O2 structure both in stability calculations and reactivity predictions. When these two convergence criteria are met simultaneously, the structure is considered optimal at this point. Based on the VASP calculation, the Gibbs free energy was calculated, and the zero-point energy and contributions of enthalpy and entropy were computed using the VASPKIT program57. The solvation effect and applied potential were considered using the implicit solvation model implemented in VASPsol59, and jDFTx60 was employed to validate the constant potential calculation 0.5Ir0.5O2, C-IrO2 and C-RuO2 catalyst implemented in VASP at the PBE-D3 level. In VASP, there are two approaches for the calculation of the core-level shift (CLS), the initial and final state approximation, respectively. The initial state approximation is based on the Kohn–Sham (K.S.) eigenvalues of the core states after a self-consistent calculation of the valence charge density, while the final state approximation requires removing the electron from the core and placing it into the valence61. We selected the initial state method to predict CLS because previous studies have shown that such an approach is reliable in reproducing the relative binding energy change as measured experimentally62. The work function’s effect was not considered during the calculations. For directly compared to the experimental observation, all the calculated core-level binding energies were transformed to absolute values.The XPS simulations were conducted to predict the core-level energies of 1 s orbital for RuSupplementary InformationPeer Review FileSource Data


Microcystis) and the north (Pyrodinium) of the IRL. Each toxin induced a consistent pattern of cytotoxicity in the panel of human cell lines assayed. During blooms, cytotoxicity due to a single type of toxin is obvious from this pattern. In the absence of blooms, the cytotoxicity seen reflected either a mixture of toxins or it was caused by an unidentified toxin. These observations suggest that other toxins with the potential to be harmful to human health may be present in the IRL. Moreover, the presence of toxins in the IRL is not always associated with blooms of known toxin-producing organisms.The Indian River Lagoon (IRL), a 156-mile-long estuary located on the eastern coast of Florida, experiences phytoplankton bloom events due to increased seasonal temperatures coupled with anthropogenic impacts. This study aimed to gather data on the toxicity to human cells and to identify secondary metabolites found in water samples collected in the IRL. Water samples from 20 sites of the IRL were collected during the wet and dry seasons over a three-year period. A panel of cell lines was used to test cytotoxicity. Hemagglutination, hemolysis, and inhibition of protein phosphatase 2A (PP2A) were also measured. Cytotoxic blooms were seen both in the south ( The Indian River Lagoon (IRL) is a 156-mile-long estuary located on the eastern coast of Florida, comprised of three lagoons: the Mosquito Lagoon, the Banana River, and the Indian River. The lagoon is where water from the Atlantic Ocean mixes with freshwater, resulting in brackish water that supports a wealth of biodiversity, from marsh snakes to fish and birds, as well as thousands of plants and animals . RecreatAureoumbra lagunensis that lasted from June to August was observed. Brown tide blooms were also seen in 2013 and 2015–2016. A nanoplankton bloom was reported in late 2019 and lasted until the winter of 2019, and a nonplanktonic cyanobacterium bloom was reported in the summer of 2020 -2,5-diphenyl-2H-tetrazolium bromide (MTT) used for cell viability assays was purchased from Millipore Sigma . Cylindrospermopsin was obtained from Enzo Life Sciences , brevetoxin B was obtained from Cayman Chemical , microcystin LR was obtained from EMD , and okadaic and domoic acid were obtained from Millipore Sigma. Supelco Diaion® HB-8065), the human neuroblastoma SH-SY5Y (ATCC® CRL-2266), and the “normal” African Green Monkey Vero (ATCC® CCL-81) cell lines were obtained from the American Type Culture Collection . The engineered TransportoCells™ OATP1A2 and OATP1B1 were purchased from Corning . Hep G2 cells were cultured in F12K Medium , supplemented with 10% FBS, 1X antibiotic/antimycotic (Gibco 15240-096), and 50 µg/mL gentamicin (Gibco 1570-060). OATP Cells were cultured in DMEM medium (ATCC 30-2002) supplemented with 10% FBS (not heat inactivated), 18 mM HEPES, 1X antibiotic/antimycotic (Gibco 15240-096), and 50 µg/mL gentamicin (Gibco 1570-060). SH-SY5Y were cultured in RPMI 1640 Medium supplemented with 10% FBS, 2 mM L-glutamine, 18 mM HEPES, 1X antibiotic/antimycotic (Gibco 15240-096), and 50 µg/mL gentamicin (Gibco 1570-060). Vero cells were cultured in RPMI 1640 Medium supplemented with 10% FBS, 55 mg sodium pyruvate, 4.5 g/L D-glucose, 2 mM L-glutamine, 18 mM HEPES, 1X antibiotic/antimycotic (Gibco 15240-096), and 50 µg/mL gentamicin (Gibco 1570-060).Cell Culture. The human hepatocellular carcinoma Hep G2 , and the LC-HRMS analysis was often dominated by contaminants ) which overwhelmed the LC-HRMS analysis. On the mass spectrometer used in this study, PEG gives a very strong signal, which can obscure minor components. Many of the PEG contaminants could be traced to the filter paper or the resin used in our various extraction processes, although the m/z observed for some PEGs did not match these known contaminants and are most likely from the water samples themselves. PEGs are ubiquitous in sunscreens, cosmetics, personal care products, industrial chemicals, and medicines. In some cases, to allow for direct comparison of extracts from the same site, the original extraction method was used, recognizing that some PEG contamination could be introduced. Methods used for each sample can be found in Extraction. Three replicate water samples (1 L) were collected at each of the 20 sites during the wet  and dry (spring) seasons over a three-year period . The pri3OH (3 × 100 mL) followed by a mixture of CH3COOCH2CH3 in CH3OH . The extracts were filtered and concentrated to dryness under reduced pressure to yield a residue. The dried residue was desalted by the addition of 2 mL of H2O and 2 mL of CH3COOCH2CH3, followed by placing the sample into a −20 °C freezer to freeze the water phase. Then, the CH3COOCH2CH3 phase was removed by pipette and dried under a stream of nitrogen to yield the final extract used for testing.Method 1: Extracts were prepared by filtering 1 L of water (3 replicates) collected at the surface through Whatman Number 3 filter paper and then extracting the filter with CH® HP-20 resin was added. The samples were frozen overnight and then allowed to come to room temperature the following day. The water sample was then filtered using a glass Büchner funnel fitted with Whatman No. 3 filter paper, and the resin and filter were washed with methanol (3 × 100 mL) followed by CH3OH:(CH3)2CHOH . The combined washes were concentrated to dryness under reduced pressure. The samples were then desalted as described above.Method 2: Extracts were prepared as follows: 1 L water samples collected at the surface (3 replicates) were sonicated using a sonic dismembrator (Fisher Scientific Model 500) for 2 min to lyse cells, then 2 g of washed Diaion® HP-20 resin was added. The samples were frozen overnight and then allowed to come to room temperature the following day. The water sample was then filtered using a fritted glass funnel (no filter paper), and the resin and filter were washed with CH3OH (3 × 100 mL) followed by CH3OH:(CH3)2CHOH . The combined washes were concentrated to dryness under reduced pressure. The samples were then desalted as described above.Method 3: Extracts were prepared as follows: 1 L water samples collected at the surface (3 replicates) were sonicated using a sonic dismembrator for 2 min to lyse cells, then 2 g of washed Diaionn-butanol (3 × 100 mL). The organic phase was dried under reduced pressure and then desalted as described above.Method 4: Extracts were prepared as follows: 1 L water samples collected at the surface (3 replicates) were sonicated using a sonic dismembrator for 2 min to lyse cells, then frozen overnight. After thawing, the samples were partitioned with 2. After this incubation, 25 μL of a 5 mg/mL solution of MTT were added to each well. Cells were incubated for 3 h at 37 °C. After centrifugation and removal of the supernatant, 100 μL of acidified isopropyl alcohol (1:500 hydrochloric acid to isopropanol solution) was added to each well. The crystals were dissolved by shaking or pipetting. The absorbency of each well was measured at 570 nm with a plate reader . The resulting absorbencies were normalized against the solvent control and plotted using Microsoft Excel (Microsoft Office 365).Cytotoxicity Assay (MTT). Cells were plated on a 384-well tissue culture plate at a volume of 30 μL/well. The number of cells/well used was as follows: 1500 for Vero, 6000 for Hep G2 and SH-SY5Y, and 3000 for OATP cells. Cells were allowed to adhere overnight. At the end of this incubation, 30 μL of medium containing treatment was added, consisting of 10 μg/mL of the samples, media alone, or media with methanol (solvent control). Pure phycotoxins, including 1.6 µM brevetoxin, 10 nM okadaic acid, 100 nM microcystin LR, 1.5 µM cylindrospermopsin, and 500 nM domoic acid, were tested at the same time to serve as controls. The cells were incubated for 72 h at 37 °C and 5% COHemagglutination Assay. This assay was adapted from a previously published protocol by Carmichael, W. W et al. . Sheep rRed Blood Cell Lysis. Adapted from previously published protocols by Malagoli, D. et al. . Samples3VO4, 10 nM okadaic acid, and 100 nM microcystin LR. Samples were plated at a volume of 5 μL/well in duplicate, followed by the addition of 7 μL of the Phosphatase Solution to each well. This was followed by the addition of 9 μL of Chromogenic Substrate to each well. The plate was covered with adhesive film and incubated for 30 min at 37 °C. At the end of this incubation, 7 μL of Stop Solution was added to each well, and the absorbance of samples was read at 405 nm wavelength using the plate reader . Average absorbance from test samples was normalized against the average absorbance for the complete reaction, which represents 100% activity using Microsoft Excel (Microsoft Office 365).Protein Phosphatase 2A (PP2A) Assay. This assay used the Eurofins/Abraxis Kit for Microcystins/Nodularins PP2A  and was performed according to manufacturer’s instructions, except for adapting it to be used on a 384-well plate. All reagents were allowed to reach room temperature (23 ± 3 °C) before starting the assay. To prepare phosphatase solution, 3 mL of Phosphatase Dilution Buffer was added to one of the Phosphatase vials and mixed carefully by inversion. The solution was gently shaken at room temperature for 1 h to ensure that the enzyme was fully hydrated. Reaction controls include complete reaction, reaction with no substrate, and reaction with no enzyme. The kit includes four microcystin LR standards at concentrations of 0.25, 0.5, 1, and 2.5 nM. Water samples and solvent controls were tested at 10 µg/mL. Assay controls included kit standards, 10 mM Na3OH with a 5 μL injection. An internal standard of 25 ng reserpine was added to each sample. Chromatographic separation was accomplished using the following conditions: Solvent A: H2O containing 0.1% formic acid and Solvent B: CH3CN containing 0.1% formic acid; t = 0 min 80:20 A:B v/v; t = 14 min 100% B; t = 29 min 100% B t = 30 min 80:20 A:B v/v, followed by 6 min re-equilibration, flow rate = 0.3 mL/min. Mass analysis in positive ion mode was completed over a m/z range of 150–2000 amu. The settings were set as follows: needle voltage 2000 V, desolvating chamber temperature 250 °C, detector 2300 V, orifice 1 85 V and 80 °C, ring lens, 15 V, orifice 2 5 V, ion guide RF 1500 V, ion guide bias 27 V, focus 10 V, quad 20 V, left/right 0 V, top/bottom 0.6 V, focus lens −120 V, push bias 0.36 V, reflectron 990 V, push 778 V, pull −778 V, and flight tube −7000 V.Secondary Metabolite/Toxin Profiling. LC-HRMS analysis was conducted in positive ion detection mode using a JEOL Accu-TOF  connected to an Infinity II HPLC system . LC-HRMS analyses were completed using a reversed-phase HPLC column . Each sample was analyzed at a concentration of 1.0 mg/mL in CHPyrodinium bloom .Fractionation of the extract from Pyridinium bloom at site 22 in the northern IRL was extracted per the normal procedure, yielding 8 mg of extract. This material was separated by preparative HPLC on a Waters Sunfire C18 OBD 10 µm column (150 mm × 19 mm) using the following conditions: Solvent A: H2O +0.1% formic acid and Solvent B: CH3CN + 0.1% formic acid; t = 0 min A:B 90:10 v/v, t = 20 min 100%B; t = 28 min 100% B; t = 30 min A:B 50:50; flow rate =12 mL/min; detection photodiode array (PDA) and ESI MS (positive and negative mode); and carrier solvent CH3CN with 0.1% formic acid. Samples were collected by time, with a total of 8 fractions collected. No fractions showed activity against the Vero, Hep G2, OATP1A2, or OATP1B1 cell lines.First preparative HPLC separation on C-18 with formic acid. A 1 L sample collected during a 2O:CH3CN 95:5 and Solvent B: CH3CN; t = 0 min A:B 90:10 v/v, t = 20 min 100% B; t = 28 min 100% B; t = 30 min A:B 50:50 v/v; flow rate =12 mL/min; detection by PDA and ESI MS (positive and negative mode); and carrier solvent CH3CN with 0.1% formic acid. Samples were collected by time, with a total of 6 fractions collected. The fraction eluting at the solvent front showed 51% inhibition of the Vero Cell line, 94% inhibition of the Hep G2 cell line, 84% inhibition of the OATP1 A2 cell line, and 57% inhibition of the OATP1 B1 cell line.Second preparative HPLC on C-18 without formic acid. The extract used in the above experiment was separated by preparative HPLC on a Waters Sunfire C18 OBD 10 µm column (150 mm × 19 mm) using the following conditions: Solvent A: H® HP-20 resin was added. The sample was frozen overnight, thawed the following day, and then filtered through a fritted glassfunnel. The retained resin was washed with 2 × 400 mL portions of CH3OH followed by 1 × 400 mL portion of CH3OH:(CH3)2CHOH 3:1 v/v. The combined extracts were concentrated by distillation under reduced pressure to yield 189 mg of extract. This extract was desalted by partitioning between H2O (1 mL) and CH3COOCH2CH3:CH3OH (9:1 v/v) (2 mL), 4 ×. This material was dried down under a stream of N2 and after reconstitution was separated by preparative HPLC on a Phenomenex Luna 5µ NH2 100 Å Axia packed column (100 × 21.2 mm) using the following conditions: Solvent A: H2O:CH3CN 95:5 v/v, Solvent B: CH3CN; t = 0 min 100% B, t = 20 min 100% A; t = 22 min 100% A; t = 23 min A:B 50:50 v/v; flow rate =12 mL/min; detection by PDA and ESI MS (positive and negative mode); and carrier solvent CH3CN with 0.1% formic acid. Samples were collected by time, with a total of 7 fractions collected. Fraction 5 (WIN22-77-5), which eluted between 8 and 11 min, showed 78% inhibition against the Vero cell line at 10 µg/mL but had no activity against the other cell lines.Third preparative HPLC separation. A 4 L sample (9-VIII-2019-21-004) collected from the NIRL was sonicated using a sonic dismembrator for 2 min to lyse cells, and then approximately 4 g of DiaionMicrocystis bloom. A 1 L water sample was collected at the surface during a bloom of Microcystis on the lakeside immediately north of the Port Mayaca Lock, Lake Okeechobee. The sample had a dense number of cyanobacterial cells floating on the surface. The sample was filtered using a porcelain Büchner funnel (~4” diameter) fitted with two pieces of Whatman #2 filter paper. The filter paper was wetted with LC-MS grade methanol, and the methanol was discarded.  The filter, which contained the cells, was placed into a beaker containing approximately 50 mL of LC-MS grade CH3OH and steeped at −20 °C. The methanol extract of the cells was decanted and filtered using a Büchner funnel and Whatman #3 paper. The filter paper and cells from the original filtration were macerated with a spatula and extracted exhaustively with CH3OH followed by CH3OH:CH3COOCH2CH3 1:1. The combined extracts were dried by distillation under reduced pressure and then transferred to a 20 mL scintillation vial and dried under a stream of N2. The total yield of extract was 0.0876 g. A total of 2 mg was subsampled to make a 1 mg/mL solution for LC-HRMS analysis, and the remaining extract was fractionated by medium-pressure liquid chromatography (MPLC) on an Isco Combi-Flash Rf4x  using a 5.5 g Redi-Sep C18 column and a linear gradient of CH3CN and H2O followed by washes with CH3OH, CH2Cl2, and a CH3CN:H2O:CF3COOH ; 0 CV A:B (94:6 v/v) hold for 5.2 CV; then linear gradient to 100% B over 40.8 CV and hold for 7.1 CV at 100% B; switch to 100% C hold for 3.9 CV; linear gradient to 100% D over 7.1 CV, hold for 2 CV at 100% D, wash with 100% C for 5.3 CV, and then wash with CH3CN:H2O:CF3COOH (50:50:0.1 v/v/v) for 5 CV. Fractions were collected as follows: 41972: 0–9 CV; 41973 9–20 CV; 41974 20–30 CV; 41975 30–42 CV; 41976 42–60 CV; 41977 60–66 CV; 41978 66–68 CV; and 41979 68–75 CV; 41980 TFA wash. Biological activity and 1H NMR data consistent with that expected for microcystins were observed in 41972 and 41974.Extraction and chromatography of Port Mayaca Lock t-test was calculated per site by comparing the data of later samplings against the fall 2018 (year 1) data using Microsoft Excel (Microsoft Office 365). A p value less than or equal to 0.05 was considered significant. Student’s t-test was also used to compare the data per season for each year again using the year 1 data as basal levels, which is shown in Statistical Analysis: Student’s