The catalytic activity is distinct from previously described RNA-ligating deoxyribozymes. Kinetic analyses revealed the optimal incubation conditions for high ligation yields and demonstrated a broad RNA substrate scope. Together with the smooth synthetic accessibility of 5′-adenylated RNAs, the new DNA enzymes are promising tools for the protein-free synthesis of long RNAs, for example containing precious modified nucleotides or fluorescent labels for biochemical and biophysical investigations.Deoxyribozymes (DNAzymes) are small, synthetic, single-stranded DNAs capable of catalyzing chemical reactions, including RNA ligation. Herein, we report a novel class of RNA ligase deoxyribozymes that utilize 5′-adenylated RNA (5′-AppRNA) as the donor substrate, mimicking the activated intermediates of protein-catalyzed RNA ligation. Four new DNAzymes were identified by in vitro selection from an N DNA is best known in the form of the famous Watson-Crick double helix of two antiparallel phosphodiester single strands held together by hydrogen bonding and stacking interactions of complementary A–T and G–C base pairs. However, the ability of DNA to form three-dimensional structures reaches far beyond the static double helix, whose main purpose is to store genetic information and faithfully transmit it to the next generation. It is now well established that the information stored in defined sequences of single-stranded DNA can be exploited for the formation of active-site architectures that catalyze chemical transformations ,2,3. SucThe second most important reaction catalyzed by DNA is the formation of new phosphodiester bonds for the ligation of nucleic acid fragments in different linear and branched architectures . The DNA2+ as a divalent metal ion. The 9DB1 DNA enzyme was heavily investigated biochemically [The catalytic activity of RNA-ligating DNA enzymes may also be compared to ligase and polymerase ribozymes that are discussed in the context of the RNA world hypothesis ,12. Moreemically ,14, and emically . The stremically ,17,18.An important aspect for practical applications of DNA enzymes is their substrate preference in terms of accepted RNA sequences and the accessibility of donor substrates with activated phosphate groups ,19. The 2 as a cofactor, the SC9 DNA enzyme enables general RNA ligation in good yields (ca 80% ligation yield in 4 h). Future biochemical and structural investigations of the new DNA enzymes are promising to reveal more detailed insights into fundamental aspects of DNA catalysis in comparison to ribozymes and protein enzymes.In this work, we report new RNA ligase deoxyribozymes that enable native linear RNA ligation using 5′-AppRNA as a donor substrate. Using in vitro selection, four new DNA catalysts were identified and biochemically characterized. With MnCl2, as previously described [32P-labeled RNA R2 in the presence of 20 mM MgCl2 and 20 mM MnCl2 at pH 7.5. The ligated active fraction of the DNA library was separated by PAGE and amplified by PCR with a 5′-phosphorylated forward primer (D2) and a tailed reverse primer (D3). The asymmetric double-stranded PCR product was separated into single strands by PAGE, and the shorter 5′-phosphorylated strand representing the enriched DNA library was again ligated to RNA substrate R1 to initiate the next round of selection. The incubation time in rounds 1–7 was 15 h, which was reduced to 5 h in round 8, and 1 h in rounds 9–11. The fraction of ligation product in each round was quantified . In vitro selection of the 9DB1 RNA ligase was carried out at pH 9.0, and the deoxyribozyme was trained to use Mg2+ [2+ as a cofactor, but faster reaction rates were restored when Mg2+ was replaced by Mn2+ [2+ for DNA catalysis, the in vitro selection in this work was carried out at pH 7.5 in the presence of 20 mM each of MnCl2 and MgCl2, and the metal ion dependence of the SC9 deoxyribozyme was determined (similar results were obtained for the other three DNA enzymes). The ligation reaction was carried out under different conditions, and the amount of ligation product at various time points was determined by PAGE. Representative gel images and the kinetic graphs for the seven tested conditions are depicted in 2+ in the ligation buffer, and it is inactive when only Mg2+ is supplied as a divalent metal ion cofactor, up to a concentration of 100 mM. Doubling the concentration of Mn2+ ions from 20 to 40 mM resulted in about two-fold faster reaction, while a further increase to 100 mM Mn2+ dramatically reduced the ligation yield. The surprising finding that Mg2+ does not play a productive role in SC9 catalysis is supported by the observation that the reaction rates at 20 mM Mn2+ and a mixture of 20 mM Mn2+ + 20 mM Mg2+ are nearly indistinguishable.The majority of deoxyribozymes require divalent metal ions as cofactors. Exceptions are NaDNAzymes ,34,35,36DNAzymes ,38, to cDNAzymes  perform 2 at pH 7.5, and the ligation yield at various time points was determined by PAGE. The yield versus time data are plotted in RNA-ligating deoxyribozymes are useful tools for the synthesis of site-specifically labeled RNA ,10. A prFirst, we checked the variability of the 3′-terminal nucleotide of the acceptor RNA. The best ligation yields were obtained when the acceptor substrate terminated with a purine nucleotide, and slightly less but still useful yields were obtained with pyrimidines (G~A > U > C). On the other hand, only 5′-AppG was accepted at the 5′-end of the donor substrate, while all nucleotides beyond this first position in the donor RNA permitted the tested mutations. The collective changes in the binding arm to transversions-1  were very well tolerated, resulting in equally fast and efficient ligation as the parent substrate. The ligation rate and yield were only slightly reduced for transition mutations , while the RNAs generated by transversion-2  mutations turned out as poorer substrates, resulting in only 20% ligated product after 4 h incubation time.These results are comparable to the originally reported substrate scope of the 9DB1 DNA enzyme . The cry2+-dependent DNA catalyst for RNA ligation substrates. The residual activity with the 5′-AppDNA donor encourages further optimization of the catalyst, which is likely achievable by few rounds of in vitro evolution from a partially randomized SC9 library.The second aspect of the substrate scope beyond the nucleotide sequence addresses the backbone constitution. A fundamental question asks whether deoxyribozymes trained to ligate RNA may be able to tolerate the removal of the 2′-OH group in the substrate, i.e., if DNA oligonucleotides can be used as substrates. It is interesting to note that deoxyribozymes that form 2′,5′-branched nucleic acid architectures have been developed for all four combinations of DNA and RNA substrates, i.e., both the scaffold and adaptor strands can be either DNA or RNA (for a summary see ). Althou2+ ions as a cofactor, while smaller Mg2+ ions are not suitable to support the catalysis of RNA ligation. It will be interesting to investigate the metal-ion dependent folding of the deoxyribozyme to identify key structural differences that are responsible for the metal ion preference [In this work, we reported a new class of DNA catalysts for RNA ligation using 5′-adenylated donor RNA as a substrate. For synthetic RNAs that contain precious modified nucleosides, activation of the 5′-phosphate as 5′-adenylate is synthetically more easily accessible than 5′-triphosphorylation. Therefore, the SC9 and related deoxyribozymes will likely find application as synthetic tools for RNA ligation. The new catalysts currently ligate 5′-AppG RNAs most efficiently, but mutations beyond the first nucleotide are well tolerated as long as Watson–Crick complementarity to the binding arm is maintained. The SC9 deoxyribozyme can only use Mneference . In the eference ,45 and neference  to guideeference . In the eference ), or thaeference .O-TOM-protected) on CPG solid supports as previously reported [4. Gradient: linear, 0–40% solvent B, 4% solvent B per 1 CV) and HR-ESI-MS . Solid supports and phosphoramidite building blocks for solid-phase synthesis were purchased from ChemGenes. 6-FAM-azide, N6-(6-Amino)hexyl-ATP-ATTO-550, deoxyribo- and ribonucleotide triphosphates (dNTPs and NTPs) were purchased from Jena Bioscience. T4 Polynucleotide Kinase (PNK), RNase T1, T4 DNA Ligase, T4 RNA Ligase, and DreamTaq DNA polymerase were purchased from Thermo Fisher Scientific. T7 RNA polymerase was prepared in house following a published procedure with minor modifications [32P-ATP and α-32P-dATP were from Hartmann Analytic GmbH. Fluorescence gel images were recorded using a ChemiDoc MP imager with epi illumination using blue and green LEDs (emission filters 530/28 (blue), 605/50 (green)) from Biorad. Radioactive gels were dried under vacuum, exposed to phosphor storage screens, and imaged using a Typhoon Phosphorimager from GE Healthcare.DNA oligonucleotides were purchased from Microsynth and purified by denaturing PAGE prior to use. RNA oligonucleotides were prepared by solid-phase synthesis using phosphoramidite chemistry  containing 5 mM 5-FAM-azide, 5 mM CuBr, and 10 mM TBTA. After incubation at 37 °C for 3 h in the dark, the labeled oligonucleotides were precipitated and purified by PAGE. Radiolabeling reaction was performed in a total volume of 10 µL using 100 pmol of oligonucleotide carrying a free 5′-OH group. The reactions were incubated at 37 °C for 1 h in 1× PNK buffer A  in the presence of 5 µCi γ-32P-ATP and 5 U of T4 PNK. The labeled product was purified by PAGE.Acceptor substrates were synthesized as 5′-hexynyl RNA and labeled with fluorescein using CuAAC (click) chemistry or synthesized with free 5′-OH and labeled with 2, 5 mM DTT, 0.1 mM spermidine) in the presence of 1 mM ATP using 10 U of T4 PNK. After incubation at 37 °C for 4 h, the oligonucleotides were precipitated and used for the 5′-adenylation reaction without further purification. Then, 30 µL of a solution containing 100 mM AMP-imidazolide  and 50 etry see .2 and 10 mM DTT were added to yield a total reaction volume of 30 µL. After 6 h incubation at 37 °C, the ligation product was purified by PAGE.First, 1.5 nmol 5′-phosphorylated N40-DNA-pool (D1) was annealed to 1.8 nmol donor RNA (R1) , and 1 mM ATP, T4 RNA ligase (0.5 U/μL), 50 mM Tris-HCl pH 7.5, after which 10 mM MgCl32P-labeled size standard was generated by two ligation reactions. First, R2 (40 µM) and R1 (40 µM) were ligated by T4 DNA-ligase using a DNA-splint . After annealing  of the three oligonucleotides, 40 mM Tris-HCl pH 7.8, 10 mM MgCl2, 10 mM DTT, 5 mM ATP, and 0.5 U/µL T4 DNA ligase were added. The reaction mixture was incubated at 37 °C for 16 h and the ligation product was purified by PAGE. Second, the RNA thus generated was ligated to 5′-32P-phosphorylated D1 by T4 RNA-ligase as described above.The 32P-labeled and 450 pmol unlabeled D1-R1 ligation product were annealed  with 1 nmol R2 in 28 μL 1× Annealing buffer . Then, 8 µL 5× reaction buffer  were added, and the reaction was started by the addition of 20 mM MgCl2 and 20 mM MnCl2 to yield a final reaction volume of 40 µL. The reaction mixture was incubated overnight (15 h) at 37 °C, followed by PAGE purification, in which the band next to the size standard was excised and extracted. The reaction time was reduced to 5 h in round 8, and 1 h from round 9 onwards.For the first selection round, 50 pmol 2, and 20 mM MnCl2 at 37 °C. After 4 h incubation, the cleavage product was purified by PAGE and submitted to PCR amplification.To favor the enrichment of DNAzymes catalyzing the formation of native 3′-5′-phosphodiester linkages, an additional selection pressure was introduced starting from round 6. The PAGE-purified ligation products were cleaved by the 8-17 deoxyribozyme (D4), upon incubation with 10 µM 8-17 deoxyribozyme (D4) in 40 mM Tris-HCl, pH 7.5, 150 mM NaCl, 20 mM MgCl2, pH 8.3). The final reaction volume was 100 µL. Amplification was performed under the following conditions: 94 °C/5 min, 94 °C/30 s, 48.3 °C/30 s, 72 °C/30 s with 9 additional cycles from step two and final 72 °C/4 min.For amplification of the enriched pool, the isolated oligonucleotides were mixed with 2 µM 5′-phosphorylated forward primer (D2), 0.5 µM reverse primer (D3), 0.2 mM dNTPs, and 5 U Taq DNA-polymerase in the presence of 1× Taq-buffer , and 625 nM reverse primer (D3). All other reaction conditions remained identical to the first PCR amplification.After phenol/chloroform/isoamyl alcohol (PCI) extraction, 1 µL of the 10-cycle PCR product was used as input for a 30-cycle PCR reaction (40 µL), using 0.2 mM of a dNTP mixture containing α-2, and 10 mM DTT were added, and the reaction mixture was incubated at 37 °C for 3 h. The ligation product was purified on PAGE and submitted to the next selection round.The asymmetric double-stranded PCR product was separated into single strands by PAGE. The shorter strand, representing the enriched, 5′-phosphorylated DNA-library, was extracted and annealed  with 10 µM donor RNA R1. Then, 1 mM ATP, T4 RNA-Ligase (0.5 U/μL), 50 mM Tris-HCl pH 7.5, 10 mM MgClE. coli. The plasmids of 40 randomly picked colonies were isolated and tested for activity. The 15 most active clones were sequenced using Sanger sequencing, and the results are listed in After 11 rounds of selection, the enriched library was cloned using TOPO-TA cloning and transformation into 32P-labeled acceptor oligonucleotide were annealed  with 60 pmol 5′-adenylated donor oligonucleotide and 40 pmol DNAzyme. Then, 2 µL 5× reaction buffer  were added, and the reaction was started by addition of the respective divalent metal ion stock solution to a final reaction volume of 10 µL . The reactions were incubated at 37 °C, after which 1 µL aliquots were taken after certain time points, and the reaction was quenched immediately by the addition of 4 μL of stop solution and freezing. Half of each time point sample was analyzed by denaturing PAGE, and band intensities were quantified by phosphor imaging or by fluorescence imaging using blue epi illumination and 530/28 nm emission filter. The yield versus time data were fit to (fraction ligated) = Y*(1 − e−kt), where k = kobs and Y = max final yield.In 7 μL 1× annealing buffer , 10 pmol 5′-flourescein or 2 to yield a final reaction volume of 20 µL. After overnight incubation (22 h) at 37 °C, the ligation product was purified on denaturing PAGE and used for the following experiments.The ligation products were prepared by annealing 200 pmol of each RNA (R1 and R2) with 200 pmol of the respective DNAzyme in 14 μL 1× annealing buffer . Then, 4 µL of 5× reaction buffer  were added, and the reaction was started by addition of 40 mM MnClIn 5 µL of 50 mM Tris-HCl (pH 7.5), 5 pmol ligated RNA was digested with 0.5 U RNase T1 for 30 s at 37 °C. The reaction was quenched by the addition of 7.5 μL stop solution and placing the reaction mixtures on ice.First, 5 pmol ligation product was heated at 95 °C in 5 µL 50 mM NaOH. After 2.5 min, the reaction was quenched by adding 7.5 μL stop solution and placing the sample on ice. All samples were resolved on denaturing PAGE and then subjected to fluorescence imaging using blue epi illumination and a 530/28 nm emission filter.2 and 20 mM MnCl2 in 40 mM Tris-HCl, pH 7.5, 150 mM NaCl in a final volume of 5 µL. The reaction mixture was incubated at 37 °C for 5 h, quenched with stop solution, and analyzed by PAGE next to an alkaline hydrolysis and T1 digestion ladder.First, 5 pmol ligation product was annealed with 50 pmol 8-17 deoxyriboyzme (D4); then, it was incubated with 20 mM MgCl2, in 50 mM CHES buffer, pH 9.0, 150 mM NaCl, 2 mM KCl, in a final volume of 10 µL at 37 °C for up to 7 h. Then, 1 µL aliquots were removed at 30 min and then at every hour, mixed with 4 µL stop solution, and analyzed by PAGE.First, 10 pmol ligation product was annealed with 100 pmol complementary DNA (D5) and incubated with 100 mM MgCl2 to yield a final reaction volume of 10 µL. After overnight incubation (22 h) at 37 °C, the reaction mixture was precipitated and submitted to the labeling reaction without further purification.For RNA ligation, 40 pmol of DNAzyme SC9, 10 pmol 5′-fluorescein-labeled R2, and 60 pmol R1 were annealed  in 7 μL 1× annealing buffer . Then, 2 µL of 5× reaction buffer  were added, and the reaction was started by the addition of 40 mM MnClN6-(6-amino)hexyl-ATP-ATTO-550 and 40 mM MgCl2. An annealing step  was performed prior to the addition of MgCl2 and the ATP substrate. The reaction mixture was incubated at 37 °C. Then, 1 µL aliquots were taken after certain time points, and the reaction was quenched immediately by the addition of 99 µL TEN buffer. All time point samples were precipitated and dissolved in 5 µL stop solution. Half of each time point sample was analyzed by denaturing PAGE, and band intensities were quantified by fluorescence imaging using blue epi illumination (530/28 nm emission filter) and green epi illumination (605/50 nm emission filter).Half of the ligation sample was mixed with 60 pmol of the corresponding FH14 ribozyme in 5 μL of ribozyme reaction buffer , including 200 µM of


G93A mice exhibit a KCC2-related alteration in chloride homeostasis, i.e., the EGABAAR is more depolarized than in WT littermates. Here, using immunohistochemistry, we found that the SOD1G93A lumbar spinal cord is less enriched with 5-HT descending fibres than the WT lumbar spinal cord. High-performance liquid chromatography confirmed the lower level of the monoamine 5-HT in the SOD1G93A spinal cord compared to the WT spinal cord. Using ex vivo perforated patch-clamp recordings of lumbar MNs coupled with pharmacology, we demonstrated that 5-HT strongly hyperpolarizes the EGABAAR by interacting with KCC2. Therefore, the deregulation of the interplay between 5-HT and KCC2 may explain the alteration in chloride homeostasis detected in prenatal SOD1G93A MNs. In conclusion, 5-HT and KCC2 are two likely key factors in the presymptomatic phase of ALS, particular in familial ALS involving the SOD1G93A mutation.Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron degeneration and muscle paralysis. The early presymptomatic onset of abnormal processes is indicative of cumulative defects that ultimately lead to a late manifestation of clinical symptoms. It remains of paramount importance to identify the primary defects that underlie this condition and to determine how these deficits lead to a cycle of deterioration. We recently demonstrated that prenatal E17.5 lumbar spinal motoneurons (MNs) from SOD1 Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a rapidly progressive neurodegenerative disease that targets motor neurons. It is one of the most common and most devastating neurodegenerative diseases. In 90% of cases, ALS is idiopathic and sporadic (sALS). Only 10% of ALS cases are familial in origin (fALS) and are inherited in an autosomal dominant manner. Approximately 20 genes are associated with ALS, with the most common causes of typical ALS being mutations in SOD1, TARDBP, FUS, and C9orf72 . The serThe central neurons that produce the biogenic amine serotonin  form a small population of 20–30,000 neurons in the rat central nervous system (CNS)  called t−]i in lumbar motoneurons (MNs) and rendering GABA unable to provide excitation [−]i during the course of CNS development has been extensively studied, and it has been shown that it mainly relies on the differential ontogenic expression of Na+-K+-2Cl− cotransporter isoform1 (NKCC1), which takes up chloride ions i) in E17.5 SOD1G93A lumbar spinal MNs because of a specific reduction in KCC2 efficacy [2AR hyperpolarizes the reversal potential of inhibitory postsynaptic potentials in rat spinal MNs through an increase in KCC2 function [G93A mice than in the lumbar SC of WT littermates. We also show that exogenous applications of 5-HT exert potent hyperpolarizing effects on the ECl, assessed here as the reversal potential of GABAAR (EGABAAR). This leads to the conclusion that the lack of 5-HT innervation in the prenatal SC may be responsible for the alteration in chloride homeostasis in SOD1G93A spinal lumbar MNs and may explain the neurodevelopmental features of ALS that we recently described in the SOD1G93A mouse [Using the SOD1patients , we prevefficacy . Interesfunction . In the 3A mouse .p > 0.05, Mann-Whitney test) between the WT (VH: 2.21 ± 0.21 AUs (arbitrary units), IA: 3.0 ± 0.25 AUs, CGM: 1.59 ± 0.22 AUs, n = 11) and SOD1G93A  mice , IA , and CGM   mice B4–B6. Hiumbar SC C1–C6. Thvical SC D1 and they test) D2.G93A and WT lumbar SCs. This HPLC analysis revealed that the 5-HT content was significantly reduced in SOD1G93A mice  compared to WT littermates    analysis of the spinal content of endogenous 5-HT, a biogenic amine, in the SOD1ey test) .GABAAR is more depolarized in E17.5 SOD1G93A lumbar MNs than in the E17.5 lumbar MNs of WT littermates due to KCC2 downregulation [GABAAR. In MNs voltage-clamped at a holding potential of −70 mV  , dropped to −67.2 ± 5.2 mV in the presence of 10 µM 5-HT  (G93A mice and those from WT littermates (83.7 ± 10.4 pA (n = 18) and 92.3 ± 17.4 pA (n = 15) in SOD1G93A and WT mice, respectively, p > 0.05, Mann-Whitney test). Finally, we quantified the intensity of EGABAAR hyperpolarization in both genotypes and did not find any difference (−14.9 ± 1.7 mV (n = 13) and −19.5 ± 3.1 mV (n = 13) in SOD1G93A and WT mice, respectively, p > 0.05, Mann-Whitney test).In a recent report, we showed that the Egulation . Becausegulation , we testolarized A1. Our qGABAAR modulation by 5-HT by performing a series of experiments in which 5-HT antagonists that block at least 5-HT2R (10 µM methysergide + 10 µM ketanserine) were applied in addition to exogenous 5-HT (10 µM). This pharmacological protocol revealed that both the inward current and EGABAAR hyperpolarization were reversed when the 5-HTR antagonists were bath-applied in the presence of 5-HT  and reversed to −51.4 ± 5.2 mV when the 5-HTR antagonists were applied  and reversed to −57.2 ± 6.1 mV when the 5-HTR antagonists were applied  and reversed to −47.3 ± 3.4 mV when the KCC2 blocker was added  and reversed to −37.0 ± 4.4 mV when VU0240551 was applied  involved in the early hyperpolarization effect of 5-HT on MNs.Our study using methysergide and ketanserin confirms that the effects of exogenous 5-HT on inward currents are related to 5-HTRs. The involvement of 5-HTs likely ,26,27. Afunction ,29, althn on MNs . The cocd 5-HT7R ,32. Thisd 5-HT7R  and high2B/C receptor in SOD1 (G37R) mice [G93A ALS mouse model.The involvement of 5-HT in ALS disease progression has been questioned ,35. The in SOD1 GR mice [37R) mice , suggesthttp://jaxmice.jax.org/strain/002726.html). Heterozygous B6SJL-TgN(SOD1-G93A)/1Gur/J mice (referred to as SOD1G93A mice in the present report) were maintained by crossing heterozygous transgene-positive male mice with B6SJL F1 hybrid females . The gestational period of SOD1G93A mice was ~18.5 days, and embryonic day 0.5 (E0.5) was defined as the day after mating. Experiments were performed on E17.5 foetuses, i.e., foetuses collected 1 day before birth.All procedures were carried out in accordance with the local ethics committee of the University of Bordeaux  and European Committee Council directives. All efforts were made to minimize animal suffering and reduce the number of animals used. B6SJLTgN(SOD1-G93A)/1Gur/J mice expressing the human G93A Cu/Zn superoxide dismutase (SOD1) mutation (Gly93→Ala substitution) were obtained from the Jackson Laboratory  (2 and 5% CO2 mixture. The composition of the aCSF was (in mM): 114.5 NaCl, 3 KCl, 2 CaCl2-2H2O, 1 MgCl2-6H2O, 25 NaHCO3, 1 NaH2PO4-H2O, and 25 D-glucose, pH 7.4, osmolarity 307 mosmol/kg H2O). The selected foetuses were then decapitated, and their tails were preserved for subsequent genotyping. The SC was opened dorsally, and the meninges were removed. The preparations were placed in a recording chamber  and kept open under a nylon mesh. This exposed the MNs and made them accessible to the patch-clamp electrode . The SC preparations were placed in a recording chamber and continuously superfused (~1.5 mL·min−1) with oxygenated aCSF. All experiments were carried out at constant temperature (30 °C). The experiments were performed blindly without knowledge of the genotype of the animals. The foetuses were genotyped at the genotyping facility of Neurocentre Magendie (Bordeaux Neurocampus). Genotyping was performed by standard PCR of mouse tail samples using established primers and a protocol provided by the Jackson Laboratory .Pregnant mice were sacrificed by cervical dislocation. A laparotomy was performed, and the foetuses were removed after cutting the uterine muscle. Foetuses of either sex were removed from their individual embryonic sacs and transferred to cooled artificial cerebrospinal fluid (aCSF) oxygenated with a 95% OG93A and WT embryos were fixed in 4% paraformaldehyde (PFA) for 2 h at room temperature. They were rinsed three times with 0.1 M phosphate-buffered saline (PBS) and then cryoprotected in 15% sucrose for 24 h followed by 30% sucrose for another 24 h. After they were placed in Tissue-Tek®  and frozen, the samples were sliced at the cervical 5–6 (C5–C6) and lumbar 4–5 (L4–L5) levels using a Leica 3050 S Cryostat. The sections (20 µm thick) were affixed to gelatinized slides and preserved at −25 °C until use. Each slide was rinsed three times with 0.1 M PBS, blocked with solution containing 2% bovine serum albumin (BSA), and then incubated for 48 h with primary antibodies prepared in PBST (1% Triton and 0.2% BSA). We processed the sections with an anti-serotonin (5-HT) antibody  secondary antibody (A-11008)  and then rinsed with 0.1 M PBS. After rinsing, the slides were mounted with an anti-fade reagent  and stored at 4 °C in darkness until observation under a confocal microscope. For better clarity, inverted images are presented.Immunohistochemistry was performed on coronal sections of lumbar SCs prepared as follows. Lumbar SC samples from E17.5 SOD1city see ). After https://imagej.nih.gov/ij/).Samples were visualized in the laboratory with a BX51 Olympus Fluoview 500 confocal microscope. Serial optical sections (0.2 μm thick) were obtained using a 60× oil-immersion objective. Lasers were selected according to the wavelength required for visualization. The global 5-HT staining density, which was used as an approximate estimation of fibre labelling, was assessed using ImageJ  using a two-stage vertical microelectrode puller . The patch electrode resistances ranged from 3 to 5 MΩ. All recordings were made with an Axon Multiclamp 700B amplifier . The data were low-pass filtered (2 kHz) and acquired at 20 kHz on a computer via an analog-to-digital converter  and data acquisition software .Motorized micromanipulators  were used to position the patch-clamp electrode on a visually identified lumbar 4–5 (L4–L5) MN using a CCD video camera (Zeiss Axiocam MR). The recorded MNs were located in the lateral column  and wereGABAAR assessment, gramicidin perforated patch-clamp recordings were conducted. For perforation, a gramicidin stock solution (Millipore Sigma) was dissolved in DMSO (Millipore Sigma) at a concentration of 10 μg·ml−1 and diluted to a final concentration of 10–20 μg·ml−1 in intracellular medium composed of (in mM): 130 KCl, 10 HEPES, 10 EGTA, and 2 MgATP, 284 mosmol/kg H2O, adjusted to pH 7.4 using 1 M KOH. The gramicidin stock solution and dilutions were prepared <1 h before each experiment. Pipette tips were filled with ~3 μL of filtered intracellular medium and subsequently back-filled with gramicidin solution. The GABAAR-specific agonist isoguvacine  was pressure ejected  via a puff pipette placed in the vicinity of the motoneuron somata with a PicoSpritzer II  driven by a programmable Master 8 Stimulator/Pulse Generator . Isoguvacine application was repeated at different membrane voltages, allowing the assessment of the reversal potential of evoked GABAAR-related currents. The EGABAAR was considered the ECl value because Cl− ions are mainly involved in GABAAR currents in foetal MNs [GABAAR was calculated to be 1.5 mV. Thus, to confirm that the perforated-patch configuration had not ruptured during the experiment, repetitive EGABAAR tests were performed. Measurements that produced an EGABAAR close to 0 mV were considered whole-cell recordings, and the cells were discarded. The Clampex membrane test was used to monitor the input resistance (Rin) and membrane capacitance (Cm); a −60 mV holding membrane potential and 5 mV steps  were chosen. The reversal potential of GABAAR (EGABAAR) was calculated by a linear fit . Changes in the current amplitude were determined using Clampfit 10.3 (Molecular Devices).For Eetal MNs ,40). MeaG93A and WT embryos were quickly removed, weighed, and placed in a tube at −80 °C for biochemical analysis. On the day of the analysis, tissue samples were homogenized using sonication in 0.1 N perchloric acid (HClO4) and centrifuged at 13,000 rpm for 30 min at 4 °C. The tissue content of the monoamine 5-HT was measured by a sensitive HPLC coupled to electrochemical detection (ECD) system according to previous protocols with some modifications [2PO4, 0.1 disodium EDTA and 2 octane sulfonic acid plus 7% methanol, adjusted to a pH of 4.2 with orthophosphoric acid and filtered through a 0.22 µm Millipore filter. 5-HT was detected using a coulometric cell  coupled to a programmable detector . The potential of the electrodes was set at +350 mV for oxidation and −270 mV for reduction. Output signals from the oxidation channel were acquired on a computer via an analog-to-digital converter  and data acquisition software . We measured the height of the signals corresponding to the time of elution of 5-HT and calculated the concentration in the sample using an appropriate calibration curve. Each day, standard solutions were injected before spinal cord samples were analysed to verify the HPLC system [Lumbar samples from E17.5 SOD1ications ,41. AliqC system . Under tAR responses were isolated by using a cocktail of drugs containing 0.2 µM tetrodotoxin , 4 mM kynurenic acid (Millipore Sigma), 10 µM (+)-tubocurarine (Millipore Sigma), 5 µM dihydro-β-erythroidine hydrobromide , and 3 µM strychnine (Millipore Sigma), which blocked voltage-dependent Na+ action potentials and glutamate, cholinergic, and glycinergic inputs to MNs, respectively. VU0240551  (10 µM) was applied to specifically block KCC2. Serotonin  was purchased from Millipore Sigma. Methysergide maleate (10 µM) and ketanserin tartrate (10 µM) (Bio-techne) were used to evaluate the involvement of 5-HTRs in the effect of 5-HT. Combined, these antagonists are known to efficiently block 5-HT2A-CR [In the gramicidin perforation experiments, GABA-HT2A-CR ,32.n is the number of MNs or SCs used in the analysis . p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) were considered significant. The significance of differences between two data sets was assessed by the Mann-Whitney test or Wilcoxon matched-pairs signed rank test for nonparametric data.GraphPad Prism 7 software was used to analyse all the data. The results are presented as the means ± the standard errors of the mean (SEMs).


A compound is named Castacrenin E and is produced by Castacrenin D oxidation. Castacrenin D is a vescalagin with an additional aromating ring to the C-1 through a C-C bond. These compounds are not only found under laboratory conditions but also in commercial oak wood representing different toasting methods and sizes. Their levels are conditioned by oak wood dimensions and toasting degree. The wood pieces with the smallest size present almost two times more compounds A and B. Moreover, the compound B is the only compound to be present in medium toasting temperatures of the smallest wood pieces. Both of them can influence either astringency sensation or bitterness taste.In the courses of studies on ellagitannin changes during oak wood toasting, two C-glycosidic ellagitannins were isolated from the french oak wood for the first time. These two compounds exhibited [M−H] C‒glycosidic ETs are characterized by the open-chain form of their glucose core and the presence of a C‒C linkage between the C1 of the open-chain glucose and C2 of the galloyl derived moiety of a 2,3‒HHDP (hexahydroxydiphenoyl) or a 2,3,5‒NHTP (nonahydroxyterphenoyl) unit  and solvent B [methanol/formic acid (99.6/0.4)]. The gradient elution was 0–3% B in 5 min, 3–12% from 5 to 35 min, and 12–100% from 35 to 40 min with a flow rate fixed at 1 mL/min and a detection wavelength maintained at 280 nm. Each ellagitannin was quantified via its molecular ion, based on external calibration curve of castalagin standard and the results are expressed as equivalents of castalagin.The above fraction obtained enclosing the eight main ellagitannins was dry heated in a laboratory oven for 60, 120, and 180 min at 220 °C. Neither the humidity nor the oxygen were controlled. After cooling, it was additionally fractionated on a C-18 column before being injected in UPLC-HRMS (High Resolution Mass Spectrometry).2O) and B (MeOH), the rate of flow was 300 μL/min, and eluent B varied as follows: 0 min,1%; 3 min, 5%; 7 min, 8%; 25 min, 12.5%; 30 min, 15%; 35 min, 20%; 40 min, 99%; 42 min, 99%; 46 min, 1%. The injection amount was 20 μL. This UPLC was connected with an HRMS, described below.The UPLC platform Agilent 1290 Infinity included a binary pumping system (1290 Infinity), a compartment thermostat column (1290 Infinity) autosampler (1290 Infinity) coupled with Diode-Array Detector (1290 Infinity). An Eclipse Plus C18 column  was employed for the chromatographic separation. UV detection was carried out at 220 and 270 nm. The mobile phases employed were solvent A . For a simple identification the fragmentor voltage was fixed at 150 V and the collision energy used for MS2 varied between 10% and 60% according to the compounds. The data obtained were analyzed by MassHunter Qualitative Analysis.UPLC was connected with an ESI-Q-TOF-MS (Agilent 6530 Accurate Mass). Mass acquisitions were realized for 42 min in negative HRMS ionization mode at 3 kV. The vaporizer temperature of the source was established at 320 °C, the capillary temperature at 350 °C, the nitrogen gas at 300 °C with a 9 L/min flow, the nitrogen sheath gas at 350 °C with a flow rate of 11 L/min, the auxiliary gas at 18, and the sweep gas at 0 (arbitrary units). The capillary voltage and the skimmer voltage were set at 3500 and 65 V, respectively. A mass range of 100–3000 Th was acquired in full scan MS mode with a mass resolution of 13,446 (v/v). Sodium  propionate (TSP-d4) served as an internal reference for proton chemical shifts. NMR experiments were recorded at 293 K. Molecule assignments were achieved by two-dimensional 1H-1H COSY, 1H-1H TOCSY, 1H-1H ROESY, 1H-13C HSQC, and 1H-13C HMBC experiments. All 2D experiments were carried out with 2048 data points × 400 increments and a spectral width of 8417 Hz and 33,209 Hz in proton and carbon dimension, respectively, and 1.5 s for relaxation delay. Mixing time was 300 ms and the spinlock time was 100 ms for ROESY and TOCSY experiments, respectively.All 1D and 2D NMR experiments were carried out with a Bruker Avance 600 MHz NMR spectrometer  operating at 600.3 MHz and equipped with a 5 mm TXI probe. Data were processed using TOPSPIN software version 3.2 . All NMR spectra were acquired in methanol-d4/D2O 1:1 (2O) and B (MeOH), each solvent containing 0.025% of TFA. The flow rate was set at 2.5 mL/min, and eluent B varied as follows: 1–3% during 25 min; 3–100% during 10 min, 100% during 5 min, 100 to 0% during 2 min, and 0% during 2 min. The injection volume was 200 μL. The data obtained were analyzed by LC Open Lab. Once ellagitannins compounds were isolated, they were characterized by LC-MS/MS and 1D/2D-NMR spectroscopy.Semipreparative HPLC was realized with an Agilent system having a quaternary pump (1260 Infinity), a compartment thermostat column (1290 Infinity), a sample injector (1260 Standard Autosampler), and a diode array detector (1260 DAD VL+). Chromatographic separation was carried out on a Prontosil column C18 column . UV detection was set at 280 nm. The mobile phases were A , 1 h and 30 min at 165 °C; light plus (LT+), 2 h at 170 °C; medium (MT), 2 h at 180 °C; medium plus (MT+), 2 h and 25 min at 190 °C; Special 2 h at 180 °C and heavy toast (HT), 2 h and 30 min at 200 °C. In the case of Special, a watering process happened just 30 min before the end of the toasting process. Two types of oak wood pieces were used, the oak chips (3–25 mm) and thin oak wood pieces (2–4 mm). Untoasted (UN) and fresh oak wood pieces were also employed for the experiment. UN wood pieces were air-dried seasoned in the seasoning park.The sensory evaluation was organized in standardized rooms . For the tasting sessions, ISO black wine tasting glasses  were used. To initiate the judges with bitterness taste and astringency sensation, a training was performed as previously described crenin E . Since in family .C-glycosidic ellagitannin (H 4.66 (Cp-1) in the 1H-NMR spectrum and a carbonyl δC 196.9 (Cp-4), an olefinic δC 138.4 (Cp-2) and two aliphatic carbon signals δC 48.8 (Cp-1) and 90.9 (Cp-5) in the 13C-NMR spectra were similar to those due to the cyclopentenone ring of Mongolicain A, which is complex tannin, isolated from Querçus species a et al. . UnfortuCastacrenin E was probably generated by oxidation of Castacrenin D. Castacrenin D is a vescalagin with an additional aromating ring  to the C-1 through a C-C bond . During The compounds A and B found in this experimental study were further searched in commercial oak wood expressing various toasting methods and sizes. Their levels were dependent on oak wood size and toasting intensity . DehydroMore precisely, in the larger oak wood pieces Castacrenin E varies between 0.40 and 1.62 mg/g of oak wood for untoasted and light toasted oak chips, respectively. The B compound is formed only with the toasting and its concentration stays around 1.50 mg/g for both light toastings in the oak chips. In the thinner oak wood pieces, Castacrenin E ranges from 0.48 to 2.15 mg/g for untoasted and light toasted modalities. Compound B exhibits its largest concentration (2.65 mg/g) in light toasted pieces whereas in untoasted presents its minimum (0.43 mg/g) and its intermediate concentration appears in medium toasted (1.38 mg/g) thin oak wood pieces. Moreover, the compound B is the only compound to be present in medium toasting temperatures of the smallest wood pieces.1H NMR spectroscopy; 1.6 and 1.8 mg/L of Castacrenin E and compound B, respectively, were tested in aqueous solution and in model wine solution for the taste/sensation identification. From the triangle test, 13 judges found significant differences in aqueous solution with the addition of Castacrenin E (p ≤ 0.01), but no significant differences were found when this compound was added in model wine solution (p ≥ 0.05). On the contrary, significant differences were perceived by 12 judges with the addition of compound B in model wine solution (p ≤ 0.01). When the compound B was added in aqueous solution no significant difference was noticed by the panel neither for astringency nor bitterness taste (p ≥ 0.05) (p ≤ 0.05). The results obtained indicate the importance of matrix for the sensation of astringency and bitterness. Previous studies have shown that ethanol decreases astringency sensation


Using mortality as an endpoint in studies comparing different reperfusion strategies in STEMI requires increasingly large sample sizes to test advances with existing therapy. Thus, there is rising interest in surrogate endpoints allowing a reduction in sample size. One of those measures is myocardial salvage which can be assessed retrospectively by T2-weighted and delayed enhancement images as shown in animal studies . CurrentTo assess the reproducibility of myocardial salvage measurements in a group of patients with acute myocardial infarction scanned at two subsequent days with a second contrast agent injection.In 20 patients with acute STEMI transverse electrocardiographically gated T2-weighted triple-inversion-recovery images were acquired to assess the area at risk and delayed enhancement (DE) images using a 3D inversion recovery gradient echo sequence assessed the infarct size. All patients underwent a repeated measurement the subsequent day starting with new scout images and using the same dose of the contrast agent. Area at risk and infarct size were determined as the percentage of left ventricular mass (%LV) by two independent observers and subsequently the myocardial salvage index was calculated as area at risk-infarct size/area at risk. In addition extent of microvascular obstruction was assessed. Inter-, intraobserver variabilities and reproducibility data were calculated according to standard definitions and compared by the method of Bland and Altman. Image quality was assessed by a score ranging from 0–4 .The mean difference between percutaneous coronary intervention and MR imaging was 2.8 ± 1.3 days. All images were suitable for analyses with a mean image quality of 3.2 ± 0.6 for DE-images and 2.5 ± 0.6 for T2-weighted-images. The area at risk was as high as 47.4 ± 11.7%LV and the infarct size 20.2 ± 9.6%LV. The corresponding myocardial salvage index was 57.7 ± 14.9 (range 23.4–75.8). In 17 (85%) patients there was evidence of microvascular obstruction. Area at risk difference (bias) between scan I and scan II was 1.2%LV and limits of agreement were ± 7.5%LV. The results for infarct size were 0.5%LV ± 2.6%LV limits of agreement. The resulting bias for myocardial salvage index was -0.3 with limits of agreement of ± 5.0. Intra- and interobserver variability was low with a mean bias of -0.7 (limits of agreement ± 4.6) and -0.0 (limits of agreement ± 4.7), respectively.The multimodal MRI approach is an excellent tool for myocardial salvage assessment with excellent reproducibility in acute STEMI. It has therefore the potential to serve as a surrogate endpoint to uncover advantages of new reperfusion strategies and allows a further reduction in the sample size of clinical trials. The previously shown excellent reproducibility for infarct size measurement could be confirmed in this trial.


Chemokines are important protein-signaling molecules that regulate various immune responses by activating chemokine receptors which belong to the G protein-coupled receptor (GPCR) superfamily. Despite the substantial progression of our structural understanding of GPCR activation by small molecule and peptide agonists, the molecular mechanism of GPCR activation by protein agonists remains unclear. Here, we present a 3.3-Å cryo-electron microscopy structure of the human chemokine receptor CCR6 bound to its endogenous ligand CCL20 and an engineered Go. CCL20 binds in a shallow extracellular pocket, making limited contact with the core 7-transmembrane (TM) bundle. The structure suggests that this mode of binding induces allosterically a rearrangement of a noncanonical toggle switch and the opening of the intracellular crevice for G protein coupling. Our results demonstrate that GPCR activation by a protein agonist does not always require substantial interactions between ligand and the 7TM core region. Chemokine receptors are GPCRs involved in immune responses and regulated by small protein ligands known as chemokines. A structural study of the human CCR6/CCL20–Go complex reveals that CCL20 binds in a shallow extracellular pocket, and suggests that activation of CCR6 by CCL20 binding involves an allosteric effect on a noncanonical toggle switch. According to the spacing of the conserved N-terminal cysteine residues, chemokines are structurally grouped into the CC, CXC, CX3C, and C chemokines2. Through chemokine receptors, chemokines can regulate leukocyte migration, activation, differentiation, and survival during the immune response, and therefore are involved in numerous diseases including inflammation, cancer, and autoimmune disorders2. To date, three chemokine–receptor complex crystal structures have been reported5. However, all chemokine ligands in the crystal structures are either an antagonist or inverse agonists to the co-crystallized receptors. On the other hand, the structural elucidation of agonist-bound GPCRs coupled to G proteins has provided a tremendous amount of insight into GPCR activation by small molecule or peptide ligands6. Nonetheless, the molecular basis of GPCR activation by protein agonists remains elusive. Thus, structural elucidation of a cognate chemokine–chemokine receptor complex is not only important for understanding the molecular basis of chemokine receptor activation by native agonists, but it could also expand our knowledge of the mechanism of GPCR activation by ligands of a distinct chemical nature.Chemokine receptors are GPCRs regulated by small protein ligands known as chemokines, which are 8–10 kDa proteins with a globular core structure stabilized by 1–2 conserved disulfide bridges1. It primarily couples through Gi/o proteins upon activation. CCR6 plays an important role on skin and mucosal surfaces under homeostatic and inflammatory conditions, particularly through the recruitment of proinflammatory IL17-producing T-cells to sites of inflammation. Therefore, CCR6 is a potential target for several inflammatory and autoimmune diseases including psoriasis7 and inflammatory bowel disease8. In human and mouse, CCR6 is exclusively activated by the chemokine CCL20, which is known to interact only with CCR67. Intrigued by the unique exclusivity of this chemokine–chemokine receptor pair, we sought to understand the molecular basis of the ligand recognition specificity exhibited by this receptor.CCR6 is a CC chemokine receptor that is selectively expressed in immature dendritic cells and memory T-cellsTo this end, we solved a cryo-EM structure of the human CCR6 in complex with CCL20 and a Go protein. Our structure reveals several interactions that are likely to be important for CCL20-mediated CCR6 activation, supported by previous mutagenesis studies and molecular dynamics simulations. Notably, CCL20 binds to a shallow extracellular pocket of CCR6, in contrast to the deep agonist-binding sites observed in other class A GPCRs. Thus, we propose that CCL20 activates CCR6 by a unique allosteric mechanism.9 to the N terminus of the receptor which comprises 38 amino acid residues before TM1 11 in the complex formation to slow down the dissociation of the final nucleotide-free CCR6/CCL20–Go complex. The complex was formed by incubating all the protein components in the presence of apyrase to remove residual GDP. Subsequently, the complex was subjected to affinity purification using a FLAG-tag engineered to the N terminus of CCR6 12. CCL20 binds to the endogenous pocket located at the extracellular side of CCR6, making substantial interactions with all three ECLs. In addition, the N-terminal residues Y27 to L38 from CCR6 wrap onto the globular core of CCL20, serving as another critical docking site for chemokine binding , which provides additional binding surface for CCL20. Supporting this structural observation, disruption of this disulfide by mutating either C36 or C288 has been shown to impair CCR6 response to CCL20 stimulation13. The N terminus of CCL20 binds in the extracellular crevice of the 7TM core of CCR6, making major interactions with the receptor’s ECL2 and N terminus. Similar to the previously solved chemokine antagonist-bound GPCR structures CXCR4/vMIP-II, US28/CX3CL1, and CCR5/[5P7]CCL55, the CCR6/CCL20 complex reveals a highly extensive binding interface which is not spatially isolated, different than that predicted by the two-site paradigm where two spatially separated contact sites are proposed to mediate chemokine–chemokine receptor interactions15.The cryo-EM map reveals that CCR6 possesses a canonical GPCR topology, consisting of seven transmembrane (7TM) α-helices, three extracellular loops (ECLs), three intracellular loops (ICLs), and a short helix (helix 8) that runs parallel to the membrane Fig. . ECL2 ading Fig. . There i4 and US28/CX3CL3 complexes  of CCR6, interacting with these two side-chains via a hydrogen bond and a salt bridge, respectively. These polar interactions likely direct binding of the N terminus of CCL20 to the 7TM core of CCR6 and therefore could be crucial for CCR6 activation15. In support of this, a dramatic decrease in chemotactic potency was observed for CCL20 variants bearing D5A or D5K mutations15. The extreme N terminus of CCL20 (NH2–AS) binds in close proximity to E19845.51 and K2987.35, both of which are CCR6-specific side-chains  and CCL20 (2–70), both of which exhibited similar potency toward CCR618. Furthermore, an engineered CCL20 variant starting with N3 (3–70) retained chemotactic activity, although with lower potency. Conversely, further truncations beyond the third residue converted the chemokine into a potent antagonist capable of inhibiting full-length CCL20-mediated chemotaxis19. The extended conformation adopted by the N terminus of CCL20 in the complex suggests that the α-amino group of the N-terminal S2 or N3 in the truncated isoforms may also be able to reach down to E19845.51 and form the same salt bridge as full-length CCL20, whereas truncations beyond N3 would prevent the N-terminal residue from reaching this site, converting the chemokine into an antagonist. To test this hypothesis, we performed molecular dynamics simulations of the binding of four CCL20 N-terminal truncation variants to CCR6. In a simulation of over 240 nanoseconds, the N terminus of CCL20 (1–70) remained close to E19845.51, within a distance that facilitated stable salt bridge formation –CCR6 complex. Instead, a new salt bridge was observed between the side-chains of E19845.51 and K2987.35 in CCR6  and several other conserved residues nearby, including Y1253.32 and N2716.52. In both the CCR7 and CCR9 inactive-state structures, hydrogen-bonding interactions are observed between Y3.32, N6.52 and Q6.48, which could stabilize TM3 and TM6 in a close-contact conformation  and K200ECL2 (CCR6), and D33 (CCL20) and R286ECL3 (CCR6). The interaction between E30 and K200ECL2 is specific to the CCR6/CCL20 complex  insect cells (ATCC) using the Bac-to-Bac Baculovirus Expression System (Invitrogen). Sf9 cells were infected at a cell density of 4 × 106 cells/ml with high titer viral stock multiplicity of infection (MOI) of 5.0. At 48 h post infection, cells were collected and resuspended in hypotonic buffer containing 10 mM HEPES, pH 7.5, 10 mM MgCl2, 20 mM KCl, and EDTA-free cOmpleteTM protease inhibitor cocktail (Roche). Cells were lysed by dounce homogenization and membranes were subjected to successive hypotonic and hypertonic membrane washes followed by ultracentrifugation. The washed membranes were resuspended in buffer containing 10 mM HEPES pH 7.5, 10 mM MgCl2, 20 mM KCl, and 40% glycerol and stored at −80 °C for future use. To purify CCR6, the washed membranes were thawed and resuspended in buffer containing 50 mM HEPES pH 7.5, 500 mM NaCl, and 1 mg/ml iodoacetamide (Sigma). Membranes were then solubilized by adding 1% (w/v) lauryl maltose neopentyl glycol , 0.2% (w/v) cholesteryl hemisuccinate  for 4 h at 4 °C. The sample was clarified by ultracentrifugation and the supernatant was batch bound to TALON cobalt-affinity resin (Takara) in the presence of 1 M NaCl and 5 mM imidazole overnight at 4 °C. The resin was washed with successively lower concentrations of detergent, and eluted in buffer containing 50 mM HEPES pH 7.5, 500 mM NaCl, 0.003% LMNG, 0.0006% CHS, 300 mM imidazole, and 10% glycerol. The elution fractions were concentrated and desalted on a PD MiniTrap G-25 column  equilibrated in buffer containing 50 mM HEPES pH 7.5, 500 mM NaCl, 0.003% LMNG, 0.0006% CHS, and 10% glycerol. The desalted protein was concentrated, flash-frozen in liquid nitrogen, and stored at −80 °C for future use.Human wild-type CCR6 from S2 to M374 was cloned into a pFastBac1 vector (Invitrogen) with a haemagglutinin (HA) signal sequence followed by a N-terminal FLAG-tagged BRIL fusion and a C-terminal 10 × His-tag. The CCR6 protein was expressed in Spodoptera frugiperda 36 was inserted between calmodulin and CCL20 . The construct was ligated into pET29b and transformed into Origami 2 cells (Novagen). Cultures derived from single colonies were grown in Luria–Bertani medium supplemented with 50 µg/ml kanamycin and 12.5 µg/ml tetracycline to mid-log phase. Protein expression was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside for 18 h at 16 °C. Cells were resuspended in chilled lysis buffer consisting of 20 mM HEPES pH 7.5, 100 mM NaCl, and 10 mM imidazole and lysed by three passes through a microfluidizer (Microfluidics). Insoluble cellular material was pelleted by centrifugation, and the clarified supernatant was applied to a prepacked nickel-NTA column  and washed extensively with lysis buffer supplemented with 500 mM NaCl and 40 mM imidazole. CCL20 was eluted in a linear gradient of 400 mM imidazole, and elution fractions were pooled and digested with TEV protease overnight at 20 °C. After digestion, the pooled elution was dialyzed with buffer consisting of 50 mM sodium acetate pH 4.5 and 150 mM NaCl, during which a white precipitate formed. The dialyzed sample was clarified by centrifugation and applied to a HiTrap S column  equilibrated in buffer containing 20 mM sodium acetate pH 4.5 and 150 mM NaCl. The sample was washed extensively and eluted in a linear gradient of 20 mM sodium acetate pH 4.5, 1 M NaCl. The purified CCL20 was buffer exchanged into 20 mM HEPES pH 7.5, 150 mM NaCl using a 3 kDa centrifugal filter (Amicon). The protein was concentrated and stored at −80 °C for future use.CCL20 expression and purification was facilitated by an N-terminal calmodulin (Origene) fusion to stabilize CCL20 during bacterial expression1γ2 was expressed as a heterotrimer and purified using an N-terminal 6× His-tag fused to miniGo10. Individual constructs for each protein were cloned into a pFastBac1 vector and viruses were prepared using the Bac-to-Bac system. ATCC Sf9 cells at a density of 4 × 106 cells/ml were co-infected with the three viruses in a 2:1:1 miniGo:β1:γ2 ratio, and expressed for 48 h. Cells were lysed by dounce homogenization in a hypotonic buffer containing 10 mM HEPES, pH 7.5, 10 mM MgCl2, 5 mM TCEP, 50 µM GDP, and cOmpleteTM protease inhibitor. The membranes were pelleted by ultracentrifugation followed by resuspension in buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM TCEP, 5 µM GDP, 1% LMNG, 0.2% CHS, and cOmpleteTM protease inhibitor for solubilization. The G protein heterotrimer was purified from the soluble fraction by cobalt-affinity chromatography. The heterotrimer was further purified by size exclusion chromatography using a Superdex 200 10/300 column equilibrated in buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 0.003% LMNG, 0.0006% CHS, 5 mM TCEP, 5 µM GDP, and 10% glycerol. The G protein-containing elutions were pooled and stored at −80 °C for future use.The miniGoβ37 was modified to include an mIgκ leader sequence to promote secretion to the media. Virus was generated using a pFastBac1 vector and the Bac-to-Bac system. At 48 h post infection, the media was collected and batch bound to cobalt-affinity resin37. The beads were collected in a gravity-flow column and washed with high-salt buffer consisting of 20 mM HEPES pH 7.5, 500 mM NaCl, and 20 mM imidazole, and followed by a wash with low-salt buffer consisting of 20 mM HEPES pH 7.5, 100 mM NaCl, and 20 mM imidazole. The protein was eluted in buffer containing of 20 mM HEPES pH 7.5, 100 mM NaCl, and 250 mM imidazole, and further purified by size exclusion chromatography using a Superdex 200 10/300 column equilibrated in buffer containing 20 mM HEPES pH 7.5 and 100 mM NaCl. Peak fractions were pooled, supplemented with 10% glycerol, and stored at −80 °C for future use.The N terminus of scFv16To form the complex, CCR6, G protein heterotrimer, and CCL20 were mixed in a 1:1.2:10 molar ratio and incubated at 20 °C for 2 h. Apyrase (NEB) was added to remove any residual GDP present in the sample and scFv16 was added to a 1:1.2 CCR6:scFv16 molar ratio. The complex was incubated on ice for 1 h, then batch bound to M2 anti-FLAG affinity resin (Sigma) equilibrated in 20 mM HEPES pH 7.5, 100 mM NaCl for 1 h at 4 °C. The complex slurry was applied to a gravity-flow column (Bio-Rad) and washed with buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 0.001% LMNG/0.0002% CHS. The complex was eluted in elution buffer consisting of 20 mM HEPES pH 7.5, 100 mM NaCl, 0.001% LMNG/0.0002% CHS, 0.2 mg/ml FLAG peptide, and concentrated to 7.5 mg/ml in preparation for cryo-EM analysis.−/Å2/s, with an accumulated dose of 81.0 electrons per Å2.Immediately prior to vitrification, 3 mM fluorinated Fos-Choline-8 (Anatrace) was added to the complex. The freshly purified complex  was applied to freshly plasma-cleaned Quantifoil R1.2/1.3 300 mesh Au holey-carbon grids (Quantifoil) and vitrified using a Vitrobot Mark IV (FEI). The grids were imaged using a Titan Krios (FEI) equipped with a Gatan K2 Summit direct-electron detector (Gatan) and a Gatan Quantum LS Imaging Filter  with a slit size of −20 eV. Data were collected in super-resolution mode at a magnification of ×165,000 and a pixel size of 0.435 Å. A total of 5303 movies were obtained with a defocus range of −0.6 to −2.0 μm. Each movie was recorded for a total of 8 s with 0.16 s per frame. The dose rate was 10.13 e38, and the contrast-transfer function (CTF) for each micrograph was estimated using CTFFIND-4.139. Particle selection, 2D classification, initial model generation, 3D classification, and 3D refinement were carried out in Relion3.0.540. 1,000,382 particles were subjected to reference-free 2D classification and averaging. 600,456 particles from the selected 2D classes were subjected to 3D classification. One class containing 230,450 particles showed detailed features for all subunits and was subjected to masked 3D refinement. Iterative cycles of masked 3D refinement, Bayesian particle polishing, and CTF refinement were performed, yielding a final map with a global resolution of 3.3 Å. The reported resolution is based on the gold-standard Fourier shell correlation (FSC) using the 0.143 criterion. All maps were corrected for the modulation transfer function of the K2 summit direct detector and sharpened by applying a temperature factor estimated during post-processing in Relion. Local resolution was determined using ResMap41.Movie frames were subjected to beam-induced motion correction using MotionCor21B receptor structure solved by cryo-EM (PDB code 6G79), and model fitting was guided by bulky amino acid residues such as Phe, Tyr, and Trp. The N terminus of CCR6 was manually built up to Y27; no density was observed N-terminal of this residue , so no model was built in this region. Coordinates of the engineered Go heterotrimeric protein from the 5-HT1B receptor cryo-EM structure (PDB code 6G79) were rigid-body fit into the map using Chimera42. scFv16 from the cryo-EM structure of the μ-opioid receptor–Gi protein complex (PDB code 6DDE) was also fit into the map in this manner. The crystal structure of CCL20 (PDB code 1M8A) was used to rigid-body fit the chemokine into the map. After docking into the EM density map, iterative manual adjustment and real-space refinement was performed using Coot43. A round of global refinement and minimization in real space was carried out using phenix.real_space_refine in Phenix44, followed by restrained refinement using Refmac545 in the CCP4 software suite46. The final model was visually inspected for fit to the map, and geometry was validated using Molprobity47 from the Phenix software suite. Model overfitting was evaluated through refinement against one cryo-EM half map . FSC curves were calculated between a map generated from the model and the full map, half map 1, and the other half map not used in model refinement . Figures were generated using Chimera 1.1442 and PyMol 2.2.048.The initial template of the CCR6 transmembrane domain was derived from the active serotonin 5-HT49  using CCR6 embedded in pre-equilibrated POPC lipid bilayers and complexed with either WT CCL20 or various N-terminal CCL20 truncations. The final system was neutral and solvated with 150 mM NaCl in the water phase. For each system, MD simulations of 240 ns duration were carried out at 300 K using Desmond50 (Schrödinger) under standard parameters and settings. Statistical analysis of the simulation trajectories was prepared with GraphPad Prism version 8.0.2.MD simulations were performed in MaestroFurther information on research design is available in the Supplementary InformationReporting Summary


Their unique bicyclic system with a quaternary stereocenter, and the potent biological activity exerted by these homotropane alkaloids, make them attractive synthetic targets. This work aims briefly to review the chemical ecology of Adalia bipunctata and the recent methodologies to obtain adaline (1), euphococcinine (2), and N-methyleuphococcinine (3).The 9-azabicyclo[3.3.1]nonane ring system is present in several insect- and plant-derived alkaloids. (−)-Adaline ( Coccinellid beetles contain a variety of defensive alkaloids that makes them unpleasant for various predators . Over 50The fluid toxicity and bitterness, added to the characteristic odor of these insects, have been regarded as a protection against insect or vertebrate predators . Bicycli1) was isolated from ladybird Adalia bipunctata myristic acid incorporated this precursor in (−)-adaline, supporting fatty acid origin for this alkaloid.Individuals of r attack –32. Thispunctata . This dipunctata –34 showeA. bipunctata eggs, once the maternal and paternal aposematic phenotype had the most significant effect on egg traits if compared to the maternal responses to offspring predators. Thus, the phenotype can also contribute to the aposematic signal variation in a ladybird’s early life, in addition to genetic factors. In this way, it should consequently lead to success in the species’ survival.Reflex bleeding is costly due to energy expended in chemical synthesis and fluid loss. Therefore, it is only deployed when other strategies have failed, and the ladybird is in severe danger –36. A ma1 in A. bipunctata. When A. bipunctata was infected by the microsporidian pathogen Nosema adaliae, larval development was significantly delayed. At elevated temperatures, developmental delays caused by infection were reduced, spore counts and infection decreased, and there was an increase in the content of 1 decane (+)-23a in 54% yield. Treatment of (+)-23a with Raney nickel resulted in the cleavage of the sulfinyl group and the N–O bond, providing the bicyclic alcohol 24, which was oxidized with PCC to give (+)-euphococcinine (2). The same protocol was applied to (+)-21b, furnishing the tricyclic adduct (+)-23b, a precursor to (−)-adaline (1).Starting from secondary amines, Murahashi et al. developed a method for preparing homochiral β-sulfinyl nitrones . Accordi2) in 7 steps from piperidine (17), in an overall yield of 2.1%. Specific rotation for (+)-2 was [α]D24 +7.43 ; {lit. [α]D +7.5 , D +6 , nonane system observed in (±)-35.Ikeda et al. prepared azabicycle (±)-nine (2) . The aut34 was prepared from N-boc-pipecolinate, following a methodology previously described by the authors nonane ring present in (±)-euphococcinine (2).Although being racemic, Ikeda's synthesis employed an innovative “6-1). Their approach had as key steps SN2-type alkynylation, activated by lithium ion in a tricyclic N,O-acetal (−)-46, and an olefin metathesis (RCM) of a dialkenylpiperidine (−)-50 for the construction of an azabicyclononane system D28 −11.4  comparable to that found in the literature {[α]D20 −13 (CHCl3), 2 providing alcohols 60a and 60b, after 12 h at −20 ºC. In addition to the good yields in this step, both intermediates were obtained with excellent enantiomeric excesses (97% for R = n-C5H11 and 90% for R = CH3).Treatment of  and 59b . The key60a and 60b were converted to azido ketones 61a and 61b by Mitsunobu reaction, and then these azido ketones underwent cyclization to furnish tetrahydropyridines 62a and 62b after treatment with Ph3P at 20 °C in diethyl ether. 62a and 62b were converted to 63a and 63b through an intramolecular allylic transfer reaction. After several attempts to perform this cyclization, the best conditions found were by using 1.1 equivalents of trifluoromethanesulfonic acid in toluene. After 5 minutes, 1.2 equiv of tributyltin fluoride was added to intermediate A, and at the end of the process, 63a and 63b were obtained after chromatographic purification with 81% yield for 63a and 74% yield for 63b. Finally, the alkenes were oxidized in the presence of osmium tetroxide and potassium periodate, to provide (−)-adaline (1) and (−)-euphococcinine (2).Compounds 1) with good yields and high enantiomeric excess using catalytic asymmetric allylation from commercially available 58. Additionally, intramolecular allylic transfer led to the enatiopure azabicycles. This 6-step sequence was successfully completed and (−)-adaline (1) and (−)-euphococcinine (2) were prepared in overall yields of 11.9% and 15.2%, respectively. Specific rotation measured for (−)-adaline (1) was [α]D20 −12.2 ; {lit. [α]D20 −11 , [2) was [α]20D −6.1 ; {lit. [α]D +6 , for natural (+)-euphococcinine (2) D25 −13.0  {lit. [α]D −13 (CHCl3), D20 +5.4 ; {lit. [α]D +7.5 , D20 = −11.2 ; {lit. [α]D –11 , D20 −6.4 ; {lit. [α]D20 −6.5 , [2) and [α]D20 −12.6 ; {lit. [α]D −13 (CHCl3), [1).In this work, (−)-euphococcinine , } for (−)(CHCl3), } for (−)N-methyleuphococcinine ((±)-3), exploring the use of arylboronic acids as catalysts for C-allylation of unprotected oximes with allyl boronates [Kürti et al. developed racemic oronates .103 was converted to α-tertiary acetal-protected hydroxylamine (±)-104 in the presence of 3,5-difluorophenylboronic acid and diisopropyl allyl boronate (108) in 95% yield after 18 h -105 in 57% yield over two steps.After screening to find the best reaction conditions, oxime ter 18 h . Hydroxy105 was alkylated with excess methyl iodide to form the corresponding ammonium salt (±)-106 in 73% yield. The N–O bridge of ammonium salt (±)-106 was reduced with zinc. The resulting diastereomerically pure amino alcohol (±)-107 was then oxidized in the presence of Dess–Martin periodinane to deliver N-methyleuphococcinine ((±)-3).(±)-N-methyleuphococcinine ((±)-3) in good yields. Besides, arylboronic acids proved to be efficient catalysts for the C-allylation of unprotected oximes. Using this method, the authors accessed the racemic form of N-methyleuphococcinine (3) in 6 steps with a total yield of 17.2% from oxime 103.Although Kurtis' synthesis was racemic, it presented a few steps and led to A. bipunctata), besides the fact that the insect releases these substances in minimal quantities, make these targets highly relevant when considering total synthesis. Since Tursch's pioneering work, several total and formal syntheses of homotropane alkaloids released by coccinellids have been carried out, contributing to a more accurate chemical and biological understanding of these alkaloids. Specifically, in this review, the main points in the synthesis of coccinellid alkaloids are: i) dipolar cycloaddition; ii) olefin metathesis; iii) intramolecular Mannich reaction. Cyclization steps, summarized in The peculiar structural factors of homotropane alkaloids added to the intriguing biological activity exerted by ladybirds (as demonstrated in this review for Therefore, homotropane-based compounds continue to attract the attention of researchers involved in the progress for new synthetic methodologies to reproduce these natural products and synthesize their analogs, improving existing methods.


Chloroflexi have been identified as thermophilic nitrite-oxidizing bacteria (NOB). Among these, Nitrolancea hollandica was isolated from a laboratory-scale nitrifying bioreactor operated at 35°C with a high load of ammonium bicarbonate. However, its distribution remains cryptic as very few closely related environmental 16S rRNA gene sequences have been retrieved so far. In this study, we demonstrate how such thermophilic NOB can be enriched using modified mineral media inoculated with samples from a wastewater side-stream reactor operated at 39.5°C. Distinct cultivation conditions resulted in quick and reproducible high enrichment of two different strains of Nitrolancea, closely related to Nl. hollandica. The same cultivation approach was applied to a complex nitrite-oxidizing pre-enrichment at 42°C inoculated with biomass from a geothermal spring in the Copahue volcano area in Neuquen, Argentina. Here, an additional distinct representative of the genus Nitrolancea was obtained. This novel species had 16S rRNA and nitrite oxidoreductase alpha subunit (nxrA) gene sequence identities to Nl. hollandica of 98.5% and 97.2%, respectively. A genomic average nucleotide identity between the Argentinian strain and Nl. hollandica of 91.9% indicates that it indeed represents a distinct species. All Nitrolancea cultures formed lancet-shaped cells identical to Nl. hollandica and revealed similar physiological features, including the capability to grow at high nitrite concentrations. Growth was optimal at temperatures of 35–37°C and was strongly enhanced by ammonium supplementation. Genomic comparisons revealed that the four Nitrolancea strains share 2399 out of 3387 orthologous gene clusters and encode similar key functions. Our results define general growth conditions that enable the selective enrichment of Nitrolancea from artificial and natural environments. In most natural habitats these NOB apparently are of low abundance and their proliferation depends on the balanced presence of nitrite and ammonium, with an optimal incubation temperature of 37°C.Nitrification is a key process for N-removal in engineered and natural environments, but recent findings of novel nitrifying microorganisms with surprising features revealed that our knowledge of this functional guild is still incomplete. Especially nitrite oxidation – the second step of nitrification – is catalyzed by a phylogenetically diverse bacterial group, and only recently bacteria of the phylum  As important part of the global nitrogen cycle, nitrification converts reduced nitrogen species into oxidized forms, namely ammonium (−III) to nitrite (+III) and further to nitrate (+V). In wastewater treatment plants, nitrification is essential to remove N-compounds like ammonium and nitrite, and to provide nitrate as electron acceptor for denitrification. In agricultural, but also natural systems, nitrification causes nitrogen loss due to leaching of negatively charged nitrate ions and emission of gaseous N-compounds produced by denitrifying bacteria.Nitrospira, which can perform the complete oxidation of ammonia to nitrate  from six genera belonging to four different phyla have been isolated so far. These are ectively , Nitrospte phyla , and theoroflexi , 2014. Toroflexi . Indicatoroflexi .Nitrolancea hollandica has been isolated from a lab-scale nitrifying bioreactor receiving a high load of ammonium bicarbonate and operating at 35°C  Nitrocaldera (Nitrospira (Nitrospira-type NXR is closely related to that of anaerobic ammonium-oxidizing (anammox) bacteria (Nitrobacter-type NXR is closely related to dissimilatory nitrate reductases (NAR) of nitrate-reducing microorganisms  = 1 mM; The low substrate affinity of Nitrospira  systems and an N-removal plant in China  in Hamburg, Germany, and one from the geothermal Las Máquinas area in Argentina. The obtained species were compared to the type strain Nl. hollandica with regard to their genomic, phenotypic and physiological features.In the present study, three new representatives of 3) at the full-scale WWTP Hamburg-Dradenau was sampled  amounted to 550 mg l–1 and 1750 mg l–1, respectively. Subsequent to this “Store and treat” (SAT) procedure, patent-registered by the HSE (Hamburger Stadtentwässerung), the reactor effluent was applied for anammox treatment.On May 22, 2017, a centrate treatment side-stream reactor .The Copahue volcano , the Rio Agrio (pH 0.5–7) and Caviahue Lake (pH 2.1–3.7) are geographical landmarks that frame a geothermal field, which is composed of five hydrothermal sites: Las Máquinas, Las Maquinitas, Anfiteatro, Termas de Copahue (in Argentina) and Chancho-Co (in Chile). The Las Máquinas area contains many thermal fluid emission systems, consisting of fumaroles, boiling-bubbling water and mud pools with wide ranges of pH (2–7) and temperature (30–90°C). The chemical composition of the fumaroles, which are mainly fed by meteoric water , showed in situ in each sample point using specific electrodes . Water samples for metal analysis were filtered using 0.2 μm membrane filters  and their concentrations (2 (In December 2008 (sample E2) and in March 2011 (sample A4), two mud pools in the geothermal area Las Máquinas, Argentina, were sampled. Both sampling sites were characterized by high sulfate concentrations . For samtrations  determintrations . To quantions  were supplied as energy source. The trace element solution (1 ml l–1) contained 33.8 mg l–1 MnSO4 × H2O, 49.4 mg l–1 H3BO3, 43.1 mg l–1 ZnSO4 × 7 H2O, 37.1 mg l–1 (NH4)6Mo7O24, 973.0 mg l–1 FeSO4 × 7 H2O, and 25.0 mg l–1 CuSO4 × 5 H2O. The pH was adjusted to 8.0 and changed to 7.4–7.6 2 days after autoclaving. In order to enrich Nitrolancea, NH4Cl was added in concentrations between 0.5 and 10 mM (–1): 100 mg l–1 MnCl2 × 4 H2O, 30 mg l–1 H3BO3, 144 mg l–1 ZnSO4 × 7 H2O, 36 mg l–1 Na2MoO4 × 2 H2O, 2.1 g l–1 FeSO4 × 7 H2O, 2 mg l–1 CuCl2 × 2 H2O, 190 mg l–1 CoCl2 × 6 H2O, and 24 mg l–1 NiCl2 × 6 H2O.The enrichments were performed in 0.05 to 0.15 l batch cultures in mineral salts medium with the following composition: 0.007 g lnd 10 mM . When bi2 and 3 mM NH4Cl were inoculated (1% v/v) with the centrate sample Z2 on June 22, 2017, and incubated without agitation at 42°C and 46°C, respectively. A transfer was performed on October 23, 2017, and the incubation temperature was lowered to 37°C. In the following, the cultures grew very well and nitrite concentrations supplied to the medium were continuously increased  containing 3 mM NaNOncreased .4Cl solution. Ammonium measurements were done with Quantofix test stripes . The pH was manually adjusted to approx. 7.4 with 5% (w/v) NaHCO3 solution. For sample A4, 1 ml was inoculated in 100 ml mineral medium for NOB with 0.3 mM nitrite as substrate in a 250 ml screw cap Schott bottle. Both cultures were grown at 42°C under static conditions and evaporation was compensated for with sterile distilled water. Both cultures were regularly transferred with 1% (v/v) inoculum into mineral NOB media containing different concentrations of ammonium and nitrite with or without addition of 0.5 mM bicarbonate, and incubated under different conditions, as detailed in A 0.5 ml aliquot of sample E2 (spring water with sediment) was inoculated into a 100 ml Erlenmeyer flask with 50 ml mineral unbuffered AOB medium  containi2, 0.5 mM NH4Cl and 0.5 mM NaHCO3) in 1.5 ml Eppendorf tubes, and incubated at 37°C. A total of 10 and 20 tubes for enrichments Z2.3.3 and A4.5.1, respectively, were inoculated with single cells or short chains resembling Nitrolancea by morphology. After nitrite consumption, active cultures were transferred twice to 5ml tubes (10% inoculum) and lastly into 100 ml Erlenmeyer flasks with 75 ml NOB medium. Additional attempts to eliminate accompanying heterotrophic bacteria were serial tenfold dilutions for cultures Z2.3.3 and A4.5.2 in three to four parallels in mineral NOB medium containing 0.5 mM NaNO2, 0.5 mM NH4Cl and 0.5 mM NaHCO3, and incubated at 37°C. All investigations of Nitrolancea were done on enrichment cultures.Cells from the enrichment cultures Z2.3.3 and A4.5.1  were man–1 bactopeptone, 0.5 g l–1 yeast extract, 0.5 g l–1 meat extract, 0.584 g l–1 NaCl in distilled water, pH 7.4), and on solid RT , R2A  or mixotrophic NOB medium .The purity of the cultures (in terms of absence of heterotrophs) was tested repeatedly by incubating culture aliquots in complex liquid medium . To test for nitrite consumption as indication for growth, the Griess-Ilosvay spot test was used  with UV detection in an automated system . Data acquisition and processing of nitrite and nitrate concentrations was performed with the integrated software EZChrom Elite 3.3.2.–1) as well as protein and cell yield per mmol nitrite. Cells were counted using a Thoma cell chamber (0.02 mm depth), and protein content was quantified by fluorescence using the NanoOrange protein quantitation kit .Nitrite consumption, nitrate production, cell numbers and protein content of cultures of A4.5.2 and Z2.3.4 were determined to calculate generation times (dV = (Vmax × [S])/(Km + [S]), where V is activity, Vmax is maximum specific activity (μmol mg protein–1 h–1), Km is the half-saturation constant for nitrite oxidation (μM), and [S] is concentration of nitrite (μM). For cell specific Vmax, the calculated maximum activity was translated to fmol NO2– cell–1 h–1.Nitrite-dependent oxygen consumption of cells from culture of A4.5.2 (1 replicate) and culture Z2.3.4  was measured following g for 15 min), fixed with 2.5% (v/v) glutaraldehyde as well as 2% (w/v) osmium tetroxide, and embedded in a mixture of Spurr and acetone as described previously  NaCl and fixed with 1:1 (v/v) 96% ethanol:PBS as described previously , lysozyme (4 mg ml–1) and RNase A (1 mg ml–1) were added. The vortexing step was extended to 30 min at 37°C.DNA for PCR and Illumina sequencing was extracted with the PowerSoil DNA isolation Kit  according to manufacturer’s instructions with slight modifications except for cultures E2 and A4.1: Before cell disruption via vortexing (step 5), Proteinase K (1 mg mlNitrolancea was tested for with specific primer sets for 16S rRNA and nxrA genes (Nitrospira was checked using the nxrB-targeted primer set NxrB-169f/NxrB-638r (nxrA) or 56°C (nxrB) and 3.5 min (for Nitrolancea primers) or 45 s (Nitrospira primers) elongation at 72°C; final elongation at 72°C, 20 min.The presence of rA genes  and subsxrB-638r . The folde novo OTU picking workflow in qiime (v1.9.1) . For the cultures E2 and A4.1, amplicons were generated using the primers 341F and 785R , followe(v1.9.1) , and sumy MR DNA . In shorDNA was fragmented using a Bioruptor . Libraries for Illumina HiSeq sequencing were generated using the NEBNext Ultra DNA Library Prep Kit for Illumina  as per manufacturer’s recommendations. Size and quality of the libraries were visualized on a Bioanalyzer High Sensitivity Chip . Diluted libraries were multiplex sequenced on the Illumina HiSeq 2500 instrument  by paired-end sequencing . For sequencing of the metagenomes A2, Z2, and Z4, library preparation was done using the Nextera XT kit  according the manufacturer’s instructions. Tagmentation was performed starting with 1 ng of DNA, followed by incorporation of the indexed adapters and amplification of the library. After purification of the amplified library using AMPure XP beads , libraries were checked for quality and size distribution using the Agilent 2100 Bioanalyzer and the High sensitivity DNA kit. Quantitation of the library was performed by Qubit with the dsDNA HS Assay Kit . The libraries were pooled, denatured and sequenced on a Illumina MiSeq . Paired end sequencing of 2 × 300 bp was performed using MiSeq Reagent Kit v3  according to the manufacturer’s protocol.k = 23, mink = 11, hdist = 1, ktrim = r, tbo = t, maxns = 0, tossjunk = t) and only reads with a length of ≥150 bp were kept. The corrected reads were assembled using metaSPAdes v3.11.1 . Terminal base calls with a quality score < Q17 were trimmed and the reads with a mean quality score of ≤Q20 were discarded. The processed HiSeq reads with a length of ≥70 bp were then corrected for sequencing errors using BayesHammer . Additio v3.11.1  with def v3.11.1 , employi v3.11.1 . The seq v3.11.1 . The MiS v3.11.1  and taxo v3.11.1 . SubsequNitrolancea genomes and the genome of Nl. hollandica was calculated using orthoANI (The average nucleotide identity (ANI) between the recovered orthoANI .nxrA) were aligned using the multiple sequence alignment web server T-Coffee , Bacteriodetes (22%) and Firmicutes (10%). Chloroflexi were present at low amounts , but no known nitrite oxidizer could be identified.As shown by amplicon sequencing, the microbial community of the original centrate treatment reactor sample Z2 was dominated by unts 2%; , but nonNitrolancea could be selectively enriched from 1.4 to 24%  revealed a very high enrichment of lancet-shaped cells , followed by Deinococcus-Thermus (28%) and Proteobacteria (22%) according to 16S rRNA amplicon sequencing (Nitrolancea abundances increased to 14.1% after the culture had been repeatedly replenished with nitrite over a period of 4 years and increased further to 44% after using high substrate concentrations (30 mM) and adding ammonium (–7 (A4.5.3) and the strain deemed isolated. The novel Nitrolancea strain was provisionally designated as Ca. Nitrolancea copahuensis based on 16S rRNA gene and whole-genome dissimilarity to Nl. hollandica (see below).Unlike the centrate treatment reactor sample, the primary nitrite-oxidizing enrichment of Las Máquinas (A4.1) grown at 42°C was dominated by members of the phylum quencing . This enammonium . Manual ammonium  and onlyNitrolancea-like NOB were also co-enriched with unknown ammonia-oxidizing microorganisms. This culture was dominated by Chloroflexi (34%), Proteobacteria (40%) and Actinobacteria (21%), while Nitrolancea-like bacteria constituted nearly 20% of the microbial community after 3.5 years of incubation at 42°C. Contrastingly, no Nitrolancea-like sequences could be detected in a parallel nitrite-oxidizing culture of E2, which was grown without ammonium addition at 42°C (not shown).Notably, in an ammonia-oxidizing culture that had been inoculated with an earlier sample from Las Maquinas (E2) with 0.5 mM ammonium, but without nitrite addition, Nitrospira were nearly absent in all centrate treatment reactor enrichments and constituted only 0.3% of the retrieved amplicon sequences in the Argentinian cultures A4.2, A4.3 and A4.4 (Nitrobacter-like 16S rRNA gene sequences amounted to 0.8% in the centrate cultures Z2.1 and Z2.2 (not shown). In the Argentinian A4 cultures, Nitrobacter was undetectable, but constituted a tiny fraction in culture E2 (0.05%).Regarding other known NOB, and A4.4 . NitrobaNitrolancea-like cells with characteristic tapered ends . Consequently, all mineral NOB media were prepared with the addition of ammonium. Fascinatingly, NHion only . In acco at 37°C . Additio 37°C (3 .Nitrolancea cultures obtained from Las Máquinas (A4.4) increased with increasing substrate concentrations (1–10 mM NaNO2) without a prolonged lag-phase  of 0.12–0.45 mM nitrite for Nl. hollandica strain Z and 0.22 mM for Nl. copahuensis (max) was relatively low (11.1–26.8 μmol nitrite mg protein–1 h–1). Growth yields ranged from 0.17 to 0.88 mg protein mmol nitrite–1.Nitrite oxidation kinetics were determined by microsensor-based oxygen consumption measurements in the different cultures . Both reahuensis . The maxNitrolancea strains described here  could be assembled into 48 to 150 contigs. These high-quality draft genomes ranged in size from 3.19 to 3.45 Mbp and had G+C contents between 62.7 and 62.9 mol%. The number of predicted coding sequences was between 3023 and 3235, including 40 to 46 tRNAs.Biomass of cultures Z2.2, Z2.3.2, and A4.4 was used for metagenomic sequencing . The draNitrolancea strains, comparative genomics revealed that two highly similar strains (Z2 and Z4) were enriched from the centrate treatment reactor sample, while the strain A2 obtained from sample A4 was clearly distinct (see below). The comparison of these metagenome-assembled genomes (MAGs) with Nl. hollandica revealed 2399 conserved clusters out of in total 3387 orthologous gene clusters within the four Nitrolancea strains . Comparison of the NxrA sequences of the novel strains and Nl. hollandica revealed high amino acid identities (Nl. hollandica had high average nucleotide identity (ANI) values of 99.2 and 99.3%, respectively, which was only 91.9% for the geothermal strain  and the cytoplasmic assimilatory nitrate reductase (nasAB) were detected in the three analyzed genomes, but not for assimilatory nitrite reductases (nirA or nirBD) or dissimilatory nitrite reduction to ammonium (nrfA).The genomic data was furthermore mined to reconstruct the core metabolic pathways of the three novel  strains . Genes ellandica . The A2 d operon . FurtherNitrolancea genomes encoded an aerobic-type CO dehydrogenase, cyanase and an [NiFe]-hydrogenase. Based on the genomic data, all strains have the potential to grow heterotrophically and to use thiosulfate for energy conservation. So far, none of these phenotypes could be observed in growth experiments with Nl. hollandica (sat), adenylyl-sulfate kinase (cysC), phosphoadenosine phosphosulfate reductase (cysH) and ferredoxin-dependent sulfite reductase (sir) could be identified in the three analyzed genomes, along with genes encoding thiosulfate sulfurtransferases  and a putative sulfite oxidase (sorA).Additionally, all llandica . Within Nitrolancea was only described in 2012  with the competing substrate O2 and thus increase carbon fixation efficiency in the absence of other CO2-concentrating mechanisms like carboxysomes  for nitrite determined for Nl. hollandica . Similarly, the representatives of Nitrolancea enriched from the Las Máquinas samples differed in the initial enrichments E2 and A4.4 in AOB and NOB medium, respectively    was measequences . TherefoNitrolancea-like NOB, as revealed by our 16S rRNA amplicon data .In conclusion, we physiologically and genomically characterized three novel representatives affiliated with the genus The strain is a mesophilic, aerobic, chemolithoautotrophic bacterium, which stoichiometrically converts nitrite to nitrate and uses carbon dioxide as carbon source. Forms short rods with tapered ends; Gram-positive. Requires ammonium supplementation and tolerates high nitrite and ammonium concentrations. Optimum growth temperature is 35–37°C. DNA G+C content is 62.7 mol%. Originated from a hot spring in the geothermal field Las Máquinas, Neuquen-Argentina.ERS4399870, ERS4399871, ERS4399872, ERS4647270-74, ERS4648393-96, and LR794103-5. Strain availability will be performed upon request.The datasets generated for this study are available in the European Nucleotide Archive (ENA) under accession numbers Nitrolancea enrichments. ES and KS isolated the nitrite oxidizers and performed FISH. SK did physiological experiments. DI performed genome sequencing. MS, LK, and SL analyzed the data. ES, KS, SK, LK, AG, and SL wrote the manuscript. All authors read and agreed on the final manuscript.AG performed the sampling. ES, SK, and SL designed the research. SH worked on the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The sheet was then tested for its dynamic permeability by irradiating with electromagnetic waves with frequencies up to 50 GHz. Two different originally developed methods were used for the high-frequency permeability measurements, a short-circuited microstrip line method and a microstrip line-probe method. It is challenging to measure the dynamic permeability of magnetic thin films/sheets beyond 10 GHz because of the low response signal from these materials. However, the two methods produced essentially equivalent results. In the frequency dependent permeability profile, the maximum position of the profile, 0.5Zn0.5Fe2O4 powder/polymer composite sheet thus exhibits high performance at GHz frequencies, and should be applicable e.g. as an anisotropic electromagnetic wave-interference material.We have fabricated a high quality magnetic Ni On the other hand, magnetic materials exhibiting Current telecommunication systems and electronic devices with super-high functionalities are operated by GHz-frequency electromagnetic waves. Consequently, magnetic materials working in the high GHz-frequency region are indispensable for e.g. sensing and spintronic torque-oscillation devices, electromagnetic wave absorbers, and magnetic sheets for radio-frequency identification (RFID). For the practical use of these magnetic materials, dynamic permeability behaviors under the irradiation of high-frequency electromagnetic waves is a key issue, because permeability determines the applicable frequency bands of magnetic-based devices. For instance, the resonance frequency , coaxial and coplanar waveguides are commonly used. The measurement results obtained from experiments are usually analyzed by the lumped-element approximation12 because of their ferromagnetic resonance (FMR) in the MHz- to GHz-frequency region. High resistivity but low conduction losses in the high-frequency region is another prominent advantage of ferrites. Magnetoplumbite-structured ferrites with a chemical formula of AFe12O19  have been widely studied, as 15. For example, in a Sm-doped high purity single-crystal SrFe12O1916. However, keeping an eye on the recent high-frequency technologies, the electromagnetic wave interference (EMI) may become a serious problem. In this regard, the magnetoplumbite-structured AFe12O19 ferrites17, spinel-structured Ni1-xZnxFe2O4 ferrites, and soft magnetic alloy materials mixed with polymers18 are expected to be attractive materials, especially in the field of EMI at GHz bands.Ferrites are promising materials for high-frequency devices19, and (ii) MSL-probe20 methods. Our aim is to provide a comprehensive understanding of the dynamical magnetic behaviors of ferrites in the GHz region. For both measurement methods, the same sample, i.e., Ni0.5Zn0.5Fe2O4 powder/polymer composite sheet from the same sample batch, was used. We used polyvinyl alcohol (PVA) as a polymer source. It should be noted that the spinel-structured Ni0.5Zn0.5Fe2O4 is widely commercialized because of its simpler crystal structure compared to the AFe12O19 ferrites with the magnetoplumbite structure. Therefore, it is an optimal material for the present study.In this study, we evaluate the dynamic permeability behavior of ferrites under the irradiation of an electromagnetic wave of GHz frequency through two different originally developed measurement techniques, (i) short-circuited MSL0.5Zn0.5Fe2O4 ceramic powder. First of all, the XRD pattern shown in Fig. The high sample quality was an essential requirement for the present study. Hence we started the study by confirming the phase purity and the fundamental structural and physical properties of the synthesized Ni0.5Zn0.5Fe2O4 ceramic powder was obtained from the microstructural study. From the FE-SEM image  and perpendicular to (⊥) the surface of the sheet  magnetic anisotropy.The magnetic properties of the Nisee Fig. f, and th0.5Zn0.5Fe2O4-powder/polymer composite sheet measured by the short-circuited MSL method19 is shown in Fig. 2MoO4)–0.7(Ni0.5Zn0.5Fe2O4) sample mixed with PVA, with a composition similar to the present sheet, the following values were reported: 4.0 (24. Our sample showed 6.8 for 2MoO4)–0.7(Ni0.5Zn0.5Fe2O4) composite exhibited 8.8 (2MoO4)–0.7(Ni0.5Zn0.5Fe2O4) composite, measured using a different measurement system and method24. This confirms the reliability of the present evaluation method.The complex permeability of the NiThe high-frequency electromagnetic wave loss phenomena should be addressed. The frequency dependence of the complex permittivity Fig. b and com″ε′ Fig. d and \do″μ′ Fig. e, respec25, the decrease in permittivity at low frequencies is attributed to the interfacial polarization between two different phases; Ni0.5Zn0.5Fe2O4 and PVA in the present case. Differing permittivies and conductivities between Ni0.5Zn0.5Fe2O4 and PVA induce space charges under the application of an electric field on the composite sheet. The space charges accumulate at the interface between Ni0.5Zn0.5Fe2O4 and PVA, leading to interfacial polarization26.We inferred that the permittivity Fig. b and \donδε Fig. d can be ity Fig. b and \donδε Fig. d rapidly25,25 according to Eq.  would be the most probable origin of For 0.5Zn0.5Fe2O4-powder/polymer composite sheet . Although the methods (i) and (ii) represent different approaches (see Method section), we emphasize that the dynamic 34. In the present case, the linearity is concluded for the entire range of 0.5Zn0.5Fe2O4-powder/polymer composite sheet could be applicable in a wide range of GHz-operational frequencies35 even at small Note that the measurement condition of Fig. From Eq. , the valentclass1pt{minima36 for calculating the damping constant,We studied a relationship between the Gilbert damping constant, α, and }fr Fig. . For thi0.65Zn0.35Al0.8Fe1.2O4 possessing a similar composition as the present sample, low damping constant values have been reported; 37. These values are one-to-two orders of magnitude lower than our sample.From Fig. 38. In the case of spinel-structured ferrite films, the constituent of cations39 and high density of crystal defects generated by dislocations and/or antiphase boundaries42 affect the damping. This results in a defect-mediated damping37. These factors are potentials for “extrinsic” damping, which leads to high 48. However, from microstructural investigations in this study, we observed neither remarkable antiphase boundaries nor misfit dislocations  substrate, the 49. Our 49, though the present sheet consists of polycrystalline Ni0.5Zn0.5Fe2O4 particles.By taking into account the discussions above, we suggest that the damping of the present Ni0.5Zn0.5Fe2O4 powder/polymer composite sheet composed of an environmentally friendly ferrite with cost-effective elements applicable over GHz bands. Another prominent significance of this study is showing the reliability of the novel evaluation methods (i) and (ii). This study gives hope for the establishment of high-frequency technologies beyond several tens of GHz bands.Therefore, the significance of the present study is the high potential of the present Ni19 and MSL-probe20 methods, to a Ni0.5Zn0.5Fe2O4 powder/polymer composite sheet prepared from high-quality Ni0.5Zn0.5Fe2O4 ceramic powder. Although the two techniques are based on different experimental setups/systems, the dynamic permeability behaviors at GHz bands are essentially consistent.In conclusion, we applied two different originally developed techniques, short-circuited MSL37. Further study on the damping is necessary, but the damping process of the current Ni0.5Zn0.5Fe2O4 powder/polymer composite sheet could be dominated by a strong coupling between 3d electrons, 3d spins, and phonons.A linear relationship between 0.5Zn0.5Fe2O4, the sheet showed soft magnetism exhibiting parallel magnetic anisotropy, high surface resistivity, small required 49. These results indicate that high-performance devices that can be utilized in frequency bands up to several tens of GHz. For example, the present Ni0.5Zn0.5Fe2O4-powder/polymer composite sheet is magnetically anisotropy, such that “anisotropic suppression” of electromagnetic waves with GHz frequencies could be possible50; the present sample can be expected to be useful as an anisotropic-EMI material in the GHz band, which is essential for high-frequency electronic devices.Although the present sheet is composed of polycrystalline Ni0.5Zn0.5Fe2O4 powder was mixed with a polymer to attain a Ni0.5Zn0.5Fe2O4 powder/polymer composite sheet. First of all, Ni0.5Zn0.5Fe2O4 ceramic powder was synthesized by a solid-state reaction method. High purity starting materials, i.e., stoichiometric quantities of α-Fe2O3 (99.9%), NiO (99.97%), and ZnO (99.999%), were mixed and calcined in air at 1000 °C for 4 h. The calcined powder was ground and pressed to pellets with the diameter of 10 mm, by applying a mechanical pressure of 10 MPa. Finally, the pelletized sample was annealed in air at 1400 °C for 4 h. The annealing temperature was chosen to achieve the most superior sample quality. To avoid peroxidation, we annealed the sample in air instead of an O2 atmosphere.In the present study, Ni0.5Zn0.5Fe2O4 after the final annealing was ground carefully using a grinder to obtain Ni0.5Zn0.5Fe2O4 particles with a homogeneous size-distribution. We classified the Ni0.5Zn0.5Fe2O4 particles using sieves with different mesh sizes. After that, the Ni0.5Zn0.5Fe2O4 powder with homogeneous size was mixed with a PVA (–(CH2CH(OH)–)500) solution. The ceramic Ni0.5Zn0.5Fe2O4 powder and PVA solution were well mixed in a crucible, resulting in slurry with proper viscosity. By spreading the slurry on a flat PET-film and applying the Doctor-Blade method, we obtained sheet-shaped Ni0.5Zn0.5Fe2O4 powder/polymer composite. After drying the sheet under ambient conditions in air, the sheet was removed from PET film, and was heat-treated to dry completely at 105 °C for 1 h; this is a monolayered sheet with the thickness of 166 μm on the average. Afterward, we piled two monolayered sheets, and performed a heat treatment at 150 °C for 6 min by applying a mechanical pressure to attain higher density of the Ni0.5Zn0.5Fe2O4 powder/polymer composite sheet. We focus only on the double-layered Ni0.5Zn0.5Fe2O4 powder/polymer composite sheet instead of a monolayered one in this study.The pelletized Ni0.5Zn0.5Fe2O4 powder was embedded in a polymer matrix  on a Fe-free Al foil, and room-temperature 57Fe Mössbauer spectra were collected at 12 mm/s  using a 50 mCi Cyclotron 57Co/Rh source. The microscopic crystal structures of the sample were observed by field-emission scanning electron microscopy  and field-emission transmission electron microscopy , respectively.The phase purity and crystallinity were evaluated by X-ray diffraction . The magnetic field dependent magnetization behaviors were measured in the range of ± 10 kOe at room temperature using a vibrating sample magnetometer . The Ni19 and (ii) MSL-probe20 methods were employed and their results were compared. In the case of (i), originally developed sample jigs were used to produce electromagnetic radiation of suitable frequencies to irradiate the sample for the measurements. The sheet sample was cut into a rectangle and placed at the short end of the MSL. In method (i), a sufficiently strong 0.5Zn0.5Fe2O4 powder/polymer composite sheet using a permanent magnet  short-circuited MSLnet Fig. a. \docum20. The MSL probe was set on the surface of sheet sample with a gap between the sample and the probe of approximately 200 μm. Before the measurements, a calibration of the network analyzer had been carefully performed by applying 20. After that, the permeability measurements were carried out. In this method, the overall experimental system is also regarded as an electric circuit model. The output signal from the measurements is 20.In method (ii), a “self-developed” probe composed of a MSL on a flexible substrate was used for the measurements