Biologically important, chiral natural products of butenolides, (−)-blastmycinolactol, (+)-blastmycinone, (−)-NFX-2, (+)-antimycinone, lipid metabolites, (+)-ancepsenolide, (+)-homoancepsenolide, mosquito larvicidal butenolide and their analogues were synthesized in very good yields in a sequential one-pot manner by using an organocatalytic reductive coupling and palladium-mediated reductive deoxygenation or organocatalytic reductive coupling and silica-mediated reductive deamination as the key steps. Developing a simple, sustainable, one-pot protocol by utilizing readily available starting materials for synthesizing a library of natural products, drugs and their analogues is a challenging task for the synthetic chemistry community ,2,3. TheOrgRC) reaction for the selective C-alkylation of cyclic/acyclic CH-acids with different carbonyls in the presence of Hantzsch ester and this reaction has been utilized by many other synthetic chemistry groups in the total synthesis of natural products and drugs [H] furanone) core as our synthetic target to synthesize through an organocatalytic sequential one-pot manner.Recently, our laboratory discovered the three-component organocatalytic reductive coupling -homoancepsenolide R have been known for their cytotoxicity and antimicrobial activity D25 = +1.2° } in very good yield/conversion. After the completion of the OrgRC reaction, two equiv. of N,N-diisopropylethylamine (DIPEA) was added to the reaction mixture, followed by 1.5 equiv. of Tf2O addition at −78 °C, and this was stirred for 1 h. After quenching the reaction with saturated NH4Cl solution and work-up, the crude product was used directly for the reductive deoxygenation reaction with Pd(OAc)2, 1,3-bis(diphenylphosphino)propane (DPPP) and polymethylhydrosiloxane (PMHS) in DMF at 60 °C for 8 h to furnish the desired monobutenolide (+)-2a and C {[α]D25 = +36.1° , lit.: [D22 = +79.2° } in 43% overall yield -2b and D {[α]D25 = +24.9° } and (+)-2c and E {[α]D25 = +24.8° , lit.: D22 =ll yield . The sam), lit.:  [α]D25 =ectively . To our 2° c = 0., CHCl3}  methods ,56,57,582a/C, (+)-2b/D and (+)-2c/E have their own biological activity and were also used as key intermediates in the expeditious asymmetric synthesis of antifungal and antitumor polyketide metabolites K-P by the Node and Comasseto groups D25 = +18.3° } in 99% conversion. Interestingly, the compound (+)-3d was highly polar and slightly insoluble in the reaction solvent DCM. After completion of the in situ OTf protection reaction, the solvent was evaporated and the crude solid material was dried and used for the next step as such. Following Pd-mediated reductive deoxygenation reaction sequence as above on the crude material, (+)-ancepsenolide (+)-2d and Q {[α]D25 = +30.5° , lit.: D25 =ee steps . We are 5° c = 0., CHCl3} ee steps .2e and R following the OrgRC/O-triflate and reductive deoxygenation reactions in a sequential one-pot manner, but unfortunately the desired bis-butenolide (+)-2e was isolated in poor yield. Hence, we followed Pashkovsky’s [3e {[α]D25 = +1.2° } into the corresponding bis-enamine derivative followed by the reductive deamination through NaBH3CN-mediated reduction and SiO2-mediated amine-elimination to furnish the required (+)-homoancepsenolide (+)-2e/R {[α]D25 = +11.4° } in 42.5% with satisfactory overall yield -homoancepsenolide (+)-kovsky’s  approachll yield .S, an important natural product showing significant mosquito larvicidal properties (LC50 = 0.41 ppm), along with its partially reduced counterpart T (LC50 = 0.47 ppm) D25 = +8.1° } in 90% yield. In our efforts to obtain the required deoxygenated product (+)-2f and S, we followed Pashkovsky’s approach. The desired OrgRC product (+)-3f was converted into the corresponding enaminone derivative 7f followed by reducing the conjugated double bond and the elimination of the amine moiety furnished the required butenolide (+)-2f and S {[α]D25 = +30.6° , lit.: D25 =ll yield . This thll yield . Anothero et al. .3 and the natural products (+)-2 by IR/NMR/mass analysis and the absolute configuration was confirmed by correlation with the published literature data including optical rotation (see We established the structure of the intermediates (+)-tion see .1H NMR and 13C NMR spectra were recorded at 400 or 500 MHz and 100 or 125 MHz, respectively. The chemical shifts are reported in ppm downfield to TMS (δ = 0) for 1H NMR and relative to the central CDCl3 resonance (δ = 77.0) for 13C NMR. In the 13C NMR spectra, the nature of the carbons  was determined by recording the DEPT-135 experiment and is given in parentheses. The coupling constants J are given in Hz. Column chromatography was performed using Acme’s silica gel (particle size 0.063–0.200 mm). High-resolution mass spectra were recorded on micromass ESI-TOF MS. IR spectra were recorded on JASCO FT/IR-5300  and Thermo Nicolet FT/IR-5700 . For thin-layer chromatography (TLC), silica gel plates Merck 60 F254  were used and compounds were visualized by irradiation with UV light and/or by treatment with a solution of p-anisaldehyde (23 mL), conc. H2SO4 (35 mL), acetic acid (10 mL) and ethanol (900 mL) followed by heating.The 1H NMR, 13C NMR and HRMS) can be found in Experimental procedures and characterization data (s)-(+)-γ-methyltetronic acid 4 was prepared according to the literature procedure from (s)-(−)-ethyl lactate [All solvents and commercially available chemicals were used as received without further purification unless otherwise stated. Optically pure ( lactate .s)-(+)-γ-methyltetronic acid 4 (1.0 equiv) and Hantzsch ester 6 (1.0 equiv.) was charged with DCM (0.2 M), followed by addition of aldehyde 5a–f (1.0 equiv.), and the resulting mixture was stirred at room temperature until the completion of the reaction as monitored by TLC. After completion of the OrgRC reaction, the organic layer was washed with brine, dried over Na2SO4 and concentrated. The crude product 3a–f was used for the next step without purification.A vial equipped with a magnetic stirbar containing proline (0.05 equiv.), , (s)-(+)-γ-methyltetronic acid 4 (1 equiv) and Hantzsch ester 6 (1 equiv) was charged with DCM (0.2 M), followed by addition of aldehyde 5 (1 equiv), and the resulting mixture was stirred at room temperature until the completion of the reaction as monitored by TLC. After completion of the OrgRC reaction, DIPEA (2 equiv) was added to it. The reaction mixture was cooled to −78 °C and Tf2O  was added to it drop wise. After 30 min. (or completion of the reaction), the reaction mixture was quenched with saturated NH4Cl solution and partitioned between DCM (15 mL × 3) and water. The organic layer was washed with brine, dried over Na2SO4 and concentrated. The crude enol-triflate product was used for the next step without purification.Step 2: General procedure for the palladium-catalyzed reduction of enol-triflate: The enol-triflate was dissolved in dry DMF (0.2 M). Pd(OAc)2 (0.01 mol%), 1,3-bis(diphenylphosphino)propane (DPPP) (0.01 mol %) and poly(methylhydrosiloxane) (PMHS) (2 equiv.) were added to the reaction mixture and heated at 60 °C. After completion of the reaction, the reaction mixture was diluted with diethyl ether and extracted with diethyl ether (10 mL × 3). The organic layer was washed with water, brine and dried over Na2SO4. After evaporation under reduced pressure, the crude compound 2 was purified by silica gel column chromatography.Step 1: Compound 3 (1 equiv) was taken in a dry round bottom flask equipped with a Dean–Stark apparatus and reflux condenser in dry toluene (20 mL). Pyrrolidine (1.5 equiv) was added to it followed by catalytic p-TSA. The mixture was refluxed at 130 °C. After completion of the reaction, the mixture was concentrated and purified by column chromatography  to get 7.Step 2: Procedure for the reduction of enaminone derivative: Compound 7 (1 equiv) was taken in MeOH and catalytic methyl orange was added to it. The mixture was acidified with a few drops of 2N HCl in MeOH so as to retain the deep red color of the indicator. NaBH3CN (2.5 equiv) was added in portions with simultaneous addition of acid to maintain the pH. After completion of the reaction, MeOH was removed under reduced pressure. The residue was diluted with water, neutralized with 1N NaOH and extracted with EtOAc (10 mL × 3). The combined organic layer was evaporated and the crude diastereomeric mixture of aminolactones was refluxed in toluene (6 mL) with silica gel for 7 h to get the product 2.In conclusion, we have developed a common methodology for the high-yield total synthesis of important butenolide natural products, from readily available simple substrates through the combination of organocatalytic reductive coupling and palladium-mediated reductive deoxygenation or organocatalytic reductive coupling and silica-mediated reductive deamination reaction sequences as the key steps. This high-yield sequential reductive protocol is an ideal method to synthesize the entire family of butenolide natural products.


Plasmodium falciparum resistance to first-line antimalarials underscores the vital need to develop compounds with novel modes of action and identify new druggable targets. Here, we profile five compounds that potently inhibit P. falciparum asexual blood stages. Resistance selection studies with three carboxamide-containing compounds, confirmed by gene editing and conditional knockdowns, identify point mutations in the parasite transporter ABCI3 as the primary mediator of resistance. Selection studies with imidazopyridine or quinoline-carboxamide compounds also yield changes in ABCI3, this time through gene amplification. Imidazopyridine mode of action is attributed to inhibition of heme detoxification, as evidenced by cellular accumulation and heme fractionation assays. For the copy-number variation-selecting imidazopyridine and quinoline-carboxamide compounds, we find that resistance, manifesting as a biphasic concentration-response curve, can independently be mediated by mutations in the chloroquine resistance transporter PfCRT. These studies reveal the interconnectedness of P. falciparum transporters in overcoming drug pressure in different parasite strains.Widespread   •P. falciparum ABCI3 is a pleiotropic mediator of antiplasmodial drug resistance•abci3 amplification or mutant pfcrtStrain-specific resistance was attributed to •Conditional knockdown assays identified ABCI3 as a putative drug target Plasmodium falciparum resistance to chemically diverse antiplasmodial compounds. In vitro resistance selections, gene editing, drug susceptibility, conditional knockdown, drug cellular accumulation, protein localization, and heme fractionation assays identified ABCI3 as both a resistance mediator and putative drug target.Murithi et al. describe ABCI3, an ATP-binding cassette transporter that conveys  Plasmodium falciparum resistance to first-line artemisinin-based combination therapies has spread across Southeast Asia and is now threatening sub-Saharan Africa , drug cellular accumulation, protein localization, and heme fractionation assays to characterize culture-adapted P. falciparum resistance to five chemically distinct antiplasmodial compounds studied by the MalDA consortium. These data highlight an important role for the ATP-binding cassette (ABC) transporter ABCI3 (PF3D7_0319700) as a pleiotropic drug-resistance determinant in P. falciparum.The Malaria Drug Accelerator  consortium is a target-guided drug-discovery platform that applies  targets . Multipl targets ; however targets . We descP. falciparum resistance mechanisms to five chemically distinct compounds  of each compound, tested in triplicate. Resistance was obtained for all five compounds, and clones were recovered by limiting dilution. Whole-genome sequencing results of these clones segregated the compounds into two distinct categories: (1) those that generated copy-number variations (CNVs) (compounds 1 and 2); and (2) those that generated single-nucleotide polymorphisms (SNPs)  in ABCI3 . For compound 4, the ABCI3 L690I and R2180G mutants had a ∼9- to 16-fold increase in IC50 . Selections using compound 5 were performed on a Dd2-B2 background, yielding ABCI3 F689C and S696Y mutations that caused a ∼11- to 180-fold increase in IC50, i.e., 89 ± 4 nM and 1,433 ± 24 nM compared with the parental Dd2-B2 IC50 of 8.0 ± 0.9 nM  confirmed similar levels of resistance to 4 as observed with the drug-pressured line (ABCI3L690I) . The L690I mutants showed no significant difference in susceptibility to the CNV-selecting compound 1  despite conferring resistance to compound 5  conferred resistance to the three SNP-selecting compounds (1 and 2 that had selected for ABCI3 CNVs (4) showed no shift in susceptibility to 5 even though 5 selected for an adjacent ABCI3 F689C mutation  compared with the parental line when tested with compound 1   .6 indicate that genetic changes in ABCI3 can mediate compound-specific resistance.Together, these data suggest that ABCI3 constitutes a resistance pathway that is distinct from that of existing first-line drugs and provide evidence that the tested ABCI3 SNP- and CNV-selecting compounds might differ in their molecular targets. In addition, data from the SNP susceptibility assays and the cross-resistance results with compound 50 of compounds against wild-type versus ABCI3 cKD parasites. We observed a modest aTc-dependent increase in parasite sensitivity to the two CNV-selecting compounds 1 and 2, with a 2- to 3-fold increase in sensitivity at 0 nM aTc  assays on CRISPR/Cas9-edited parasites in which endogenous ABCI3 had been tagged at its C-terminal end with 3×HA (hemagglutinin) or 3×FLAG epitopes (yielding the lines 3D7-A10cis-Golgi marker ER lumen protein retaining receptor (ERD2)    D and S3Ar (ERD2)   A. These r (ERD2) . IEM anar (ERD2) . Paraller (ERD2) .1 to inhibit Hz formation in P. falciparum. As a surrogate for inhibition of heme detoxification in the parasite, we first tested the ability of this compound to inhibit the conversion of hematin to β-hematin  in a pyridine-based detergent-mediated assay designed to simulate the DV milieu (1 inhibited β-hematin formation (with a mean ± SEM IC50 of 29 ± 2.2 μM) at concentrations comparable with the 4-aminoquinoline-positive controls CQ and amodiaquine , similar to the negative controls pyrimethamine (an antifolate) and doxycycline , and Hz PPIX at different pH. Drug-heme affinities were measured by titrating compounds (0–2 mM) against 10 μM aqueous Fe(III)PPIX in 40% DMSO, either at pH 7.4 (with 0.02 M HEPES) or pH 5.6 (with 0.02 MES [2-[N-morpholino]ethanesulfonate]) and measuring the Soret band absorbance. We then calculated the binding association constant K on the assumption of a 1:1 drug/heme-binding stoichiometry. Titration using CQ as a control yielded a marked decrease in absorbance, with mean ± SEM log K values of 5.32 ± 0.03 and 5.16 ± 0.03 at pH 7.4 and 5.6, respectively (1 appeared to bind monomeric Fe(III)PPIX with moderate affinity, with mean ± SEM log K values of 4.00 ± 0.08 (pH 7.4) and 3.64 ± 0.03 (pH 5.6). Only weak binding associations were observed for compounds 3–5 (Fe(III)PPIX can be maintained as an unaggregated and monomeric state in an aqueous solution of 40% dimethyl sulfoxide (DMSO) and is spectrophotometrically quantified by measuring the absorbance of the Soret band at 402 nm . We leveectively . This agectively . Compoununds 3–5 , consist1 and 2 includes inhibition of Hz formation and heme detoxification, we next assessed whether PfCRT could affect their activity, as mutations in this DV transporter can protect parasites against Hz formation inhibitors such as CQ. These assays used recombinant Dd2 parasites expressing either the mutant Dd2 PfCRT isoform that mediates CQ resistance or the wild-type CQ-sensitive 3D7 isoform . Dose-response assays with the control drug CQ showed the expected 9-fold higher IC50 and IC90 values in Dd2Dd2 parasites compared with isogenic Dd23D7 parasites  IC90 values , L690I and R2180G (compound 4), and F689C and S696Y (compound 5) have been observed in P. falciparum field isolate genome datasets , suggesting that these compounds have modes of action unrelated to antimalarials in clinical use.Our data identify ABCI3 as a pleiotropic modulator of 4 and 5 using CRISPR/Cas9 gene editing. Cross-resistance studies showed that unlike CNVs, these mutations sometimes conferred no resistance or even hypersensitized parasites to other SNP-selecting compounds. The L690I and F689C mutations, while adjacent, produced distinct phenotypes when profiled against the three SNP-selecting compounds. L690I, selected with 4, conferred resistance to both this compound and 3 but not 5. In contrast, F689C, selected with 5, only conferred resistance to this agent but sensitized parasites to both 3 and 4. Strikingly, S696Y, located in the same transmembrane 5 helix as residues 689 and 690, conferred high levels of resistance (≥180-fold) to all three SNP-selecting compounds. These data provide evidence that ABCI3 SNP-mediated drug resistance is compound specific and suggest that this transporter interacts differently with SNP versus CNV-selecting compounds. These results also suggest that like the CNVs, some ABCI3 mutations can confer resistance to a broad set of chemotypes.We confirmed that ABCI3 L690I, F689C, and S696Y were the primary drivers of parasite resistance to compounds 1 (CNV), 3 (Y2079C and R2180P), and 4 (L690I and R2180G) in parental 3D7-A10, ABCI33 copies, and ABCI3L690I ed. parasites using a previously validated inoculum effect assay , contrasting with 50% inhibition with 5 nM of the control PKG inhibitor ML10. We also observed no significant change in IC50 of these compounds against a previously reported PKG cKD line  and the non-drug-pressured Dd2-B2 line recalls the biphasic responses to piperaquine (PPQ) seen in PPQ-resistant parasites , as opposed to 3D7 that shows a classic monophasic curve. Of note, pfcrt-edited Dd2Dd2 differs from Dd23D7 by eight amino acid substitutions in this transporter (M74I/N75E/K76T/A220S/Q271E/N326S/I356T/R371I), which collectively mediate resistance via a gain of CQ transport  in the highly acidic DV and their binding to Hz, combined with the absence of an appropriately mutated PfCRT transporter that can efflux these drugs back into the parasite cytosol  lost their biphasic dose response to 1 and 2, providing evidence that these SNPs reversed mutant PfCRT-mediated resistance. Results with our ABCI3 cKD lines  were informative in showing that reduced levels of ABCI3 caused increased parasite sensitivity to all five tested compounds. This susceptibility profile, however, differed markedly between the SNP-selecting  and CNV-selecting (1 and 2) compounds. While the cKD parasites were ∼7- to 11-fold more sensitive to 3–5 in the absence of aTc, they were only ∼2- to 3-fold more sensitive to 1 and 2. We conjecture that ABCI3 itself might be a target of 3–5. This distinction between the modes of action between the SNP- and CNV-selecting compounds is consistent with our recent observations of differences in their timing of action, with 1 showing a peak of activity in trophozoites  versus 3 and 4 that showed cumulative activity across all stages  increase in the IC50. These data suggest an intricate connection between PfCRT and ABCI3 in dictating parasite susceptibility to these compounds. We note that both transporters are apparently essential for asexual blood stage parasite growth, as previously demonstrated for PfCRT and evidenced herein with our cKD studies that showed a loss of growth upon ABCI3 depletion  and Dd23D7(CQ-sensitive) lines suggested a different mode of parasite resistance in Dd2-B2 parasites with the CQ-resistant Dd2 PfCRT isoform able to confer resistance, also presumably through gain of transport properties resulting from PfCRT mutations. Intriguingly, point mutations in ABCI3 neutralized this PfCRT-driven resistance.P. falciparum is able to evade antiplasmodial drug action. The study also identifies ABCI3 as a pleiotropic drug transporter to consider when assessing the risk of resistance to new antimalarials arising in the discovery and development pipeline.This study highlights unique strain-dependent ways in which df2260@cumc.columbia.edu).Further information and requests for resources and reagents should be directed to the lead contact, David Fidock  AlbuMAXII (Invitrogen) and 10 μg/mL gentamycin at 37°C in flasks gassed with 5% CO2/5% O2/90% N2. The 3D7-A10, Dd2-B2 and NF54 P. falciparum parasite lines have been previously reported , and 1 and 6 were synthesized at the Drug Discovery and Development Centre (H3D) at the University of Cape Town in South Africa as part of the SoftFocusKinase 59 (SFK59) library  followed by flow cytometry  .Compounds  library . Parasitrtorius) . IC50 vaP. falciparum clones resistant to compounds 2-4 was performed by the Winzeler lab, using methodology reported in  as described previously  was used to prepare DNA libraries from samples for whole-genome sequencing using the dual index protocol . The libeviously . Single P. falciparum 3D7 genome (PlasmoDB version 48) using BWA (Burrow-Wheeler Alignment). We used Samtools and Picard to remove PCR duplicates and reads that did not map to the reference genome. Reads were realigned around indels using GATK RealignerTargetCreator and base quality scores were recalibrated using GATK Table-Recalibration. GATK HaplotypeCaller  was used with the clones to identify all possible variants, which were filtered based on quality scores  and read depth (depth of read > 5) to obtain high-quality SNPs. These SNPs were annotated using snpEFF. The list of variants from the resistant clones were compared against the 3D7-A10 parent to obtain homozygous SNPs that were present exclusively in the resistant clones. IGV was used to confirm the SNPs present in the resistant clones. BicSeq was used to discover copy number variants (CNVs) against the 3D7-A10.In the Fidock lab, whole-genome sequencing of genomic DNA from parental and resistant clones employed an Illumina TruSeq DNA PCR-Free library preparation protocol and a MiSeq sequencing platform. Briefly, 2 μg of genomic DNA were sheared to a mean length of 550 bp, end-repaired, adenylated on their 3’ ends and ligated to indexed adaptors. Samples were pooled and sequenced on Illumina MiSeq flow cells to obtain 300 bp paired-end reads. Sequence data were aligned to the Streptococcus pyogenes and was fused to the selectable marker yDHODH that confers resistance to DSM1 . The donor plasmid contained the abci3 fragment with the L690I mutation and blasticidin-S deaminase (bsd), a selectable marker that protects against blasticidin (Sigma-Aldrich). Plasmid transfections were conducted using an Amaxa nucleofector (8 highly synchronized and magnet-purified 3D7-A10 mature schizonts were first re-suspended in 100 μl of Nucleofector Solution 2 (with the supplement added) that had been pre-warmed to room temperature. This was mixed with 10 μg of plasmid DNA in a volume of 5 μl deionized water in a cuvette and electroporated using the Amaxa U-033 program. Parasites were allowed to re-invade fresh RBCs in complete media pre-warmed to 37oC. Transfected parasites were selected with 2 μg/ml blasticidin for six days starting the day after electroporation, and parasites were maintained thereafter in complete media until recrudescence. Gene editing was assessed via Sanger sequencing of PCR products amplified from bulk cultures. Edited parasite clones were obtained by limiting dilution.The ABCI3 L690I mutation was introduced into 3D7-A10 parasites using a two-plasmid CRISPR/Cas9 system. Cas9 was derived from  to DSM1 . Both Caeofector . Briefly50 values (GraphPad Prism version 9).Edited parasites were assayed for drug susceptibility by flow cytometry . BrieflyP. falciparum codon-optimized Cas9 endonuclease was derived from Streptococcus pyogenes and was expressed under a calmodulin promoter. The plasmid also carried a human DHFR (hDHFR) selectable marker (that confers resistance to WR99210) under a P. chabaudi dhft-ts promoter and the guide RNA (gRNA) sequence under a U6 promoter. Guide RNAs were selected using ChopChop 9 (https://chopchop.cbu.uib.no). 108 parasites were electroporated with purified circular plasmid DNA as described  locus by inserting the linearized pSN054 donor vector , which iTo assess the effect on parasite viability of knocking down ABCI3 expression, synchronous ring-stage parasites were cultured in the presence (50 nM) or absence of aTc, in triplicate in a 96-well U-bottom plate (Corning 62406-121). Luminescence was measured at 0, 72, and 120 h post-invasion using the Renilla-Glo(R) Luciferase Assay System  and the GloMax Discover Multimode Microplate Reader (Promega). Luminescence values were normalized to 200 nM CQ-treated samples and results were visualized on a scatter plot using Prism .50 analysis, as described above.Compound susceptibility of the cKD parasites was assessed as described above. Compounds were assayed using 2-fold dilutions with inhibition measured after 56 h. Parasite viability was determined by staining the parasites with SYBR Green and MitoTracker Deep Red followed by flow cytometry and IC2 followed by an overnight incubation at 4°C. Cells were then permeabilized on ice using 0.05% Triton X-100 in 1×PBS for 5 min. Autofluorescence was quenched with 50 mM glycine for 10 min. After two washes in 1× PBS the cells were resuspended in 1% (w/v) bovine serum albumin (BSA) in 1×PBS blocking buffer and incubated with the appropriate dilution for each primary antibody used: 1:200 for rabbit or mouse anti-Flag (Genscript), rabbit anti-ERD2 (BEI Recourses), rabbit anti-BiP (kindly provided by Dr. Min Zhang), 1:50 for rat anti-Rab5B or Rab7 (kindly provided by Dr. Gordon Langsley), or 1:200 for anti-PfCRT antibodies. This was followed by incubation with the corresponding species-specific secondary antibodies  diluted 1:2000 in 1% BSA in 1× PBS. Thin blood smears of stained RBCs were prepared on microscope slides and mounted with cover slips using Prolong Diamond Antifade Mount with DAPI (Thermo Fisher). Parasites were imaged using a Nikon Eclipse Ti-E wide-field microscope equipped with a sCMOS camera (Andor) and a Plan-apochromate oil immersion objective with 100× magnification . A minimum of 27 Z stacks (0.2 μm step size) were photographed for each parasitized RBC. NIS-Elements imaging software  was used to control the microscope and camera as well as to deconvolve the images . ImageJ (Fiji) (version 2.0.0-rc-68/1.52 h) was used to crop the images, adjust brightness and intensity, overlay channels, and prepare montages.Indirect Immunofluorescence assays (IFAs) were performed in suspension as described . Briefly3 copies (resistant to 1) and ABCI3 L690I ed. (resistant to 4) parasite lines were exposed to serially diluted MMV compounds or CQ at parasitemias ranging from 0.25% - 4%. The inoculum size equals the parasitemia × hematocrit  (50 minus the absolute IC50) /   . The drued RBCs) . This raP. falciparum cultures were fixed in 4% paraformaldehyde (Polysciences Inc.) in 100 mM PIPES/0.5 mM MgCl2, pH 7.2 for 1 h at 4°C. Samples were then embedded in 10% gelatin and infiltrated overnight at 4°C with 2.3 M sucrose/20% polyvinyl pyrrolidone in PIPES/MgCl2. Samples were trimmed, frozen in liquid nitrogen, and sectioned with a Leica Ultracut UCT7 cryo-ultramicrotome (Leica Microsystems). 50 nm sections were blocked with 5% fetal bovine serum (FBS)/5% normal goat serum for 30 min and subsequently incubated with rabbit anti-HA antibody (Sigma) at 1:100 for 1 h, followed by secondary anti-rabbit IgG antibody conjugated to 18 nm colloidal gold (Jackson ImmunoResearch Laboratories) for 1 h. Sections were stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA) equipped with an AMT 8 megapixel digital camera and AMT Image Capture Engine V602 software (Advanced Microscopy Techniques). All labeling experiments were conducted in parallel, with controls omitting the primary antibody. These controls were consistently negative at the concentrations of colloidal gold-conjugated secondary antibodies used in these studies.To immunolocalize HA-tagged ABCI3, 2O, 20% (v/v) acetone and 2 M HEPES buffer (pH 7.4) and 50% pyridine) was added to each well to give a final pyridine concentration of 5% (v/v). Acetone (60 μL) was then added to assist with hematin dispersion. The UV-vis absorbance of the plate wells was read at 405 nm on a SpectraMax P340 plate reader. The β-hematin inhibitory IC50 values for each compound were computed from the blank-corrected absorbance values at 405 nm using sigmoidal dose-response curve fitting analysis .A solution containing water + 305.5 μM Nonidet P-40 (NP-40) + DMSO at a v/v ratio of 70% + 20% + 10%, respectively, was prepared and 100 μL added to all wells in columns 1-11 in a flat-bottomed 96-well plate. Working stocks of test compounds and controls were constituted to 10 mM, from which 20 μL of each was added to wells in the final column (column 12) together with distilled water (140 μL) and 305.5 μM NP-40 detergent (40 μL). This effectively lowered the final drug concentration to 1mM. Each compound (100 μL) was then serially diluted from columns 12 to 2 (column 1 served as a blank). A 25 mM hematin stock solution was prepared by sonicating hemin in 100% DMSO for 3 min and 178.8 μL of this solution was suspended in 20 mL acetate buffer  and thoroughly mixed. The homogenous suspension (100 μL) was then added to all wells to give final hematin concentrations of 100 mM and a drug concentration of 0.5 mM in column 12. Plates were covered and incubated at 37°C for 5 h after which 32 μL of 50% pyridine solution (20% (v/v) H1, 3, 4, 5, CQ and pyrimethamine was determined in 72 h assays as described above. IC50 values were used to initiate heme fractionation studies (50 concentrations (based on the 72 h chemosensitivity assay) ranging from 0.5-4× at 5% parasitemia and 2% hematocrit. A no-drug control was included. After 28 h, late trophozoites were harvested by lysing RBCs with 0.05% saponin followed by multiple washes with 1×PBS (pH 7.5) to remove traces of RBC Hb. Pellets were then resuspended in 1×PBS (pH 7.5) and an aliquot of the trophozoite suspension was stained with 1×SYBR Green and 100 nM MitoTracker Deep Red and quantified via flow cytometry to determine the total number of trophozoites. The remaining trophozoites were then released from their host RBCs by hypotonic lysis and sonication. The fractions corresponding to digested Hb, free heme and Hz were then carefully recovered through centrifugation and treatment with HEPES buffer (pH 7.4), 4% SDS, 25% pyridine solution, 0.3M HCl and 0.3M NaOH. The UV−visible spectrum of each heme fraction was measured as a Fe(III)heme−pyridine complex using a multi-well SpectraMax P340 plate reader. The total amount of each heme species was quantified using a heme standard curve where the mass of each heme-Fe species per trophozoite (fg/cell) was calculated by dividing the total amount of each heme species by the corresponding number of parasites in that fraction, as determined by flow cytometry. Statistical comparisons and analyses for trends were made using Prism  using Students’ t-tests with Welch corrections.NF54 parasite susceptibility to compounds  studies , 2015). 0 is the initial absorbance (no test sample), A∞ is the final limiting absorbance for the Fe(III)-drug complex, and K is the conditional association constant. Titrations were performed in triplicate and the average association constant (K) values reported with the standard error of the mean.A stock solution (1.2 mM) of hemin (Molecular weight 651.94 g/mole) was prepared in 100% DMSO. A 10 μM working solution was then prepared in 40% DMSO and 0.02 M HEPES buffer (pH 7.4) constituted in ultrapure HPLC-grade water, to maintain the heme in a strictly monomeric state. A 10 μM working solution of hemin at pH 5.6 was prepared in an identical manner except that HEPES was substituted with 0.02 M 2-[N-morpholino]ethanesulphonate (MES). Test compounds were diluted in 40% DMSO and 0.02 M HEPES buffer (pH 7.4) to obtain 2 mM working stocks while a 2 mM CQ diphosphate stock was also prepared in 40% DMSO and 0.02 M HEPES buffer (pH 7.4). All working solutions were covered in aluminum foil to avoid any photodegradation and used immediately upon preparation. Hemin solutions  at the two pH values were titrated with increasing concentrations of the different compounds and absorbance values were recorded at 402 nm subsequent to each addition of a known volume of compounds. Data were corrected for dilution and analyzed using a standard non-linear least squares fitting method, assuming a 1:1 complexation model in which the change in absorbance (A) as a function of drug concentration in the hemin solution [D] is given by the equation below:U tests throughout, except for the heme fractionation studies reported in Details regarding statistical tests are reported in the legends to


KCCs exhibit significant variations in K+ affinities, yet recent atomic structures demonstrated that K+- and Cl−-binding sites are highly conserved, raising the question of whether additional structural elements may contribute to ion coordination. The termini and the large extracellular domain (ECD) of KCCs exhibit only low sequence identity and were already discussed as modulators of transport activity. Here, we used the extracellular loop 2 (EL2) that links transmembrane helices (TMs) 3 and 4, as a mechanism to modulate ECD folding. We compared consequences of point mutations in the K+-binding site on the function of WT KCC2 and in a KCC2 variant, in which EL2 was structurally altered by insertion of a IFYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHAAA (3xHA) tag (36 amino acids). In WT KCC2, individual mutations of five residues in the K+-binding site resulted in a 2- to 3-fold decreased transport rate. However, the same mutations in the KCC2 variant with EL2 structurally altered by insertion of a 3xHA tag had no effect on transport activity. Homology models of mouse KCC2 with the 3xHA tag inserted into EL2 using ab initio prediction were generated. The models suggest subtle conformational changes occur in the ECD upon EL2 modification. These data suggest that a conformational change in the ECD, for example, by interaction with EL2, might be an elegant way to modulate the K+ affinity of the different isoforms in the KCC subfamily.K They belong to the amino acid/polyamine/organocation superfamily and therefore adopt the so-called LeuT-fold (+–Cl− cotransporters (KCC1-4), the Na+–K+–Cl− cotransporters (NKCC1 + 2), and Na+–Cl− cotransporters, the CCC-interacting protein CIP1, and the polyamine transporter CCC9  family consists of secondary active membrane transporters mediating the symport of cations , flanked by intracellularly located N- and C-termini and a large extracellular domain (ECD) , 29, 30.ications , 35, 36.ications . As in aications , 41, 42.− coupling and K+ transport (+ and Cl− affinities vary among CCCs (+-binding site comprises five strictly conserved amino acid residues located in TM1 (Asn133 and Ile134), TM3 (Tyr218), and TM6 (Pro432 and Thr435) (−-coordinating sites described in KCCs and NKCCs (B).Recent three-dimensional reconstruction from single-particle cryo-EM provided insight into the highly conserved ion-binding sites in KCCs and NKCC1 , 46, 47,ransport , 48, 49.nd NKCCs B.Figure HA). EL2 is located close to the ECD and we hypothesized that structural modification of this loop will affect folding of the ECD. While KCC2WT and KCC2HA showed same functionality, we observed striking differences when residues coordinating the K+ binding site were mutated. The structural modification of EL2 in KCC2HA yielded a variant which despite mutation of conserved sites could still transport K+. Homology modeling suggested that insertion of the HA tag is indeed inducing conformational changes in the ECD because of a modified EL2 interaction. These changes most likely are transduced via the extracellular interaction network . To investigate the impact of the ECD on KCC2-mediated transport, we used KCC2b with a 3xHA tag (IFYPYDVPDYAGYPYDVPDYAG-SYPYDVPDYAAHAAA) in EL2 of the KCC2b clone (KCC2bHA). The tag with a length of 37 amino acid residues is inserted at the end of EL2 reaching into TM4 . In parallel, we monitored their transfection rates to provide similar transfection rates for every construct used in our flux measurements. Tl+ flux measurements demonstrated that KCC2aWT (100% ± 9%), KCC2bWT , and KCC2bHA  possess similar transport activities .KCC2 is present in the two splice variants KCC2a and KCC2b that differ in the first exon coding the distal part of the N terminus. To analyses whether these two splice variants differ in their transport activity, we performed Tlinto TM4  . However, resolution in the mmKCC2a cryo-EM structure , KCC2aY218F (TM3), and KCC2aP432H, and KCC2aT435A (TM6). All mutants showed transfection rates in HEK-293 cells similar to KCC2awt (B). Tl+ flux measurements revealed significantly reduced transport activity for each of the five mutants by an average of 53% ± 9% for KCC2aN133S (p = 1.44 × 10−12), 51% ± 9% for KCC2aI134T (p = 2.33 × 10−10), 54% ± 15% for KCC2aY218F (p = 9.3 × 10−8), 39% ± 5% for KCC2P432H (p = 5.3 × 10−10), and 35% ± 8% for KCC2T435A (p = 5.66 × 10−10) compared with KCC2awt (100% ± 9%) : KCC2bN110S in TM1, KCC2bY1958F in TM3, and KCC2bT412A in TM6. All mutants showed a transfection rate in HEK-293 cells similar to KCC2bwt (B). Tl+ flux measurements revealed similar reduced transport activity for the each of the three mutants by an average of 51% ± 8% for KCC2bN110S (p = 1.89 × 10−5), 67% ± 17% for KCC2bY195F (p = 0.0012), and KCC2bT412A  compared with KCC2bwt (100% ± 10%)  between TM3 and TM4. The tag with a length of 37 amino acid residues is inserted at the very end of EL2 in front of TM4 . Importantly, this variant supports transport activity indistinguishable from KCC2bWT (A). We first probed whether the insertion has a direct impact on the coordinating tyrosine residue in TM3, which is directly connected to EL2. Replacement of this tyrosine (Y195 in KCC2bHA) by phenylalanine had no effect on KCC2bHA transport activity  and threonine (KCC2bHA-T412A) in TM6. Again, these mutants showed flux activities indistinguishable from KCC2bHA , 7d99 (hsKCC4), 6y5r (hsKCC3), 6y5v (hsKCC3), and 6m1y (hsKCC3)) to determine a set of homology models of mouse KCC2b (B). We included the 3xHA tag between Ala225 and Met226. Moreover, we included K+ and Cl− from KCC2-4 during homology modeling according to our mutagenesis study. We created more than 30 models in an inward-facing state, which were ranked based on energy minimalization. From the best scoring models, we selected four models which showed significant variations in their ECD conformation (A and B). Insertion of the HA tag did neither alter the overall conformation of mmKCC2 nor the coordination of K+ and Cl− (B and 6C). This is in agreement with the functional data showing that the HA-tag KCC2 exhibits WT activity . The orientation and conformation of the HA tag changed significantly within the different homology models (A and B). In all models, the EL2 + HA-tag approaches the ECD, thereby introducing subtle changes in its overall fold. Changes in the ECD fold were especially pronounced between the two cysteine bridges (B). In the model exhibiting the closest interaction with the HA tag , the disulfide bridge Cys310–Cys325 was even broken.To understand the impact of EL2 modification on Kse KCC2b B. We incormation , A and B and Cl− B and 6C.activity . In the observed B. The ory models , A and B bridges B. In themodel 1, B, the di+- and Cl−-binding sites in KCC1 to KCC4, and NKCC1 (+-binding site in KCC1 (Tyr216 in TM3), KCC3 , KCC4 , and NKCC1 (Tyr305 in TM3) diminishes or abolishes transport activity (mmKCC2a) significantly reduced activity in both splice variants. Reduction of transport activity can result from intrinsic impairments or changes in post-translational modifications that affect transport activity or subcellular distribution. In the future, more in-depth analyses are necessary to determine the precise contribution of each of these putative mechanisms.Recent breakthroughs in structural analysis of CCCs by cryo-EM revealed evolutionary highly conserved Knd NKCC1 , 48, 49.activity , 44, 46.+ vary between 5 and 50 mM within the KCC subfamily . In a similar context, mutation of the glycosylation site Asn312 in the ECD reduces mmKCC4 activity . The KCC2bHA construct had the tremendous advantage of exhibiting WT transport activity, which allowed for a systematic investigation of the K+-binding sites by mutagenesis. Our modeling experiments confirmed that HA-tag insertion did not alter the overall structure or the dimerization of KCC2. The models suggest that the HA tag extends away from the dimer center, whereas in some of the models, EL2 moves into the interaction range to the ECD. We observed changes in the ECD especially in the region flanked by the two stabilizing disulfide bridges. Interestingly, this region of the ECD shows the lowest sequence conservation. Our substitution experiments of K+-coordinating residues in KCC2bwt or KCC2bHA, respectively, revealed a striking effect of the HA insertion. Mutations in K+-binding sites, which had an unambiguous effect on transport activity in both splice variants, did not change transport activity when EL2 was prolonged by the HA tag in KCC2bHA. This observation suggests that the coupling mechanism that underly the K+ transport is affected by subtle conformational changes of the ECD.However, with our previous approaches of disrupting the ECD integrity, it was not possible to draw a conclusive line from the ECD fold to modified Kn KCC2WT B. The KC+ can be transported in KCC2bHA when the conserved K+ coordination site is corrupted. Despite the electroneutrality of the transport in KCCs, one potassium and two chloride ions are observed in most KCC structures, which were also included into our homology modeling (B). The first coordinating site (CL1) exploits conserved residues mainly in TM1  (B) (hsKCC2 structure (PDB entry code 6m23) reveals an alternative coordination for K+ (K’) , which is close to the Cl1 site in KCC3 . Such an alternative site would be easily achieved considering that K+ and two Cl− ions, respectively, are coordinated via a main chain network in the flexible regions of TM1 and TM6 (B). For chloride binding, main chain nitrogen of the flexible regions in TM1 and TM6 wrap around these ions.At this point, we can only speculate why Kmodeling B. The fi Met138) , 48, 49, Met138) B  (B) , 48. How K+ (K’) C, left,  and TM6 B. For chB and 6C) is Thr435, which coordinates K+ by its side chain and Cl− by its main chain. In comparison, in the alternative site, the main chain oxygens now coordinate K+ together with Pro432 and Ser433 in TM6 and Tyr469 in TM7. This replaces the former coordination by Asn133, Tyr218, and Thr435, which we have mutated in our study. Obviously, Thr435 is no longer coordinating both ions simultaneously, which might affect the conformational flexibility of TM1 and TM6 at this position. To switch to an alternative K+ coordination, only subtle changes in TM1 and/or TM6 are required. It is possible that the interaction of HA tag in EL2 with the ECD can serve this purpose. In such a scenario, mutation of the conserved sites might have no effect when the alternative sites are present due to conformational changes in TM1 and TM6.Critical for coupled ion binding in transport to the conserved site B and 6C C) show that ion-binding sites, both for K+ and Cl−, depend on several main chain coordination in TM1 and TM6. Changes in ion coordination therefore require main chain movements of TM1 and TM6. Following a similar line of argumentation as for KCC4, we hypothesize that TM1 and TM6 move because of changes in the fold of the ECD by HA-tag insertion to cause a switch from a conserved K+-binding site  to an alternative site . This would explain why our point mutations in the conserved site did not inactivate KCC2bHA. Future experiments have to clarify whether these alternative K+-binding sites are important in ion translocation by using site-directed mutagenesis and activity measurements.In a recent publication on the structure of monomeric KCC4 in nanodiscs , an elecFig. 6+–Cl− cotransport.In summary, our data suggest that conformational changes in the ECD by interactions with other extracellular loops might trigger the coordination switch, supporting the notion of a regulatory role for the ECD in KSite-directed mutagenesis of mouse KCC2a NM_001355480.1) and mouse KCC2b (NM_020333.2) with an HA tag in EL2 and rat KCC2b (NM_134363.1) was performed according to the QuikChange mutagenesis system (Stratagene) , 54, 55.80.1 and +–Cl− cotransporter activity, HEK-293 cells were transiently transfected with the respective construct, using TurboFect (Fermentas). Four hour before transfection, the medium was replaced by Dulbecco's modified Eagle's medium. For transient transfection in a 6-well plate, 6-μl TurboFect (Fermentas), 150 μl Opti-MEM (Invitrogen), and 3-μg DNA were mixed and incubated for 20 min at room temperature (RT) before transfection. For K+–Cl− cotransporter activity measurements, HEK-293 cells were plated in a 0.1 mg/ml poly-L-lysine–coated black-well 96-well culture dish (Greiner Bio-One) at a concentration of 100,000 cells/well, 24 h after transfection. The remainders of the cells were plated on a 0.1 mg/ml poly-L-lysine–coated glass coverslip. After ∼18 h, coverslips were proceeded for immunocytochemical analyses to determine the transfection rates, which were routinely between 20 and 30%.For immunocytochemistry and measurements of KFor immunocytochemistry, all steps were performed at RT. HEK-293 cells grown on poly-L-lysine–coated coverslips were fixated for 10 min with 4% paraformaldehyde in 0.2 M phosphate buffer. After three washing steps in PBS, cells were incubated with the blocking solution  for 30 min. The primary antibody solution  was added in the carrier solution  for 1 h. After washing three times in PBS, the secondary antibody, which was conjugated to a fluorescent probe , was incubated for 1 h. After three washes in PBS, cells were mounted onto glass slides with Mowiol (Roth) and 4′,6-diamidin-2-phenylindol . Photomicrographs were taken using a BioZero fluorescence microscope (Keyence).−-dependent uptake of Tl+ in HEK-293 cells as described previously  with 2 μM FluoZin-2 AM dye (Invitrogen) plus 0.2% (wt/vol) Pluronic F-127 (Invitrogen) and incubated for 48 min at RT. Cells were then washed three times with 80-μl preincubation buffer and incubated for 15 min with 80 μl preincubation buffer plus 0.1 mM ouabain to block the activity of the Na+/K+ ATPases. Afterward, the 96-well plate was placed into a fluorometer , and each well was injected with 40 μl 5x thallium stimulation buffer . The fluorescence was measured in a kinetic-dependent manner  across the entire cell population in a single well. By using linear regression of the initial values of the slope of Tl+-stimulated fluorescence increase, the transport activity was calculated.Transport activity of KCC2 was determined by Cleviously , 54, 56.t test after Student's t test for similar variances between samples or the two-sample t test after Welch in the few cases where SDs differed by more than a factor of two . For crosswise paired comparisons of means of transport activities among KCC2awt, KCC2bwt, KCC2bHA, and mock (Transport activities of each of the mutants were tested against the control sample (mock and WT), using two-sample r of two . Becauseg method , which cand mock , we compand mock .HA, five templates with the PDB entry 6y5r, 6y5v, 7d99, 6m1y, and 6m23 were selected according to the quality estimation by Global Model Quality Estimation and quaternary structure quality estimate. The template structures all adopt an inward-facing conformation. The generated models were constructed as homodimers. Homology modeling and model refinement for KCC2 with an additionally inserted 3xHA tag IF-YPYDVPDYAG-YP-YDVPDYAG-S-YPYDVPDYAAHAAA was performed in Modeller 9.12 in UCSF Chimera 1.14 individually for each template. Multitemplate alignment with position-specific scoring matrix was done in Jalview. From the outputs of 30 models, the final model with the lowest evaluation scores of normalized Discrete Optimized Protein Energy was selected for further refinement. For the no-template aligned part, the additional N-terminal and C- terminal of amino acids were deleted and nonconserved loop areas were optimized by loop modeling. The clash score was reduced by doing an energy minimization step and optimizing the rotamers of side chains. The homodimers were manually realigned in the two-fold axis between the monomers according the superposition with the template dimer. The final refinement and validation of model was done in the Phenix and Coot based on the Ramachandran plot and MolProbity score.For the comparative modeling of KCC2bAll relevant data are available from the corresponding author upon reasonable request.The authors declare that they have no conflicts of interest with the contents of this article.


In the present study, we developed and optimized a new approach that combines SULT-dependent biotransformation using recombinant and permeabilized fission yeast cells (enzyme bags) with PAPS production in situ applying quality by design principles. In the initial application of the procedure, yeast cells expressing human SULT1A3 were used for the production of 4′-hydroxypropranolol-4-O-sulfate from 4-hydroxypropranolol. The optimized protocol was then successfully transferred to other sulfonation reactions catalyzed by SULT2A1, SULT1E1, or SULT1B1. The concomitant degradation of some sulfoconjugates was investigated, and further optimization of the reaction conditions was performed in order to reduce product loss. Also, the production of stable isotope labelled sulfoconjugates was demonstrated utilizing isotopically labelled substrates or 34S-sulfate. Overall, this new approach results in higher space-time yields while at the same time reducing experimental cost.For  After administration drugs are either directly excreted unchanged or metabolized first. The main specimen for excretion of most drugs and metabolites is urine. The parent drugs often show shorter detection windows due to extensive metabolism. Therefore, in toxicological, forensic or doping control analysis metabolites are often used as target analytes in urine samples . This fact might contribute to the limited number of sulfonation studies as compared with research on glucuronidation. In biological sulfonation, conversion of inorganic sulfate into the high-energy cofactor PAPS is a prerequisite. In this pathway, adenosine triphosphate (ATP) sulfonation is initially catalyzed by ATP-sulfurylase to generate adenosine-5′-phosphosulfate (APS), which is subsequently phosphorylated by APS kinase to yield PAPS . AfterwaSaccharomyces cerevisiae ATP sulfurylase and APS kinase are encoded by the genes MET3 and MET14, respectively . KH2PO4, CuSO4 • 5 H2O, H3BO3, potassium hydrogen phthalate, Na2SO4, nicotinic acid, MnSO4 • H2O, and KI were from Merck . ZnSO4 • 7 H2O was purchased from Acros . Tris, agar, NH4HCO3, NH4Cl, FeCl3 • 6 H2O, MgCl2 • 6 H2O, glucose, Triton-X100, and biotin were from Roth . Inositol was from Th.Geyer , and MoO4 • 2 H2O was purchased from Alfa Aesar . Dehydroepiandrosterone (DHEA) was obtained from Steraloids . 4HP, ATP, and citric acid were purchased from Sigma Aldrich . 7-Hydroxycoumarin (7HC) and formic acid were purchased from TCI . 34S labelled (NH4)2SO4 and D9-Salbutamol (D9-SA) were purchased from Sigma-Aldrich , D6-DHEA was obtained from Sigma-Aldrich . Acetonitrile was from Fischer Scientific , and HCOONH4 was from VWR Chemicals . Ultrapure water was prepared with a Milli-Q water purification system LaboStar 2-DI/UV from SG Wasseraufbereitung und Regenerierstation GmbH . All other chemicals and reagents used were also of the highest grade available.NaS. pombe were carried out as described (4Cl (93.5 mM), glucose (2% w/v), Na2HPO4 (15.5 mM), potassium hydrogen phthalate (14.7 mM) and standard amounts of salt, vitamin and mineral stock solutions. Liquid cultures were kept shaking at 230 rpm.The recombined fission yeast strains YN3, YN4, YN20, and YN25 used in this project were described before . The preescribed . Briefly4HCO3 buffer  and directly used for SULT-dependent reactions. Enzyme bags were resuspended in 200 µL of aqueous NH4HCO3 buffer  or phosphate buffer  containing PAPS or ATP, ammonium sulfate, magnesium chloride and substrate as indicated. Biotransformations were carried out at 37°C in a shaking incubator (300 rpm). Enzymatic reactions were stopped by short sharp centrifugation at 14,100 rcf for 2 min and 200 µL of sample in 1.5 ml micro centrifuge tubes were directly frozen at –20°C. After defrosting, samples were centrifuged again . Supernatants were directly analyzed by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) or diluted with a mixture of acetonitrile and ultrapure water  prior to analysis. Negative control samples were incubated without cofactors 2SO4, and MgCl2) or without cells, respectively.This was essentially done as described before  with sli4)2SO4 concentration was always kept to the half of ATP concentration), incubation time (3–72 h), cell number per incubation, pre-incubation time, and magnesium chloride concentration (1–100 mM) were altered. Results of the pre-screening disclosed some limits and trends of the factors. For further optimization a Box-Behnken design was used to investigate the effect of the five dependent variables in biotransformation and to optimize the experimental conditions to achieve the highest yield. The variables in this design involved ATP concentration (11–20 mM), magnesium chloride concentration (10–100 mM), pre-incubation time , incubation time (3–24 h), and cell number  software program.The optimization process was performed applying quality-by-design (QbD) principles. Design of experiments (DoE) was used for multivariate statistical analysis aiming to ensure robust protocol conditions. It started with a broad systematic screening of the influence of several factors on the incubation of 4HP with YN20 (SULT1A3): ATP concentration  and D9-SA (100 µM) were used as substrates in enzyme bags experiments. Enzyme bags experiments were also carried out with DHEA (100 µM) and SA (100 µM) utilizing (NH4)234SO4 (5.5 mM) and ATP (11 mM) as educts for the cofactor PAPS. Production of labelled sulfoconjugates was monitored by UHPLC-MS/MS.DDegradation of an already sulfonated metabolite in enzyme bags experiment was tested by incubating 7-hydroxycoumarin sulfate (7HCSU) with either YN3 or YN4 for 5 h at 37°C. All experiments were carried out in duplicates.8 precultured and pelleted fission yeast cells that are permeabilized using 200 µL of Triton-X100 [0.3% in Tris-KCl buffer ] at 30°C for 60 min at 230 rpm. After washing with NH4HCO3 buffer  enzyme bags were resuspended in 200 µL of aqueous NH4HCO3 buffer  and supplied with ATP at 11 mM, ammonium sulfate at 22 mM, and magnesium chloride at 20 mM. Following substrate addition  mixtures are incubated at 37°C at 300 rpm. Sharp centrifugation at 14,100 rcf for 2 min followed by a freeze-thaw cycle at −20°C and a second centrifugation at 14,100 rcf for 2 min yielded the sulfoconjugates in the supernatant.Based on the optimization experiments the final enzyme bag method used 2.5 × 10Separation was conducted on a 1290 Infinity UHPLC System  with an Agilent InfinityLab Poroshell 120 Phenyl Hexyl .42- and optimization of the product yields multifactorial screening supported by Minitab software was started with five factors: ATP concentration, MgCl2 concentration, number of cells, reaction incubation time, and time of preincubation without substrate. Initial results indicated that a reduction of yield was correlated with longer preincubation time  in comparison to the initial reaction with external PAPS 2SO4, 100 µM substrate, 20 mM MgCl2, and 2.5 4HCO3 buffer (pH 7.8). Substrates were incubated with SULTs reported in literature to metabolize the respective substrates (As proof of concept the above-mentioned standard protocol developed with the SULT1A3 (YN20) and 4HP was then applied to 7HC, DHEA, salbutamol (SA) and 4HP using various SULTs . Experimbstrates . DHEA wa6-DHEA, D9-salbutamol (D9-SA), and (NH4)234SO4 were conducted in the present study (6-DHEA were subjected to SULT2A1-dependend enzyme bag biotransformations either with (NH4)2SO4 or (NH4)234SO4. Results of UHPLC-MS/MS analysis proved same retention time of DHEASU, D6-DHEASU, and DHEA-34S-SU (9-SA with SULT1A3 (YN20) as well  could not be shown using above mentioned conditions, even though the enzyme is known to metabolize this substrate . It was 4HCO3 buffer (at pH 7.8 or 7.4) or in phosphate buffer . The results showed that in pH 7.8 phosphate buffer 7HCSU displays the smallest amount of degradation (4)2SO4 concentrations , and also in phosphate buffer (pH 7.8). The results demonstrated that the peak area of 7HCSU reached the highest levels at 1 mM substrate concentration, while the influence of the (NH4)2SO4 concentration on the yield was minor 2SO4 in a recombinant sulfoconjugate biosynthesis system is reported. While production and regeneration of PAPS were previously described in a chemoenzymatic approach (4)2SO4 resulted in a more than doubled yield of product  led to a significant decline of yield in sulfonated product formation. As a structural analogue of PAPS, ATP has been reported to competitively inhibit the sulfonation of human M and P phenol sulfotransferase  . This pr6-DHEA, D9-salbutamol (SA), and (NH4)234SO4 were achieved applying the established protocol as well (4HCO3 buffer (pH 7.8).Afterwards, the standard protocol was applied to additional substrates and SULTs . Further as well , 5. The  as well  and Sun  as well . However as well . Degrada as well . FurtherE. coli. Highest yields were obtained when the sulfate concentration dramatically exceeded that of ATP. Consequently, we increased the (NH4)2SO4 concentration to 44 mM. However, the yield of 4HPSU did not show a significant rise with increasing (NH4)2SO4 concentrations. This might indicate that the concentration of PAPS is not the main limiting factor of 4HPSU yield in this case.In a previous study by Being a known hydroxysteroid converting SULT, SULT2A1 shows low affinity to phenolic compounds like 7HC , which mThe developed assay allows to determine whether a substrate is sulfonated by any one of the 14 human SULTs and also permits a comparison of their sulfonation activity. Due to the fact that the biosynthesized sulfoconjugates are not available as references, a quantitation of the results by UHPLC-MS/MS is not possible. Therefore, metabolite formation rates cannot be given in absolute values in this study.The successful development of an optimized PAPS replacement protocol (details in


Herein, we developed a facile solvothermal and subsequent annealing process to synthesize SnS2/RGO composite, in which SnS2 nanosheets are bonded on RGO, and investigated their potential as anodes for Na+ and K+ storage. When used as an anode in SIBs, the as-prepared SnS2/RGO displays preeminent performance (581 mAh g−1 at 0.5 A g−1 after 80 cycles), which is a significant improvement compared with pure SnS2. More encouragingly, SnS2/RGO also exhibits good cycling stability (130 mAh g−1 at 0.3 A g−1 after 300 cycles) and excellent rate capability (520.8 mAh g−1 at 0.05 A g−1 and 281.4 mAh g−1 at 0.5 A g−1) when used as anode for PIBs. The well-engineered structure not only guarantees the fast electrode reaction kinetics, but also ensures superior pseudocapacitance contribution during repeated cycles, which has been proved by kinetic analysis.To date, the fabrication of advanced anode materials that can accommodate both Na With the intensification of environmental pollution, green renewable energy has currently become an active area of research ,2. Among2) possesses unique two-dimensional (2D) layered structure with large interlayer spacing and high specific capacity based on both conversion and alloying processes [2 also has the low intrinsic conductivity and the dramatic volume and structural changes, which will easily lead to the poor sluggish kinetics and rapid capacity reduction in the process of Na+ and K+ insertion/extraction [2 and various conductive carbonaceous composite has become one of the most effective approaches to ameliorate these problems [2 nanosheet composite fabricated by Li et al., delivered a reversible capacity of ~420 mAh g−1 at a current density of 500 mA g−1 after 100 cycles for Na+ storage [2 and rGO composite synthesized by Glushenkov, displayed a reversible capacity of 250 mAh g−1 at 25 mA g−1 after 30 cycles for PIBs [2 electrode with long cycle life is still a huge challenge for SnS2.Specifically, the hexagonal tin (IV) sulfide  for SIBs. Moreover, SnS2/RGO also displays a high-rate capacity (520.8 mAh g−1 at 50 mA g−1 and 281.4 mAh g−1 at 500 mA g−1) and good cycling stability (130 mAh g−1 at 300 mA g−1 after 300 cycles) for K-ion storage. The fast electrode reaction kinetics and superior pseudocapacitance contribution may be the reason for the excellent performance, which has been proved by kinetic analysis. Thus, the present study provides a novel strategy to prepare SnS2 and rGO composite with excellent performance for both sodium and potassium storage and this strategy which may be extended to fabricate other high-performance electrode materials for energy storage.Herein, SnS2∙2H2O and 0.761 mmol (62.4 mg) 2-methylimidazole (2-MIN) were added into the above solution and stirred vigorously for 12 h at 25 °C. After that, 0.8 mmol (60 mg) thioacetamide (TAA) was added into the solution under stirring. After stirring for another 2 h, the solution was transferred into a Teflon-lined autoclave (25 mL) and kept at 120 °C for 24 h. The solid product was collected and dried. Finally, the precursor annealed at 350 °C with a heating rate of 2 °C min−1 in Ar gas for 1 h to gain the final products of SnS2/RGO. The SnS2 was produced by similar route, but with no added organic molecules and GO in the solvothermal process.In a typical preparation, 10 mg GO (prepared by the modified Hummers method) was first dispersed in 8 mL isopropanol. Then 0.172 mmol (38.8 mg) SnCl2/RGO and SnS2 were tested by a transmission electron microscope , the high-resolution TEM  and a field emission scanning electron microscope . The components of SnS2/RGO and SnS2 were analyzed by energy dispersive spectrometry (EDS). Crystallographic phases of SnS2/RGO and SnS2 were measured by powder X-ray diffraction  using a Cu Kα radiation. Raman spectroscopy  with 532 nm laser excitation. X-ray photoelectron spectroscopic (XPS) surveys were performed on an X-ray photoelectron spectrometer , which uses an Al Kα as the excitation source.The morphologies of SnS−2. A sodium foil with a diameter of about 14 mm was prepared in a glove box under the protection of high-purity argon as a counter electrode using sodium block . The glass fiber (Whatman) was used as the separator and 1 M NaPF6 dissolved in the diethyleneglycoldimethylether acted as electrolyte. To study the electrode performance of SnS2/RGO for PIBs, CR 2016 coin batteries was built using SnS2/RGO as anode, potassium metal was used as counter electrodes. The electrolyte was 3.0 M potassium bis(fluorosulfonyl)imide (KFSI) in TGM. The anode was prepared by casting slurries of active material, super P and PVDF binder in a mass proportion of 7:2:1 onto copper foil with an active material loading of around 1.05–1.4 mg cm−2. The galvanostatic charge/discharge tests were conducted on battery test station  from 0.01–3.0 V. Cyclic voltammetry (CV) tests (0.01–3.0 V) were carried on the CHI 760E electrochemical workstation.For the Na-ion half cells, 2032 coin-type cells were employed. The electrodes were obtained by applying a slurry mixture consisting of active materials (70 wt.%), Super-P (20 wt.%), and polyvinylidene difluoride , to a copper foil and drying it under vacuum at 60 °C for 6 h. The loading of the active material on each disc was about 1.05–1.4 mg cm2/RGO and SnS2. The Sn-based nanosheets in-situ grown on graphene oxide (GO) substrate can be obtained via solvothermal reaction by added 2-methylimidazole (2-MIN) and GO substrate in the first step. In the absence of 2-MIN and GO substrate, flower-like three-dimensional microspheres consisting of larger nanosheets are obtained. After a calcination process under Ar atmosphere, SnS2/RGO and SnS2 are obtained.2 nanosheet is tightly grown on microns GO nanosheets, indicating the synthesized sandwich structure integrates the features of micro- and nanostructures. 2/RGO, the d-spacing of 0.338 nm corresponding to the (100) plane in SnS2. Moreover, the selected-area electron diffraction (SAED) pattern  elemental mapping images of SnS2/RGO are shown in 2 sphere.The FESEM and TEM are carried out to investigate the morphological features of as-prepared materials.  pattern f shows t2/RGO and SnS2 show similar diffraction peaks (2 with P-3m 1 (164) space group (JCPDS no. 23-0677), corresponding to the above mentioned HRTEM and SAED results. There are no obvious diffraction peaks for the carbon phase due to its poor crystallinity. 2, with an interlayer spacing of 0.59 nm. The Raman spectra of SnS2/RGO and GO are shown in 2/RGO and GO have distinct characteristic peaks at ~1344 and 1602 cm−1, which can be attributed to the D (disordered carbon) and G (graphitic carbon) bands of graphite. Moreover, the intensity ratio of D-band to G-band (ID/IG) is 1.06, 1.37 for GO, and SnS2/RGO, respectively, indicating more defects in SnS2/RGO [2/RGO exhibits a peak located at 313 cm−1, which corresponds to the A1g vibration of SnS2 [2 adsorption-desorption isotherm profiling of SnS2/RGO. The relatively large surface area (33.885 m2 g−1) and abundant pores (0.159 cm3 g−1) of SnS2/RGO can provide adequate active sites and promotes the rapid transport of electron and ions. X-ray photoelectron spectroscopy (XPS) was further carried out to investigate the surface electronic states and the chemical compositions of SnS2/RGO. The characteristic peaks of Sn 3d, S 2p, C 1s, and O 1s are observed in the wide survey spectrum (5/2 and Sn 3d3/2 of Sn4+ in SnS2/RGO [3/2 and S 2p1/2 of S2− (2 [The composition of two samples is investigated by X-ray diffraction (XRD). It can be seen that SnSon peaks a and theSnS2/RGO ,22. In a of SnS2 . Figure spectrum . Figure SnS2/RGO ,26. The 2 of S2− e, which f S2−  layers on the electrode surface, which was a common phenomenon in tin-based sulfide anode materials [2/RGO electrode. For further comparison, the cycling performance of SnS2/RGO and SnS2 was measured at 0.5 A g−1 and shown in 2/RGO displays the better performance than pure SnS2 electrode and maintains a high capacity of 581 mAh g−1 after 80 cycles. The SnS2/RGO exhibits excellent sodium storage performance that exceeds many previously reported anode materials for SIBs  than pure SnS2 after 10 cycles at 0.2 A g−1, indicating that the improvement of conductivity benefiting from the introduction of RGO. Moreover, the Warburg coefficient of SnS2/RGO is 46.6 Ω s−0.5 (2 (316.1 Ω s−0.5), showing that Na-ion has a faster diffusion ability in SnS2/RGO. These results are coincident with their sodium storage performance.Inspired by their special structure, the electrochemical performance of the obtained SnSaterials ,32. Aftefor SIBs . Beside formance d. It can Ω s−0.5 f, which 2/RGO, the charge storage behavior and reaction kinetics of SnS2/RGO and SnS2 are further analyzed according to CV and GITT tests. 2/RGO and SnS2 at multiple scan rates from 0.1 to 0.6 mV s−1. It should be pointed out that the curves keep its shape even at a high scan rate of 0.6 mV s−1. The dependency between the peak current (i) and the sweep rate (ν) is based on Equation (1): 2/RGO and SnS2, respectively. It can be seen that SnS2/RGO electrode shows a larger b-values than SnS2 electrode, indicating the pseudocapacitive contribution ratio of SnS2/RGO is larger than that of the SnS2 electrode. The ratios of capacitive contribution can be evaluated by Equation (2) [kV)1(v and the kV)2. In addition, the ratios of pseudocapacitive contribution are all increased as the scan rate increases but from 62.76 to 83.16% for SnS2/RGO, 44.56 to 74.13% for SnS2  ,37,38: and GITT tests. As shown in 2/RGO also displays the excellent Li-ion storage performance  and good cycling stability with a high capacity of 581 mAh g−1 at 0.5 A g−1 over 80 cycles when applied as anode for SIBs. Simultaneously, it delivers a high capacity of 130 mAh g−1 at 0.3 A g−1 after 300 cycles as anode for PIBs. These results indicate that the SnS2/RGO is a promising anode material for electrochemical energy storage.In summary, the well-designed sandwich-like hierarchical SnS


Here, Pd-doped Co3O4 loaded on carbon nanofiber materials through electrospinning and heat treatment was efficiently synthesized, and used as a free-standing electrode. Benefiting from its abundant active sites, high surface area and effective ionic conduction capability from three-dimensional (3D) nanofiber framework, Pd-Co3O4@CNF works as bifunctional oxygen electrode and exhibits superior activity and stability superior to commercial catalysts.Self-supporting electrodes usually show excellent electrocatalytic performance which does not require coating steps, additional polymer binders, and conductive additives. Rapid  To date, a wide range of electrochemistry technologies have received extensive attention in both academia and industry. In the extensive literature, most reported catalysts for electrochemistry are synthesized in powder form, and then coated on the current collector (electrode substrate) by adding a polymer binder (such as Nafion or polytetrafluoroethylene) . Unfortuin situ synthesis of highly active ingredient on self-supporting electric conductors is identified as a straight forward path to prepare such binder-free and integrated electrodes. Thanks to its high surface area, outstanding flexibility and electrical properties, one-dimensional (1D) structure of carbon nanofibers have been extensively applied for energy storage and conversion equipment as high efficiency electrodes  electrode. Considering the simplicity and efficiency of electrospinning to prepare nanofibers, combined with the heat treatment process, we have achieved the preparation of the Pd- Co3O4@CNF electrode. Such electrode shows extraordinary oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) performance in alkaline solution, and exhibits superior zinc-air battery (ZAB) performance to commercial catalysts.In our work, we designed a Pd-doped Co3O4@CNF was prepared by electrospinning and subsequent thermal treatment (10H14O4Pd) and 0.23 g cobalt acetate (Co(AC)2·4H2O) in 10 g dimethylformamide (DMF) under magnetically stirring for over 5 h. The obtained precursor was filled in a syringe with 23-gauge blunt tip needle. Electrospinning was carried out under the electrostatic filed of 22 kV . We used X-ray photoelectron spectroscopy  to study the surface chemistry of these samples.The crystal structure of samples was investigated by an X-ray diffractometer . A scanning electron microscope , transmission electron microscopy (TEM), and high-resolution transmission electron microscopy (HR-TEM) were used to analyze the microstructure. Distribution of elements in pd-CoERHE = EHg/HgO + 0.098 + 0.059×pH. All powder catalysts for comparison will be prepared as slurry coated on the substrate as a working electrode. To test ORR performance, the prepared electrode film was cut into a circle with diameter of 5 mm and fixed on the surface of the rotating disk electrode by Nafion solution. To study the OER performance, the catalyst film was cut into rectangular pieces of 1.5 × 0.5 cm2, and used directly as a working electrode.All the electro-catalytic tests were tested using electrochemical workstations (Shanghai Chenhua Instrument Co. Ltd.CHI660E and CHI760E). The electrolyte is 1 M KOH solution. The counter electrode and reference electrode are graphite rod and Hg/HgO (filling with 1M KOH) respectively. All potentials are calibrated to reversible hydrogen electrodes (RHE) on base of the next equation: −1 in N2 saturated electrolyte for activation. Linear sweep voltammetry (LSV) test was then performed at 1600 RPM in an O2 saturated electrolyte with scanning rate of 10 mV s−1. The accelerated durability test (ADT) was carried out at voltage from 0.57 to 1.07 V (vs. RHE) for 5000 cyclic voltammetry cycles with sweep rate of 100 mV s−1. Nyquist plots were tested by electrochemical impedance spectroscopy (EIS) measurements in O2-saturated electrolyte at 0.85 V (vs. RHE). For OER, 30 cycles CV tests were performed at a scanning rate of 50 mV s−1 in N2 saturated electrolyte for activation. LSV test was then performed in N2 saturated electrolyte with sweep rate of 10 mV s−1. The ADT were carried out at the voltage range of 1.02–1.52 V (vs. RHE) for 5000 cyclic voltammetry cycles with a scan rate of 100 mV s−1. Nyquist plots was tested at 1.6 V (vs. RHE) in N2-saturated electrolyte. The stability tests were further investigated by long-term chronoamperometry (CA) tests for 20 h.For ORR, 30 cycles of cyclic voltammetry (CV) tests were performed at a scanning rate of 50 mV s3O4@CNF sample was cut in a 1.5 × 2 cm2 rectangular piece  to make a catalyst layer. For the comparative commercial sample (Pd/C + RuO2), air electrodes loaded with equal mass of Pd were prepared by drip coating using the same ORR catalyst formulation ratio. The electrolyte was contained by 6 M KOH and 0.2 M ZnCl2 solutions. LANBTS BT-2016C system was used for cycling test (20 min for each charge and discharge period).Zn foil was used as the anode. Air cathode is assembled by an air diffusion layer, foamed nickel, and a catalyst layer. Obtained Pd-Co3O4@CNF is illustrated in 3O4@CNF was successfully obtained via an ordinary electrospinning method and subsequent heat treatment.  As a comparative sample, Co3O4@CNF was synthesized in the same way except that C10H14O4Pd was not added. In the experiment a flexible fiber membrane with a width of about 11 cm was obtained after electrospinning, and a 9.5 cm wide self-supporting electrode with mechanical strength still can be obtained by subsequent heat treatment. SEM and TEM characterization were performed to directly observe nanoscale structure of samples. As demonstrated in 3O4 are randomly incorporated in the fibrous structure. After heat treatment, nanoparticles can be observed uniformly distributed on the surface of the nanofiber  pattern was further displayed, corresponding to the crystal structure of Co3O4.  and , suggesting the high purity and crystalline. Moreover, in the XRD pattern, there are no diffraction peak which can be attributed to palladium or palladium oxides, suggesting that no large Pd-based nanocrystals exist in Pd-Co3O4@CNF sample and ultra-low doping of Pd will not change the phase of Co3O4. To further investigate the valence state of the Pd-Co3O4@CNF, together with the Co3O4@CNF, XPS measurements were performed. As shown in the Co 2p spectrum , the peaks are located at 780.0 and 795.5 eV, 781.4 and 797.2 eV, demonstrating the existence of Co2+ and Co3+ species, respectively , carbon sp3 , epoxy/hydroxyls , and carboxylates , respectively  of Pd-Co3O4@CNF is 0.81 V, which is more positive than Co3O4@CNF and commercial Pd/C catalyst. For OER, the potential corresponding to current density of 10 mA cm−2 (Ej10) is 1.48 V, compared with 1.56 V for commercial RuO2 catalysts. As shown in 3O4@CNF exhibits the lowest Tafel slopes (73.1 mV dec−1 and 65.7 mV dec−1) for both OER and ORR, revealing the enhanced reaction kinetics (3O4@CNF oxygen electrode has the lowest charge transfer resistance, indicating that the electrocatalysis process is enhanced. Moreover, as confirmed in 3O4@CNF shows high 4e− transfer selectivity in ORR process. In addition, the durability of the electrode and stability of its electrochemical performance are also the key criteria for evaluating electrocatalysts. Considering the self-supporting structure and integrated synthesis of the electrode, Pd-Co3O4@CNF exhibit excellent stability. As shown in 3O4@CNF was investigated by ADT and CA tests. The current density of Pd-Co3O4@CNF oxygen electrode at the same potential did not decrease significantly after 5000 consecutive potential cycles, respectively. In addition, the current density of Pd-Co3O4@CNF can be maintained over 89% of the initial current density after a 20 h long termed CA test, indicating that Pd-Co3O4@CNF oxygen electrode can retain most of its activity after a long period of operation, especially compared with commercial catalysts (The performance of Pd-Cokinetics . The Nyqatalysts .3O4@CNF electrode was applied to the air cathode in a rechargeable Zn-air battery as illustrated in 2, and the battery performance was tested as a comparison. As shown in 3O4@CNF exhibited lower charging voltage, higher discharge voltage, and higher peak power density compared to commercial catalysts, demonstrating the optimized performance of Pd-Co3O4@CNF electrode. In addition, constant current charge-discharge tests were performed on air cathode at a current density of 10 mA cm−2 to appraise the rechargeability of electrodes. As illustrated in 3O4@CNF based ZABs provides a lower charge-discharge gap and maintain over 240 h, and it exhibits superior stability to commercial catalysts-based battery. As mentioned above, the Pd-Co3O4@CNF air cathode exhibits battery performance in zinc-air cells, which can be attributed to its excellent electrocatalytic activity and stability for both OER and ORR.Under the proposal of oxygen electrode performance discussed above, Pd-Co3O4@CNF self-supporting electrode was successful prepared by electrospinning method and subsequent thermal treatment. Benefiting from its plentiful active sites, high specific surface area, and integrated configuration, Pd-Co3O4@CNF exhibits enhanced activity and stability for oxygen electrode reactions in 1.0 M KOH. Stimulated by the promising bifunctional activity, the ZABs with Pd-Co3O4@CNF electrode exhibit superior charge-discharge capacity and stability compared with the commercial catalysts based ZABs. This study provides an efficient and scalable method of synthesis high-performance self-supporting electrodes by effectively introducing metallic active component. This high performance and stable air cathode has been proven to be effective in ZABs applications. It also proposes a general strategy for manufacturing flexible self-supporting electrodes which can applied to energy storage and conversion devices.In summary, Pd-Co


The results showed that 10 µg/mL TA significantly improved the cumulus expansion index (CEI), cumulus-expansion-related genes  expression and blastocyst formation rates after parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) compared to the control groups, but not oocyte nuclear maturation. Nevertheless, 10 µg/mL TA dramatically enhanced the mRNA expression of oocyte-development-related genes , GSH, ATP, SOD1, PGC1α, BMP15, GDF9 and CDC2 levels and reduced intracellular ROS level in porcine oocytes. These results indicated that porcine oocyte cytoplasmic maturation was improved by 10 µg/mL TA treatment during IVM. In contrast, a high concentration of TA (100 μg/mL) significantly decreased the CEI and PTGS1, PTGS2, PTX-3 and HAS2 mRNA expressions in cumulus cells, and reduced oocyte nuclear maturation and the total cell numbers/blastocyst. In general, these data showed that 10 μg/mL TA supplementation has beneficial effects on oocyte cytoplasmic maturation and subsequent embryonic development in pigs.To investigate the effects of tannins (TA) on porcine oocyte  Next, 50 COCs were put into a 500 μL IVM culture medium comprising TCM-199 supplemented with 0.6 mM cysteine, 0.1 mM sodium pyruvate, 10% porcine follicular fluid (The cumulus expansion index (CEI) was assessed by the morphology of COCs and distinguished to five grades after 42 h IVM as described previously . BrieflyAfter 42 h of IVM, COCs were denuded with 0.1% hyaluronidase by pipetting and were then washed 3 times in Tyrode’s albumin lactate pyruvate (TALP) medium. The denuded oocytes were evaluated with a microscope  and classified as immature  or at metaphase II .To measure the intracellular GSH and ROS levels, denuded oocytes (MII stage) were sampled in Dulbecco’s phosphate-buffered saline  with added CellTracker Blue CMF2HC  and H2DCFDA . Each treatment group (using 15 oocytes for 1 replicate) was incubated (in the dark) for 30 min in 10 µM CellTracker Blue and 10 µM H2DCFDA. Then, the samples were washed 3 times with DPBS, oocytes were placed into 4 µL droplets of TALP-HEPES and fluorescence was observed under an epifluorescence microscope . The excitation and emission wavelengths were 371/464 nm for CMF2HC and 492~495/517~527 nm for H2DCFDA, respectively. The intensity of fluorescence was measured by Image J software.Denuded oocytes (using 15 oocytes for 1 replicate) were washed 3 times in DPBS and fixed with 4% paraformaldehyde for 1 h, washed 3 times and incubated in DPBS supplemented with 500 nM BODIPY FL ATP  for 1 h in the dark. Oocytes were washed 3 times in DPBS and mounted on cover slips. Images of each oocyte were captured using an epifluorescence microscope . The excitation and emission wavelengths were 504/514 nm for BODIPY FL ATP. The intensity of fluorescence was measured by Image J software.v/v) for 30 min at room temperature. Then, nonspecific sites were blocked with 2% bovine serum albumin (BSA) in PBS overnight at 4 °C. Oocytes were incubated with a rabbit polyclonal antibody against SOD1 , PGC1α (ab54481), BMP15 , GDF9 (ab93892) or CDC2 (ab18) at 37 °C for 2 h. A goat anti-rabbit fluorescein isothiocyanate-conjugated secondary antibody  or a Donkey anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody  was applied for 2 h. In addition, oocyte nuclei were labeled with 10 µg/mL Hoechst 33342 for 10 min. Oocytes were washed 3 times in PBS and then mounted on glass slides and evaluated with the same exposure times and adjustments under an epifluorescence microscope . The intensities of SOD1, PGC1α, BMP15, GDF9 and CDC2 were measured by analyzing the oocyte images with Image J software .Porcine MII-stage oocytes were fixed with 4% paraformaldehyde for 30 min and placed in PBS containing 1% Triton X-100  and activated by electric stimulation with a single direct current (DC) pulse of 1.5 kV/cm for 60 µs utilizing a BXT Electro-Cell Manipulator 2001 . Activated oocytes were washed and transferred into wells containing 500 µL porcine zygote medium-3 (PZM3), and cultured under a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 at 38.5 °C for 7 days. To analyze each blastocyst’s total cell number, the blastocysts were dyed with 10 µg/mL Hoechst 33342 for 4 min.Denuded oocytes with 1st PB were selected and transferred into a chamber with two electrodes spaced 3.2 mm apart that was filled with activation solution  droplet and equilibrated for 20 min in the incubator. Fresh boar semen was washed 3 times in DPBS with 0.1% BSA under centrifugation at 1900× 2 at 38 °C in Dulbecco’s modified Eagle’s medium  containing 20% fetal bovine serum  (v/v) and 100 IU/mL each of penicillin and streptomycin. Oocyte enucleation was performed by an aspiration pipette to remove the first polar body and the adjacent cytoplasm in the TALP medium containing 5 µg/mL cytochalasin B. Then, using an injection pipette, we injected a donor cell into the perivitelline space of each enucleated oocyte, and the couplets were equilibrated in a fusion solution , and transferred into a 20 µL droplet of fusion solution and exposed to a single DC pulse of 1.2 kV/cm for 30 µs using an electrical pulsing machine . The reconstructed couplets were equilibrated in an activation solution after incubating in the PZM3 medium for 1 h, and then lined up in an activation chamber filled with activation medium and activated with a single DC pulse of 1.5 kV/cm for 30 µs using a BTX ElectroCell Manipulator 2001. SCNT embryos were washed and placed in the PZM3 medium at 38.5 °C in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 for 7 days. To analyze each blastocyst’s total cell number, the blastocysts were dyed with 10 µg/mL Hoechst 33342 for 4 min.Adult pig ear tissues were chopped into small pieces and incubated in an atmosphere of 5% COGAPDH (for cumulus cells) or RN18S (for oocytes) using the equation R = 2−ΔΔCt. For ease of comparison, the average expression level of each gene from the control group was set as 1. Expression values were normalized to those of GAPDH or RN18S.For the analysis of gene expression, total mRNAs were separately extracted from oocytes and cumulus cells for each group using the TRIzol reagent , according to the manufacturer’s protocol, and the total mRNA concentration was quantified using a NanoDrop 2000 Spectrophotometer . Following this, complementary DNA (cDNA) was produced using amfiRivert cDNA Synthesis Platinum Master Mix . A PCR plate  was made by adding 1 µL cDNA, 0.4 µL (10 pmol/µL) forward primer, 0.4 µL (10 pmol/µL) reverse primer, 10 µL SYBR Premix Ex Taq  and 8.2 µL Nuclease-free water , and then amplified on a StepOneTM Real-Time PCR System . The amplification protocol included an initial denaturation step for 10 min at 95 °C followed by 40 cycles consisting of denaturation for 15 s at 95 °C, annealing for 1 min at 60 °C and extension for 1 min at 72 °C. The primers used for the real-time PCR are listed in t-test. A p-value < 0.05 was considered statistically significant. Each experiment was repeated at least 3 times.The data were analyzed by one-way ANOVA followed by Tukey’s test using SPSS 17.0 statistical software . The SCNT embryo development was compared by Student’s p < 0.05) and showed a significant decrease in the proportion of oocytes with CEI grade 3 compared to the control and 100 µg/mL TA . Therefore, CEI was significantly increased in 10 µg/mL TA compared to the control and 100 µg/mL TA , but there were significant differences compared to the 100 µg/mL TA  expression compared to the control and 100 µg/mL TA (p < 0.05) expression compared to the control and 100 µg/mL TA , and the level of ROS was significantly decreased in 10 µg/mL TA compared to the control and 100 µg/mL TA (p < 0.05).We then evaluated the intracellular GSH, ROS and ATP levels at the MII stage of porcine oocytes . The levp < 0.05; p < 0.05; To confirm the previous results, we analyzed the protein levels of SOD1 (anti-oxidation factor), PGC1α  and BMP15 and GDF9 (oocyte-development-related markers) in porcine oocytes. In line with the GSH and ROS results, SOD1 expression was significantly increased in 1 µg/mL and 10 µg/mL TA compared to the control and 100 µg/mL TA , supported by mRNA expression of BMP15 and GDF9. In addition, we detected CDC2  expression by immunofluorescence and found that it exhibited the highest level in 10 µg/mL TA compared to the other groups (p < 0.05; Both BMP15 and GDF9 protein expressions  were sigp < 0.05; p < 0.05; To evaluate the oocyte developmental potential, we used 521, 690 and 233 matured oocytes to perform PA, IVF and SCNT, respectively. Embryos derived from PA and IVF had a similar developmental pattern: Although there was no significant difference in cleavage rates among the groups, the blastocyst formation rates were significantly increased in 10 µg/mL TA compared to the control and 100 µg/mL TA (PA, 53.63 ± 5.44% vs. 30.62 ± 4.31% and 24.26 ± 3.67%; IVF, 23.74 ± 0.92% vs. 16.50 ± 0.57% and 16.35 ± 0.81%, In recent years, great interest has been focused on using natural antioxidants in food products. These natural antioxidant molecules are derived from coffee leaves, sweet potato, ginkgo biloba leaves and tomato pulp, such as carotenoids, anthocyanins, flavanols and lycopene, respectively . Numerouvia gap junctions [in vitro maturation and their subsequent embryo development [via cumulus expansion.It is well known that the cumulus cells surrounding the oocyte regulate its growth and meiotic maturation, with cell-to-cell communication unctions . The cumunctions  and satuunctions ,25. Therunctions . The oocelopment ,28. It ielopment . Moreoveelopment . Therefoelopment . Additioelopment . HAS2 iselopment . Moreoveelopment  and PTX-elopment . It has elopment . In the via c-Mos, thus there is tight interaction between MAPK and MPF [BMP15, GDF9, CDC2 and CYCLIN B1 expression compared to the control group, and slightly increased C-MOS. Additionally, the protein expression of BMP15, GDF9 and CDC2 showed high levels in porcine oocytes, which was in line with their gene expression. These results indicated that TA could improve porcine oocyte cytoplasmic maturation by increasing BMP15, GDF9 and MPF levels.The nuclear and cytoplasmic maturation events that occur during oogenesis up to MII confer the oocyte with developmental competence, defined as the capacity to support embryo development in all species . The MAP and MPF . The act and MPF . Zhang a and MPF . GDF9 is and MPF . Lin et  and MPF . GDF9 an and MPF . We selein vitro [2O2 formation [ROS scavenging is critical for oocyte maturation and embryo development in vitro . Generalormation . The absormation ,47. Mitoormation . PGC1α hormation . Moreoveormation . Nrf2 siormation . It has ormation . Salman ormation . In thisCytoplasmic maturation includes cytoplasmic processes that prepare the oocyte for fertilization and embryo developmental competence and occurs throughout oocyte growth and simultaneously with nuclear maturation . These pvia gap junctions in the COCs [HAS2 following 100 µg/mL TA treatment led to abnormal expression of PTX-3 and TNFAIP, resulting in low cumulus expansion and incomplete inhibition of cAMP transport from the cumulus, causing the failure of the activation of MPF and MAPK in the oocyte, thereby affecting nuclear maturation. These experiments suggested the possibility of inducing, by excessive antioxidant supplementation, a dangerous condition called “antioxidant paradox” leading to “reductive stress” that in turn may impair nuclear maturation [Surprisingly, we found that although exposure to 100 µg/mL TA significantly decreased cumulus expansion and oocyte nuclear maturation compared to the control, once these oocytes had matured, they had the same ability as the control oocytes to develop a blastocyst after PA and IVF, but there were significant differences compared to the 1 µg/mL and 10 µg/mL TA. These results agreed well with those of other authors who recorded that the polar body extrusion rate was negatively influenced in the presence of a high concentration of TA . Furtherthe COCs . Generalthe COCs ,56. Simuthe COCs . This prthe COCs . Our resturation .Our results demonstrated that 10 μg/mL of TA improved cumulus expansion, the MPF level of oocyte and subsequent embryonic development after PA, IVF and SCNT by increasing the cumulus-expansion- and oocyte-development-related gene expression, improving the level of GSH, ATP, SOD1, PGC1α, BMP15, GDF9 and CDC2, and decreasing the level of ROS. Therefore, our findings provide further insight into the function and toxicity of TA in the fields of animal reproductive physiology and human assisted reproductive technology.