Streptococcus suis serotype 2 is an important porcine pathogen and zoonotic agent causing sudden death, septic shock and meningitis, with exacerbated inflammation being a hallmark of the infection. A rapid, effective and balanced innate immune response against S. suis is critical to control bacterial growth without causing excessive inflammation. Even though interleukin (IL)-1 is one of the most potent and earliest pro-inflammatory mediators produced, its role in the S. suis pathogenesis has not been studied. We demonstrated that a classical virulent European sequence type (ST) 1 strain and the highly virulent ST7 strain induce important levels of IL-1 in systemic organs. Moreover, bone marrow-derived dendritic cells and macrophages contribute to its production, with the ST7 strain inducing higher levels. To better understand the underlying mechanisms involved, different cellular pathways were studied. Independently of the strain, IL-1β production required MyD88 and involved recognition via TLR2 and possibly TLR7 and TLR9. This suggests that the recognized bacterial components are similar and conserved between strains. However, very high levels of the pore-forming toxin suilysin, produced only by the ST7 strain, are required for efficient maturation of pro-IL-1β via activation of different inflammasomes resulting from pore formation and ion efflux. Using IL-1R−/− mice, we demonstrated that IL-1 signaling plays a beneficial role during S. suis systemic infection by modulating the inflammation required to control and clear bacterial burden, thus promoting host survival. Beyond a certain threshold, however, S. suis-induced inflammation cannot be counterbalanced by this signaling, making it difficult to discriminate its role.The online version of this article (10.1186/s13567-019-0670-y) contains supplementary material, which is available to authorized users. Streptococcus suis causes sudden death and meningitis in pigs and is responsible for important economic losses to the swine industry. Furthermore, it is also a zoonotic agent responsible for meningitis and septic shock in humans , TRIF−/− [C57BL/6 J-Ticam1Lps2/J], TLR2−/− [B6.129-Tlr2tmKir/J], TLR4−/− [B6.B10ScN-Tlr4lps−del/JthJ], caspase-1−/− [B6N.129S2-Casp1tm1Flv/J], NLRP3−/− [B6.129S6-Nlrp3tm1Bhk/J], NLRP1−/− [B6.129S6-Nlrp1btm1Bhk/J], AIM2−/− [B6.129P2-Aim2Gt(CSG445)Byg/J], NLRC4−/− [−/− [B6.129S7-Il1r1tm1Imx/J] mice on C57BL/6 background were housed under specific pathogen-free conditions alongside their wild-type counterparts. Mice were purchased from Jackson Research Laboratories , except for NLRC4−/− mice, generated by Dr. G. Núñez  [MyD88NLRC4−/− , and IL-an, USA) .l-glutamine, and 50 µM 2-mercaptoethanol  and complemented with 10% granulocyte–macrophage colony-stimulating factor. For macrophages (MΦ), hematopoietic stem cells (5 × 105 cells/mL) were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and complemented with 30% L929 cell-derived macrophage colony-stimulating factor supernatant [2 and trypsinized using 0.05% trypsin-0.03% EDTA (Gibco) prior to infection. Cell purity was at least 85% CD11c+ or F4/80+ for bmDCs and MΦ, respectively. Albeit this culture system cannot completely rule out the presence of other innate cells (such as macrophages), it represents an enriched source of bmDCs.Hematopoietic stem cells from femurs and tibias of wild-type and knockout mice were used to generate bone marrow-derived DCs (bmDCs) as previously described , 14 in Rernatant . Cells w® Non-Radioactive Cytotoxicity Assay . Cells were resuspended at 1 × 106 cells/mL in complete medium and stimulated with the different S. suis serotype 2 strains listed in Table 6 CFU/mL; initial multiplicity of infection = 1). Conditions used were based on those previously published [+, a stock solution of KCl  was prepared and appropriately diluted.All activation studies were done with endotoxin-free material and under non-toxic conditions as evaluated using the CytoTox 96ublished . At indiublished . When neBacterial DNA and RNA isolation was performed as previously described . Cells wLevels of IL-1α, IL-1β, IL-6, and tumor necrosis factor (TNF) in cell culture supernatants were measured by sandwich enzyme-linked immunosorbent assay (ELISA) using pair-matched antibodies from R&D Systems  according to the manufacturer’s recommendations.Atp5b and Gapdh, determined to be the most stably expressed using the algorithm geNorm, were used to normalize data. Fold changes in gene expression were calculated using the quantification cycle threshold (Cq) method using the CFX software manager v.3.0 (Bio-Rad). Samples from mock-infected cells served as calibrators.Cell mRNA was extracted according to the manufacturer’s instructions (TRIzol). RNA purity was assessed by spectrophotometric quantification and integrity verified by electrophoresis on denaturating agarose gel. cDNA was generated using the Quantitect cDNA Synthesis Kit  with 500 ng of RNA pretreated with DNase. Real-time qPCR was performed on the CFX-96 Touch Rapid Thermal Cycler System (Bio-Rad) using 250 nM of primers (Integrated DNA technologies), the SsoFast Evagreen Supermix Kit (Bio-Rad) and 20 ng of cDNA. No template controls were included and all samples were run in triplicate. The cycling conditions were 3 min of polymerase activation at 98 °C, followed by 40 cycles at 98 °C for 2 s and 57 °C for 5 s. Melting curves were generated after each run to confirm the presence of a single PCR product. The sequences of primers used in this study are shown in Additional file −/− mice were used. Animals were acclimatized to standard laboratory conditions with unlimited access to water and rodent chow [S. suis serotype 2 strains or the vehicle solution (sterile THB) were administered at a dose of 1 × 107 CFU by intraperitoneal inoculation. Survival was evaluated and mice monitored at least three times daily until 7 days post-infection (pi).Six-week-old male and female wild-type C57BL/6 and IL-1Rent chow . These sg for 10 min at 4 °C. For liver and spleen, extraction buffer was prepared using complete Mini, EDTA-free, protease inhibitor cocktail tablets  according to the manufacturer’s instructions and organs homogenized using a POLYTRON PT 1200E system bundle . Homogenate supernatants were collected following centrifugation at 10 000 × g for 10 min at 4 °C. Levels of IL-1α and IL-1β were determined by ELISA, while IL-6, IL-12p70, interferon (IFN)-γ, C–C motif chemokine ligand (CCL) 2, CCL3, and C–X–C motif chemokine ligand (CXCL) 9 were measured using a custom-made cytokine Bio-Plex Pro™ assay (Bio-Rad). Acquisition was performed on the MAGPIX platform (Luminex®) and data analyzed using the Bio-Plex Manager 6.1 software (Bio-Rad).For kinetics of IL-1α and IL-1β production, blood from infected mice was collected by intracardiac puncture following euthanasia and anti-coagulated with EDTA (Sigma-Aldrich) as previously described , 36. Pla−/− mice were inoculated with S. suis as described above and blood bacterial burden was assessed 12 h and 48 h pi by collecting blood from the caudal vein. For liver and spleen, organs were collected and homogenized as described above. Bacterial burden was determined by plating appropriate dilutions on THA.Wild-type and IL-1R−/− mice. Each test was repeated in at least three independent experiments. p < 0.05 was considered as statistically significant.Normality of data was verified using the Shapiro–Wilk test. Accordingly, parametric (unpaired t-test) or non-parametric tests (Mann–Whitney rank sum test), where appropriate, were performed to evaluate statistical differences between groups. Log-rank (Mantel–Cox) tests were used to compare survival between wild-type and IL-1RSystemic levels of both IL-1α and IL-1β were measured in plasma, liver and spleen following infection with the classical European ST1 strain P1/7 and the highly virulent ST7 strain SC84. IL-1β levels were barely detectable in mock-infected animals and did not change between 6 to 48 h. Similarly, IL-1β levels were scarcely detectable in plasma throughout the course of infection, including upon presentation of severe clinical signs of systemic disease, with no significant differences between strains Figures .Figure 1S. suis infection [S. suis infection was evaluated. Strain P1/7 induced modest levels of IL-1β in a time-dependent manner, with production by bmDCs being significantly greater than that by MФ at 12 h and 16 h (p < 0.001)  in both cell types. In fact, levels induced by strain SC84 16 h pi were approximately 40 times higher than those induced by strain P1/7. Interestingly, a slight delay in IL-1β production was observed by SC84-stimulated MФ in comparison to bmDCs. Moreover, IL-1α kinetics by bmDCs and MФ were similar to those obtained for IL-1β  Figure . On the ) Figure  at 8 h, S. suis strains (p < 0.01)  reduced in TLR2−/− bmDCs stimulated with P1/7 or SC84 (p < 0.01), no difference was observed with TLR4−/− bmDCs , in which absence of the lipoprotein diacylglyceryl transferase, a key enzyme in LP synthesis, renders LPs unrecognizable by TLR2 [lgt-deficient mutants induce significantly less IL-1β than those from wild-type strains (p < 0.01), but levels were in fact undetectable   Figure B, indicaS. suis-induced IL-1β production on MyD88, but only partially on TLR2 , suggested a potential role of endosomal TLRs. To evaluate this, DNA and RNA were extracted from strains P1/7 and SC84 and complexed or not with DOTAP liposomal transfection reagent, which allows phagosomal delivery. In the absence of DOTAP S. suis DNA and RNA did not induce IL-1β production from bmDCs (data not shown). Meanwhile, DNA and RNA from both strains induced significant, but similar production when complexed with DOTAP (p < 0.05) . When Alum was added, a known activator of the NLRP3 inflammasome, a significantly higher production was observed for both genetic materials . Consequently, recognition of RNA and DNA may suggest an involvement of TLR7 and TLR9, respectively, in S. suis-induced IL-1β production.Though an extracellular bacterium, dependence of S. suis-induced IL-1β production, bmDCs were pre-treated with inhibitors  or the vehicle (DMSO). Treatment with NF-κB inhibitor reduced secretion of IL-1β induced by both strains by approximately 50% (p < 0.01)   while JNK inhibitor had no effect  and extracellular-regulated kinase (ERK) are the most important . To dete) Figure D. Intere) Figure D. Finallt Figure D. ConseqS. suis-induced IL-1β requires caspase-1, bmDCs deficient for this enzyme was used. IL-1β production was reduced by more than 75% in caspase-1−/− bmDCs (p < 0.01), indicating that it is essential for maturation of IL-1β following infection by both strains , there was no significant involvement of NLRP1 or NLRC4 , with a major implication of NLRP3  efflux is a common denominator in the assembly and activation of inflammasomes [S. suis-infected bmDCs exposed to increasing concentrations of extracellular K+, used to inhibit its efflux, was evaluated. Ten mM of extracellular K+ was sufficient to significantly inhibit IL-1β induced by both strains (p < 0.05)  , suggesting that the higher levels of SLY produced by this strain are implicated in IL-1β production , with residual levels being similar to those measured for P1/7, P1/7Δsly and SC84Δsly , which was abolished following treatment with cholesterol    Figure . Followie Figure .Figure 8S. suis systemic infection, it can also lead to host death if uncontrolled. Consequently, bacterial load in blood, spleen and liver was evaluated 12 h and 48 h following infection with strains P1/7 and SC84. No differences were observed between wild-type and IL-1R−/− mice 12 h pi regardless of strain   Figure . By cont) Figure . NotablyS. suis, in comparison with other important pro-inflammatory cytokines such as TNF or IL-6 [Previous studies showed low levels of IL-1 in plasma after experimental infection with  or IL-6 , 35. As  or IL-6  and its  or IL-6 . To our  or IL-6 . TherefoS. pneumoniae [To further analyze differences in IL-1 signaling between strains, in vitro studies with bmDCs and MΦs were performed. Interestingly, production by MΦ was somewhat delayed compared to that by bmDCs following infection with both strains. This seems to be a characteristic of streptococci, as it was also reported for GBS, GAS and eumoniae –48, and eumoniae .S. pneumoniae, suggesting that recognized bacterial motifs might even be conserved amongst streptococci [S. pneumoniae (including recombinant pneumolysin) showed that production of IL-1 was TLR4-independent [S. suis infection. Finally, although considered a classical extracellular pathogen, S. suis strains P1/7 and SC84 can be internalized, albeit at low rates, and the nucleic acids can be recognized by TLR7 and TLR9 [Unlike with most other cytokines, IL-1β production is controlled by a two-step signaling process. Firstly, activation of PRRs such as TLRs leads to the transcription of pro-IL-1β. Subsequently, a second signal induces cleavage of the precursor into active IL-1β through capsase-1- and inflammasome-dependent maturation. The higher levels of IL-1β induced by strain SC84, in comparison to strain P1/7, suggested differential cell activation or processing mechanisms. However, the cellular activation leading to IL-1β production was similar for both strains, indicating that the recognized components are relatively well-conserved. In addition, production of IL-1β was MyD88-dependent and partially involved recognition of surface lipoproteins by TLR2. Comparable results were reported for GBS and ptococci , 50. Meaptococci , listeriptococci  and, morptococci , may actependent , 53. Independent , 12. Morand TLR9 . In thisand TLR9 .S. suis [Following engagement of TLRs, activation of the MAPK and NF-κB signaling pathways results in the initiation of an inflammatory response leading to cytokine production. IL-1β production by bmDCs induced by both strains was dependent on the NF-κB and ERK pathways, but independent of JNK, similar to what has previously described for other cytokines induced by S. suis  and for S. suis –57. MeanS. suis , 58. As S. pneumoniae [S. suis-dependent inflammasome activation are difficult to determine since all four inflammasomes can be activated by a wide range of molecules. Previous studies showed that NLRP1 could directly sense the protease activity of the Bacillus anthracis lethal toxin [S. suis SLY might be involved in a similar process. Moreover, strain SC84, unlike strain P1/7, also possesses a type IV secretion system encoded by its 89 K pathogenicity island [Receptors and pathways engaged by this pathogen could not explain the differences observed in IL-1β production between strains. Moreover, IL-1β gene induction confirmed that the first step involved in this production is similar. Consequently, the steps involved in its maturation were evaluated. Though IL-1β production induced by both strains depended on caspase-1, inflammasome activation was different between strains: while maturation of pro-IL-1β induced by strain P1/7 was only partially dependent on NLRP3 and AIM2, strain SC84 activated NLRP3 and, to a lesser extent AIM2, NLRP1 and, surprisingly, NLRC4. These differences in inflammasome activation between strains, both in the different inflammasomes activated and their implication levels, could explain the differential IL-1β levels produced by bmDCs. Although NLRP3 and AIM2 participate in IL-1β release by bmDCs and MΦ following infection by GBS and eumoniae , 53, theeumoniae . The speal toxin . Althougy island , which my island . Regardiy island . In accosly gene is highly conserved between S. suis strains with a maximum diversity of 1.8% at the nucleotide level [Previous studies showed that bacterial pore-forming toxins could play a role in inflammasome activation . Herein,de level , 65, Thide level . The sam+ efflux could be responsible for the mechanism by which SLY stimulates inflammasome activation, as previously described for pneumolysin and the β-hemolysin of GBS [+ inhibited S. suis-induced IL-1β production. Importantly, although only 10 mM of K+ was sufficient to reduce IL-1β production, the effect was greater for strain SC84. For strain P1/7, the K+ efflux generated could be due to other yet unknown bacterial mechanisms that are probably shared by other classical S. suis strains and responsible for “normal” inflammasome activation by this pathogen.Generation of a Kn of GBS , 51. Indn of GBS , 42. In S. pneumoniae showed a protective role of IL-1 during infection, with its absence altering bacterial clearance and survival [S. suis, IL-1 signaling also plays a central and beneficial role following infection with the ST1 strain P1/7, which represents classical strains. Indeed, IL-1 signaling induced by this strain modulates the host innate immune response by increasing production of other pro-inflammatory mediators required for control of bacterial burden in blood and organs, which if unrestricted, causes host death. However, and similar to what has been described for type I IFN, the “protective effect” of IL-1 was not observed following infection with the highly virulent ST7 strain SC84 [S. suis systemic infection, the levels of induced inflammation play a critical role. In accordance, IL-1 signaling itself cannot counterbalance the exacerbated inflammation induced by SC84 strain, resulting in host death. Interestingly, this strain possesses additional virulence factors such as the 89 K pathogenicity island, which is responsible for a greater innate immune system activation, resulting in a cytokine storm [S. suis infection, though to a significantly lower degree and rate, demonstrating the need for a balanced and controlled inflammation. S. suis-induced IL-1 did not autoregulate itself, suggesting that levels induced in the first hours of infection are sufficient to activate the immune system. This is in accordance with results obtained during systemic infection with GBS, during which levels of IL-1β are similar between wild-type and IL-1R−/− mice in kidneys, peritoneal lavage and brain [Following secretion, IL-1α and IL-1β bind their shared receptor, IL-1R, leading to cell activation, stimulation and secretion of diverse pro-inflammatory cytokines (positive feedback loop), recruitment of neutrophils and macrophages, and activation of killing mechanisms, amongst other effects . Previousurvival –24. In tain SC84 . In factne storm . It shound brain .S. suis strain induce IL-1 in vivo, but only in internal organs. While both strains similarly activate innate immune cells due to conserved bacterial components such as LPs, high levels of the pore-forming toxin SLY play an important role in IL-1β maturation via activation of the NLRP1, NLRP3, AIM2, and NLRC4 inflammasomes. Based on these results, a model of the mechanisms involved in S. suis-induced IL-1β production by bmDCs is proposed  and highly virulent (SC84) d Figure . GloballAdditional file 1.Oligonucleotide primers used in this study.Additional file 2.IL-1α release from bone marrow-derived dendritic cells (bmDCs) and macrophages (MФ) stimulated withS. suisis strain-dependent. IL-1α kinetics as measured by ELISA following infection of bmDCs (A and C) or MФ (B and D) with strain P1/7 (white bars) or SC84 (gray bars). Non-stimulated cells served as negative control (C-). Data are expressed as mean ± SEM (n = 4).Additional file 3.Addition of Alum enhancesS. suisnucleic acid-induced IL-1β production by bone marrow-derived dendritic cells (bmDCs). IL-1β production by bmDCs following activation with 1 µg of S. suis RNA or DNA from strains P1/7 and SC84 in the presence of Alum. Data are expressed as mean ± SEM (n = 3). *(p < 0.05) indicates a significant difference with negative control (elution buffer).Additional file 4.S. suis-induced TNF production by bone marrow-derived dendritic cells (bmDCs) is inflammasome-independent. Percentage of TNF secretion by caspase-1 (CASP-1), NLRP3, AIM2, NLRP1 or NLRC4-deficient bmDCs induced by strain P1/7 (white bars) or SC84 (gray bars) after 16 h, in comparison to wild-type counterparts . Data are expressed as mean ± SEM (n = 3).Additional file 5.S. suis-induced IL-6 and TNF secretion by bone marrow-derived dendritic cells (bmDCs) is independent of additional extracellular potassium (K+) concentrations. bmDCs were infected with either strain P1/7 or SC84 in the presence of different concentrations of KCl and IL-6 (A) or TNF (B) production was measured after 16 h by ELISA. Data are expressed as mean ± SEM (n = 3).Additional file 6.IL-1β production by recombinant suilysin (rSLY) is Toll-like receptor (TLR) 4-independent. IL-1β secretion by wild-type and TLR4−/− bone marrow-derived dendritic cells stimulated with rSLY (5 μg/mL) for 16 h. Data are expressed as mean ± SEM (n = 3).Additional file 7.IL-1 does not modulate its own production followingS. suisinfection. Spleen and liver levels of IL-1α (A and B) and IL-1β (C and D) in wild-type (WT) and IL-1R−/− mice 12 h following infection with strain P1/7 or SC84. Data are expressed as mean ± SEM (n = 5).


Scientific Reports 10.1038/s41598-018-23706-7, published online 03 April 2018Correction to: The Acknowledgements section in this Article is incomplete.“We are grateful for Ms. Andrea Orbán’s technical assistance. This work was funded by the Hungarian Scientific Research Fund , NVKP_16-1-2016-0017 and VEKOP-2.3.3-15-2017-00016). Árpád Ferenc Kovács is a Kerpel-Fronius Ödön Fellow. Lilla Turiák would like to acknowledge the Bolyai Janos Research Scholarship program.”should read:“We are grateful for Ms. Andrea Orbán’s technical assistance. This work was funded by the Hungarian Scientific Research Fund , NVKP_16-1-2016-0017, VEKOP-2.3.3-15-2017-00016 and EFOP-3.6.3-VEKOP-16-2017-00009). Árpád Ferenc Kovács is a Kerpel-Fronius Ödön Fellow. Lilla Turiák would like to acknowledge the Bolyai Janos Research Scholarship program.”


Both complexes exhibit a square-planar geometry, with unbound phenol moieties participating in intermolecular hydrogen bonding with co-crystallized water and methanol. The presence of both κ2 and κ3 coordination modes arising from the same solution suggest a dynamic process in which phenol donors may coordinate or dissociate from the metal center, and offers insight into catalyst speciation throughout Pd-mediated processes. The unit cell contains dichlorido]bis(methylene)}bis)palladium(II), {(L2)PdCl2}, and chloridomethyl](methyl)amino}ethyl)(methyl)amino]methyl}phenolato)palladium(II), {(L2X)PdCl}, molecules as well as fractional water and methanol solvent molecules.Bidentate and tridentate coordination of a 2,4-di- The presence of palladium(II) complexes displaying both κ2 and κ3 coordination modes arising from the same solution suggests a dynamic process in which phenol donors may coordinate or de-coordinate based on the electronic demands at the metal center.Several related complexes feature ligands similar to these amine bis(phenolate) species bound in a κtert-butylphenol) and fractional quantities of methanol and water crystallization solvents. Both metal centers adopt similar distorted square-planar geometric arrangements, in which both nitrogen atoms of the ligand are bound to the Pd center to form a five-membered ring, and either one or two chlorine atoms are present to complete the coordination sphere depending on the coordination mode of the ligand. In both complexes, the N—Pd—N bond angle is similar to those observed for related amine bis(phenolate) Pd complexes PdCl] complex. This complex is only slightly distorted from ideal square-planar geometry . An additional close contact of ∼2.460 Å is observed between Cl1 and the O3—H bond of an unbound phenol from the neighboring [PdCl2] complex.Deprotonation and coordination of O1, presumably in the presence of water during crystallization, gives rise to the [PdCl2] complex also exhibits only minor distortions from an ideal square-planar geometry  suggest that the conformations observed in the solid state are retained on the NMR timescale. Signals attributed to both the κ2 and κ3 complexes are observed, including signals attributed to the protonated phenol moieties. Signals attributed to the ligand methylene groups are rendered diastereotopic upon coordination of the distal donor atoms to the Pd center, while methylene units for unbound donors remain magnetically equivalent.Solution NMR data PdCl] subunits. Details of this interaction are illustrated in Fig. 23 species, viewed along the a axis. Additional O—H⋯Cl interactions are observed between O3—H3 and Cl1, and O6—H6 and Cl3, though neither of these interactions forms an extended network. The interaction between O3—H3 and Cl1 is of interest as it is the only observed close contact between the κ2 and κ3 complexes within the asymmetric unit. This feature is absent in the minor component, in which the [PdCl2] phenol hydroxy moiety O3B—H3D exhibits an intramolecular close contact with Cl3. Within the minor component, a related intramolecular close contact is observed between the remaining [PdCl2] phenol hydroxy group O4B—H4D and Cl2.Hydrogen bonding Table 2 is obsertert-butylphenol) ligand and bis(benzonitrile)dichloropalladium(II) in 5 mL of acetonitrile, using the method reported previously by Wile and co-workers .Both species within this unit cell are generated upon combining equimolar quantities (0.254 mmol) of the {6,6′-bis(methylene)}bis and polarization transfer (DEPT-135) experiments. 1H  δ = 8.05 , 7.51 , 7.42 , 7.36 , 7.17–7.08 , 6.83 , 6.55 , 4.64–4.50 , 3.79–3.65 , 3.59–3.48 , 3.35–3.20 , 3.00 , 2.92–2.80 , 2.73–2.64 , 2.47 , 2.37–2.29 , 2.19–2.10 , 1.62 , 1.50 , 1.47–1.38 , 1.32 , 1.30 , 1.27 , 1.19 ; 13C{1H}  δ = 158.1 (4°), 153.3 (4°), 141.6 (4°), 139.6 (4°), 136.3 (4°), 129.0 (aryl C-H), 128.7 (4°), 125.5 (4°), 124.9 (4°), 124.0 (aryl C-H), 121.0 (4°), 117.6 (4°), 65.5 (CH2), 62.6 (CH2), 62.5 (CH2), 61.1 (CH2), 55.8 (CH2), 51.5, 41.7 (CH3), 35.4 (tBu 4°), 35.3 (tBu 4°), 35.0 (tBu 4°), 34.3 (tBu 4°), 34.1 (tBu 4°), 33.8 (tBu 4°), 31.6 (CH3), 30.2 (CH3), 30.1 (CH3), 29.9 (CH3), 29.6 (CH3). m.p. 435 K (decomp.)Crystal data, data collection and structure refinement details are summarized in Table 32 and CH3 moieties, respectively. Phenol and methanol O—H distances were constrained to 0.84 Å. Methyl CH3 and hydroxyl H atoms were allowed to rotate but not to tip to best fit the experimental electron density. Uiso(H) values were set to a multiple of Ueq(C/O) with 1.5 for CH3, OH and water, and 1.2 for C—H, CH2, units, respectively.Water H atoms were restrained to have O—H bond lengths of 0.84 (2) Å, and 1.36 (2) Å H⋯H distances . All H atoms attached to carbon atoms as well as phenol and methanol hydroxyl hydrogens were positioned geometrically and constrained to ride on their parent atoms. C—H bond distances were constrained to 0.95 Å for aromatic C—H moieties, and to 0.99 and 0.98 Å for aliphatic CHSHELX, esd = 0.02 Å). Minor O atom O2B was constrained to have the same ADP as the C atom to which it is bonded. Two phenol H-atom positions were positionally restrained based on hydrogen-bonding considerations and to avoid close contacts to C-bound H atoms. Subject to these conditions, the occupancy rates of the major moieties refined to 0.917 (3), 0.857 (4) and 0.899 (4).Three of the four phenol hydroxyl groups are positionally disordered by rotation of the aromatic ring. For two of the three minor moieties, the O—C distance and the 1,3 O to C distances of the minor and major moieties were restrained to be similar . Subject to these conditions the occupancy ratio refined to 0.716 (8):0.284 (8).A Uij components of ADPs were restrained to be similar for the three methanol and the one water moiety . Subject to these conditions the occupancy ratio refined to 0.685 (8):0.315 (8) for the methanol-to-water ratio, and 0.843 (4):0.157 (4) for the methanol-to-methanol ratio.One solvate methanol molecule was refined as disordered over two orientations, and another to be disordered with a water molecule. The O—C distances of the major and minor methanol molecules were restrained to be similar , and the 10.1107/S2056989019010454/lh5914sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989019010454/lh5914Isup2.hklStructure factors: contains datablock(s) I. DOI: 1940199CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Additional C—H⋯O hydrogen bonds link the chains into layers parallel to (10The asymmetric unit of the title compound consist of two independent molecules. In the crystal, N–H⋯O and C—H⋯O hydrogen bonds form zigzag chains along the  14H11N3O3, consists of two independent molecules having very similar conformations in which the indazole moieties are planar. The independent molecules are distinguished by small differences in the rotational orientations of the nitro groups. In the crystal, N—H⋯O and C—H⋯O hydrogen bonds form zigzag chains along the b-axis direction. Additional C—H⋯O hydrogen bonds link the chains into layers parallel to (10The asymmetric unit of the title compound, C H and 2H-indazoles are well recognized for their anti-hypertensive and anti-cancer properties while other indazole derivatives are a versatile class of compounds that have found use in biology, catalysis and medicinal chemistry A⋯O5 and N4—H4A⋯O2 hydrogen bonds coupled with C16—H16⋯O1 hydrogen bonds form zigzag chains extending along the b-axis direction n Table 1. These cs Table 1. The layet al., 2016et al., 2011et al., 2000et al., 2000H-indazol-3-ol -1H-indazole ; Godbout et al., 1992Gaussian16 program  and DFT exchange utilizing the B3 functional together with the LYP correlation functional dnorm) of the title compound is shown in Fig. 6dnorm surface can be used to identify very close intermolecular interactions with dnorm being negative (positive) when intermolecular contacts are shorter (longer) than the sum of the van der Waals radii. The dnorm value is mapped onto the Hirshfeld surface by red, white or blue colours. The red regions represent closer contacts with a negative dnorm while the blue regions represent longer contacts with a positive dnorm and the white regions represent contacts equal to the van der Waals separation with dnorm equal to zero. As depicted in Fig. 6The standard resolution molecular Hirshfeld surface . Including the reciprocal contacts, the contribution of the O⋯H contacts (15.7%) for the title compound is larger than that of the N⋯H contacts (4.6%) [Fig. 7b) and 7(c)].The 2D fingerprint plots highlight particular atom-pair contacts and enable the separation of contributions from different interaction types that overlap in the full fingerprint. Using the standard 0.6–2.6 Å view with the 6-Nitro-3-(4-methoxyphenyl)-1H-indazole (I)v/v) in a microwave tube with a stir bar were added p-methoxyphenylboronic acid (1.5 equiv.), a solution of caesium carbonate (1.3 equiv.) dissolved in 0.5 mL of H2O and Pd(PPh3)4 (0.1 equiv.) under argon. The reaction vessel was sealed with a silicone septum and was subjected to microwave irradiation at 413 K with stirring. The reaction mixture was then allowed to cool to room temperature, diluted with ethyl acetate (15 mL) and water (10 mL) and extracted (3 times). The combined organic layer was dried over MgSO4 and concentrated under reduced pressure. The crude material was purified by column chromatography on silica gel (EtOAc/Ether) to give the desired final product. Yield: 74%. Orange solid, m.p. 503–505 K. 1H NMR  δ 13.74 , 8.46 , 8.24 , 7.96 , 7.92 , 7.10 , 3.82 . 13C NMR  δ 159.8, 146.1, 144.2, 140.7, 128.7, 125.3, 123.3, 122.4, 115.5, 114.9, 107.8, 55.6. HRMS (ESI) m/z calculated for C14H11N3O3 [M + H]+: 270.0834, found 270.0780.To a solution of 6-nitroindazole (0.1 g) dissolved in 1.5 mL of a mixture of 1,4-dioxane/EtOH  = 1.2–1.5Ueq.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S205698901801647X/ff2156sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S205698901801647X/ff2156Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698901801647X/ff2156Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S205698901801647X/ff2156Isup4.cmlSupporting information file. DOI: 1879920CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Myosin is a large family of actin-based molecular motors, which includes efficient intracellular transporters that move cargoes and material essential for cell’s life. Here, we describe protocols for labelling single myosin motors with quantum dots, tracking them in an in vitro reconstituted single-molecule motility assay, acquiring image stacks and analyzing them. We describe the required steps to obtain trajectories of single myosin motors from which fundamental biophysical parameters such as the motor velocity, run length and step size can be derived. We also describe protocols for an ensemble actin gliding assay, which is valuable to test the motor viability and its ensemble properties. The protocols allow probing the effect of changes in nucleotides, ions, and buffer composition on the motor properties and are easily generalizable to track the movements of different motor proteins. Specifications TableValue of the Protocol:processive, which means that they perform a large number of steps and ATP hydrolysis cycles before dissociating from actin  = 1–2 mM). : ATP regenerating system , 100 μg/ml creatine phosphokinase (Sigma C3755), prepare them fresh). ATP regeneration system is very important at low ATP concentration (< 500 μM) to restore ATP from ADP + Pi in the solution and maximize the observation time. •Bio-HMM-5B (Section 1)•Rhodamine-phalloidin labeled F-actin (Section 2)Materials:1Prepare a flow cell as described in Section 3.2Flux biotinylated BSA 1 mg/ml and incubate for 3 min.3Wash with 3 volumes of MB-KCl.4Flux streptavidin 1 mg/ml and incubate for 5 min.5Wash with 4 volumes of MB-KCl.6Flux bio-HMM-5B 0.4 μM and incubate for exactly 5 min.7While incubating, prepare IB and RM.8Wash with 4 volumes of biotinylated BSA 1 mg/ml.9Flux 23 nM rhodamine phalloidin F-Actin (in MB-KCl supplemented with 10 mM DTT), incubate for 2 min.10Wash with 4 volumes of IB.11 Check under the microscope that a reasonable number of actin filaments are firmly bound to the coverslip surface.12Wash with RM with the desired ATP concentration.13 Close carefully the chamber with silicone grease. Alternatively, leave the flow cell open to change the actin and ATP concentration on the same sample.14Acquire images.Methods:In the single molecule motility assay, bio-HMM-5B is first attached to streptavidinated QDs at single molecule concentration. Then, a flow chamber is built in which fluorescently-labelled actin filaments are attached on a bed of inactivated N-ethylmaleimide (NEM) myosin II on the coverslip surface . QD-labe•Bio-HMM-5B 0.04-0.2 μM•Streptavidin-QD 655 nm (Life Technologies Q10123MP) 0.2 μM•3, pH 7.3. Myosin-V Buffer (VB): 0.5 M NaCl, 10 mM MOPS, 0.1 mM EGTA, 3 mM NaN•D-Biotin 10 mg/ml in VB. •Binding Buffer (BB): VB supplemented with 0.1 mg/ml α-casein, 2 μM CaM, 3 mM DTT.Materials:1In a 0,5 ml tube, mix1 μl of bio-HMM-5B 0.04-0.2 μM, 1 μl of streptavidin-QDs 0.2 μM, and 2.5 μl of BB. The probability of having no more than one myosin head attached per QD increases as the bio-HMM-5B concentration decreases and, assuming Poisson statistics, it is about 91% at 1:5 bio-HMM-5B:QD molar ratio .2Put on ice for > 30 min.310 min before fluxing in the chamber, add 0.5 μl of D-Biotin 10 mg/ml to saturate all free streptavidin sites on the QDs. Leave all components on ice during assembly of the single molecule motility assay (next section).Methods•Flow chamber (Section 3)•Motility Buffer with KCl (MB-KCl): 20 mM MOPS pH 7.4, 1 mM MgCl2, 0.1 mM EGTA, 50 mM KCl, and 1 mM DTT. (Tip: filter sterilize and store it at 4 °C for a couple of weeks)•Motility Buffer with α-casein (MBα): MB-KCl supplemented with 0.1 mg/ml α-casein•Imaging Buffer (IB): MB-KCl buffer supplemented with 1.2 μM glucose oxidase, 0.2 μM catalase, 4.2 μM alpha-casein, 17 mM glucose, and 20 mM DTT. •Imaging Buffer with calmodulin (IB-C): IB buffer supplemented with 2 μM CaM•Reaction Mix (RM): IB supplemented with 0.1 mg/ml creatine phosphokinase, 5 mM creatine phosphate, 2 μM CaM and ATP at the desired concentration (Tip: RM must be prepared fresh for every assay)•2 High Salt (HIS) Buffer 2x (pH 7.5): 1 M KCl, 40 mM KPi, 2 mM EGTA, 4 mM MgCl•NEM-myosin II (20 mg/ml) in HIS 2x (see protocol in Batters and Veigel ) (Tip: s•μl of previously prepared bio-HMM-5B conjugated to streptavidinated QDs 655 (QD-HMM-5B) 40 nM (Section 5.1)•Rhodamine-phalloidin F-actin (Section 2)Materials:1Prepare a flow cell as described in Section 3 and leave it on ice 2Prepare all buffers and leave them on ice3Flux NEM-Myosin II 2 mg/ml in HIS 2x and incubate for exactly 1 min4Wash with 3 volumes of MBα5Flux 23 nM rhodamine phalloidin F-Actin (in MB-KCl supplemented with 10 mM DTT) and wait for 2 min.6Wash with 4 volumes of IB7Dilute 5 μl of QD-HMM-5B in 95 μl IB-C to get 2 nM QD-HMM-5B.  must be done)8 Dilute QD-HMM-5B prepared in step 7 to 0.1 nM in IB-C and flux it in the chamber and check under the microscope that a reasonable number of QDs are attached on actin filaments.9Dilute QD-HMM-5B prepared in step 7 to 0.1 nM in RM at the desired ATP concentration and flux it in the chamber.10 Close carefully the chamber with silicone grease. Alternatively, leave the flow cell open to change the QD-HMM-5B and ATP concentration on the same sample.11Run under the microscope for watching myosins moving along actin filaments!Methods:Images are acquired on a modified Nikon ECLIPSE TE300 inverted fluorescence microscope  equippedFor the gliding assay: F-actin Exc/Em. 532/580 nm. Laser power on the sample was about 3 mW, 200 ms exposure time and 200 EM gain are usually fine .Fig. 5ThFor the single molecule motility assay: F-actin Exc/Em. 532/580 nm, laser power on the sample was about 3 mW; QD-HMM-5B Exc/Em. 488/655 nm, laser power on the sample was about 3 mW. Exposure time is set according to ATP concentration and consequent velocity between 50 and 100 ms. EM gain was 300 .Fig. 6ThGliding assay: single filaments characterized by a travelled distance >1 μm were tracked over the frames with the ImageJ Plugin “Multitracker”. The filament velocity was calculated frame by frame and the average velocity with its standard error was associated to each filament. The final average velocity was calculated by weighted average with associated standard error. By this analysis we obtained an average gliding velocity of 163 ± 3 nm/s at 1 mM ATP (N = 20 filaments).Single molecule motility assay: single QDs were localized frame by frame with nanometer precision by a custom-made software named “PROOF” that is freely available and widely described elsewhere . Localiz


Bacillus megaterium levansucrase variants with focus on the synthesis of levan- and inulin-type FOS. Modifications were introduced at positions R370, K373 and F419, which are either part of the oligosaccharide elongation pathway or are located in the vicinity of residues that modulate polymerization. These amino acids were exchanged by residues of different characteristics, some of them being extremely low- or non-represented in enzymes of the levansucrase family . F419 seemed to play a minor role in FOS binding. However, changes at R370 abated the levansucrase capacity to synthesize levan-type oligosaccharides, with some mutations turning the product specificity towards neo-FOS and the inulin-like sugar 1-kestose. Although variants retaining the native R370 produced efficiently levan-type tri-, tetra- and pentasaccharides, their capacity to elongate these FOS was hampered by including the mutation K373H or K373L. Mutant K373H, for instance, generated 37- and 5.6-fold higher yields of 6-kestose and 6-nystose, respectively, than the wild-type enzyme, while maintaining a similar catalytic activity. The effect of mutations on the levansucrase product specificity is discussed.The connection between the gut microbiome composition and human health has long been recognized, such that the host-microbiome interplay is at present the subject of the so-called “precision medicine”. Non-digestible fructooligosaccharides (FOS) can modulate the microbial composition and therefore their consumption occupies a central place in a strategy seeking to reverse microbiome-linked diseases. We created a small library of  The microbiome makes up to 100 trillion cells, ten-fold more than cells in an higher eukaryote host2. The proliferation of beneficial bacteria known as probiotics (i.e. bifidobacteria and lactobacilli) can be modulated by the dietary consumption of prebiotics, which is often accompanied by a decline in the number of pathogenic species. Anxiolytic and antidepressant effects are among the major benefits for the host well-being that have been associated with the metabolism of probiotics3. With the advent of the ‘omics era, the treatment for microbiome-linked diseases pursues a personalized care, increasing the demand for pre- and probiotics. In consequence, the market for prebiotics and other functional food ingredients has kept a brisk pace over the last three decades, and is poised to exceed USD 750 million by 2024, stimulated by concerns towards diabetes, obesity and digestive health (https://www.gminsights.com/). Inulin (1F), a well-known functional ingredient is predominantly isolated from chicory and can be hydrolyzed to obtain mixtures of products of lower degree of polymerization (DP)4. Short chain 1F-type FOS have half the calories per gram than traditional sweeteners, being thus attractive as food additives in low-caloric products5. Trisaccharides 1- and 6-kestose as well as the tetrasaccharide nystose and oligosaccharides of the neo-series possess bifidogenic activity7.The French gastronome Jean Anthelme Brillat-Savarin wrote in his Physiologie du Gout: “Dis-moi ce que tu manges, je te dirai ce que tu es” (Tell me what you eat and I will tell you what you are). Almost two centuries later the connection between the intestinal microbiome composition and human digestive and mental health continues to amaze researchers6F) contain β2-1 and β2-6 linkages, respectively, and are synthesized by enzymes from families GH32 and GH68 using sucrose as starting material. While 1F-type FOS are well studied and are commercialized by different companies, 6F-type products such as 6-kestose and 6-nystose are less common. Some enzymes from family GH32, i.e. fructofuranosidases from yeast Schwanniomyces occidentalis or an invertase from Saccharomyces cerevisiae have been successfully used in the synthesis of 6F-type oligosaccharides9. However, most levansucrases from family GH68 synthesize polymers larger than 2 × 106 Da, and some of them produce a mixture of polymer and FOS12. 6-kestose is the first product of levansucrase-catalyzed transfructosylation reactions and is as well the best acceptor of the fructosyl-moiety during polymerization. Accordingly, it does not accumulate in substantial amounts during the reaction course, this also being the case for other short FOS with β2-6 linkages.Inulin and levan  and B. megaterium (PDB 3om2) levansucrases shows that the side chain of residue F419 (F409 in B. subtilis levansucrase) is partially buried and therefore not expected to engage with elongating FOS via CH–π interactions. Exchange of F419 by other amino acids might, however, have an impact in the orientation of the side chains of adjacent residues R370 and Y421, both known for their participation in product binding and control of the hydrolysis/transfer partition18. Substitution of residue F419 could thus indirectly provoke changes in the +1 and +2 subsites.A number of 20. These positions were revisited in this work to allow substitutions previously unexplored, and to expand the range of possible amino acid combinations. By diminishing the levansucrase ability to produce larger products, we anticipated the accumulation of short-length chain oligosaccharides, in particular 6-kestose and 6-nystose, whose biological activity requires a more in-depth study.The role of residues R370 and K373 in the modulation of the polymerization process is well establishedSingle variant K373L was employed for the synthesis of 1-kestose, 6-kestose and 6-nystose, which were received with a purity higher than 95%.B. megaterium levansucrase variants was designed to introduce changes in positions R370, K373 and F419  of sucrose when it acts as donor  containing B. megaterium levansucrase library. Cleared-cell lysates of 768 clones were screened for global activity (hydrolysis and transfer) on 0.5 m sucrose through the detection of reducing sugars after 10 min incubation at 37 °C. The product profile of active variants was analyzed on TLC after incubation of 1 µL cleared-cell lysates with 0.5 m sucrose for 20 h , while glutamine is found at this position in the levansucrases from Erwinia amylovora (PDB 4d47) and G. diazotrophicus (PDB 1w18)  is directed toward the oligosaccharide binding surface and is partially exposed. Although some orientations of the glutamic acid side chain (rotamers) could theoretically be tolerated at position 419, some other may induce a major disturbance of adjacent loops ; however, this exchange resulted in a nearly inactive variant. Alanine is found at the equivalent position in the inulosucrase from B. subtilis levansucrase single variant R360H21 (R370H in this publication) and B. megaterium levansucrase K373R19. Variants from these two groups synthesized short FOS inefficiently, with mutant K373R (1G2) producing FOS of DP 4-8 in similar yields to those of the wild-type enzyme  and 1-kestose (PDB 2yft) in the active site to retrieve information about the network of interactions needed to sustain polymerization of 1F-type products. Residue R542 in L. johnsonii inulosucrase , can interact with the glucosyl-moiety of sucrose at the +1 subsite and only with 1F-type-FOS at the +2 subsite, while R545 (K373 in B. megaterium levansucrase) can form hydrogen bonds with sucrose at the +1 subsite and with 1-kestose at the +1 and +2 subsites  resulted in a greater accumulation of 1-kestose and a higher production of an unknown disaccharide, probably formed of two fructosyl units Fig. . 1-kestoB. megaterium wild-type levansucrase increases over the reaction course and neokestose is largely hydrolyzed to blastose once sucrose is depleted18. Group 4 variants hydrolyzed neokestose only partially at the final reaction stage   for instance, produced 37- and 5.6-times more 6-kestose and 6-nystose, respectively, than the wild-type enzyme  showed that the substitution by alanine did not disturb the catalytic residues and had only a small effect on the conformation of the side chains of residues spatially close to the mutation19. The analogous mutation (K372A) in B. licheniformis levansucrase resulted in the synthesis of neokestose > 1- kestose > 6-kestose20. While arginine and histidine are expected to provide stabilization of growing oligosaccharides via hydrogen bonds, leucine is not. The product profile of mutants K373H and K373L was nonetheless similar  column chromatography and 2) a combination of a fermentative treatment of the crude oligosaccharide mixture with the yeast Hansenula polymorpha and column chromatography , which displayed the best catalytic efficiency among enzymes from group 5 was selected for the synthesis and isolation of 1-kestose, 6-kestose and 6-nystose, with special focus on the last two products. Glucose, fructose and residual sucrose are the main sub-products in oligosaccharides synthesis, often hampering the downstream processing. Frequent methods employed in fractionation and purification of FOS include ultra- and nano-filtrationphy Fig. . H. polym sucrose and 3 U mL−1 of variant K373L at 37 °C for 48 h. After this reaction time there were only traces of sucrose left, which facilitated the purification procedure and limited the first separation step to the monosaccharides. The concentrations of produced glucose and fructose in the raw product were 248 mm and 194 mm, respectively. Both fermentative and chromatographic separation of glucose and fructose were performed in quantitative yields and without noteworthy loss of FOS . Although the strategy for production and purification of these sugars was not optimized in this work and requires further development, it proved how engineered levansucrases can be applied for gaining access to custom oligosaccharides.The association of the gut microbiome composition with the occurrence and development of both gastrointestinal and extragastrointestinal diseases in humans has been in the spotlight over the last decades. Special attention has been devoted to the bidirectional communication between the central and enteric nervous systems, the so-called “gut-brain-axis”B. megaterium levansucrase as a query (D5DC07). Proteins with a similarity threshold of 0.95 were clustered with CD-Hit37 to reduced sequence redundancy. Sequences not containing the conserved GH68 sequence motifs 94WD95, 172WXGX175, 256RDP258, 349DXXER353, and 421YSX423 which are part of the central cavity of the β-propeller, were also discarded. Non-redundant sequences (372) were aligned with MUSCLE38 . E. coli BL21(DE3) was transformed for expression of the library variants. Inserts from 15 random selected clones were sequenced to validate the library quality. No unwanted mutation and an even distribution of the desired codons were obtained.The library was generated following the method described previously−1 kanamycin were used to inoculate 400 µL of ZYM505/well containing the appropriate antibiotic. The plates were covered with an air permeable foil and grown at 37 °C overnight in a TIMIX incubator at 1000 rpm. The final OD600 for each well was around 10–15. These pre-cultures were used to inoculate 1 mL ZYM505/well with appropriate antibiotic (starting OD600 around 0.05). Plates were shaken at 37 °C until an OD600 of 0.6–1.0 was reached. Expression was induced by adding IPTG at a final concentration of 0.5 mm. Cultures were incubated overnight at 20 °C. Cells were harvested by centrifugation and resuspended in 100 µL lysis-buffer/well . Deep well plates were incubated at 30 °C for 45 min in a TIMIX incubator at 1000 rpm and afterwards they were centrifuged at 3000 × g for 30 min. Cleared cell lysates were transferred to 96-well microtiter plates and used directly for screening assays.Single clones from agar plates containing 10 mg mLm Sörensen buffer pH 6.6) in 96 well plates. Sörensen buffer contains 50 mm Na2HPO4 and 50 mm KH2PO4. The reactions were stopped after 10 min by adding 100 µL DNS reagent. Plates were heated at 95 °C for 5 min and cooled down for 2 min at 4 °C. For quantification, samples were diluted 1:6 with water and the absorbance measured at 540 nm. Data was processed using a calibration curve of absorbance versus glucose concentration.Enzyme extracts were used to determine the initial activity at 37 °C by adding 1 µL crude extract to 99 µL sucrose solution (0.5 M sucrose and 50 mB. megaterium levansucrase variants on TLC. Reactions were performed in 96-well plates containing 1 µL enzyme crude extract and 99 µL sucrose solution (0.5 M sucrose and 50 mm Sörensen buffer pH 6.6). Plates were incubated at 37 °C during 20 h and reactions stopped with 100 µL of 50 mm NaOH. Standards and 1 µL of each enzymatic reaction were applied on TLC plates (silica gel 60 F254 with concentration zone), dried and run three times using WIE (water/iso-propanol/ethyl acetate 1/3/6) as eluent. The plates were dried and stained with N-(1-naphtyl)-ethylenediamine (0.3% m/v) in 5.0% (v/v) conc. H2SO4 and 94.7% (v/v) methanol.Enzyme extracts were used to determine the product profile of E. coli bearing selected plasmids was used to inoculate 10 mL LB-medium containing 10 µg mL−1 kanamycin. Precultures were incubated over night at 37 °C and used to inoculate 250 mL LB-medium with appropriate antibiotic. Expression of levansucrase was induced when cells reached an OD600 of around 0.6 by adding IPTG at a final concentration of 0.5 mm. Cultures were incubated over night at 20 °C. Cells were harvested by centrifugation and resuspended in 7 mL of 20 mm Sörensen buffer pH 6.0. After sonication, extracts were cleared by centrifugation at 13,000 × g. Cleared lysates were loaded onto a CM sepharose column and levansucrase eluted with a linear gradient of Sörensen buffer from 20 mm to 1.0 m. Purification fractions were pooled and concentrated. An extinction coefficient of 64750 M−1 cm−1 and a path length of 1 cm were used to calculate concentration of purified enzymes measured at 280 nm.One single colony of freshly transformed B. megaterium levansucrase variants. 1 mL reactions containing 50 mm Sörensen buffer pH 6.6, 0.5 mm sucrose and 2 U mL−1 of each enzyme were incubated at 37 °C. Samples (50 µL) were taken over the reaction course and enzymes inactivated by adding an equal volume of 50 mm NaOH. After centrifugation  to remove precipitated protein the samples were diluted 1,000-times and analyzed by HPAEC-PAC. Analysis of oligosaccharides was performed with a Dionex ICS-5000+ SP system utilizing a CarboPac PA10 column with pulsed amperometric detection. Elution solvents employed for separation were 100 mm NaOH (A) and 100 mm NaOH/1 M NaOAc (B). A multistep gradient was programmed as follows: 0–5 min 100% A, 5–30 min 0–50% B, 30–45 min 100% A. Appropriate standards were used for quantification of some products, while comparison of relative areas was utilized for products with unknown structures. The concentration (or peak area) of oligosaccharide products was calculated from three independent experiments.Enzyme extracts were used to determine the product profile of m Sörensen buffer pH 6.5 containing varying concentrations of sucrose and purified levansucrase variants. Initial rates were determined by the DNS method, quantifying the release of reducing sugars over the time course. KM and kcat values were calculated by fitting the data to the Michaelis-Menten equation using a non-linear curve in OriginPro (OriginLab). Experiments were performed in duplicate.Catalytic parameters of levansucrase variants were obtained from reactions carried out in 50 mm sucrose, 50 mm Sörensen buffer pH 6.6 and 3 U mL−1 of the levansucrase variant K373L were incubated at 37 °C for 48 h. The solution was stopped by heating at 95 °C for 10 minutes. After filtration of the mixture (0.2 µm exclusion size) the solvent was removed under reduced pressure to obtain 19.5 g of a colorless crude product containing mono-, di- and oligosaccharides.125 mL of an aqueous solution containing 0.5 H. polymorpha. The eluent for the silica gel column was H2O/isopropanol/ethyl acetate (1:3:6). For the fermentation of glucose and fructose, 5 g of the crude product were dissolved in 150 mL ddH2O and 50 g of immobilized H. polymorpha yeast were added to consume the monosaccharides. The mixture was incubated at 25 °C for 24 h. The fermentation reaction was inactivated by heating at 95 °C for 10 min. Yeast alginates were withdrawn by filtration and the solvent was removed under reduced pressure to yield 2.9 g of a colorless syrup. The syrup was purified over the size exclusion column Bio-Gel® P2 (eluent ddH2O) to obtain FOS with a degree of polymerization of 3–4 and impurities of glycerol. Further purification with reversed phase chromatography  led to the desired products 1-kestose , 6-kestose  and 6-nystose .For the elimination of the high glucose and fructose content two methods were tested: normal phase column chromatography with silica gel and fermentation with the yeast H. polymorpha. After incubation overnight at 25 °C the pre-cultures were used for inoculation of 250 mL YM-medium. The cells of the main culture were grown at 25 °C and 140 rpm for 24 h. The yeast cells were harvested by centrifugation at 8,000 rpm for 10 min. The supernatant was discarded and the cells were immediately used for immobilization or stored at −20 °C.The pre-cultures of 5 mL YM-medium were inoculated by selecting one single colony of YM-medium contains yeast extract (3.0 g), malt extract (3.0 g), peptone from soybeans (5.0 g) and glucose (10.0 g). It was filled up with distilled water to 1 L.2O and heated to 70 °C until a viscous solution resulted. After cooling the alginate solution to room temperature 12.5 g of H. polymorpha (wet weight) was added to give a homogeneous suspension. The alginate-yeast suspension was dropped steadily with a syringe pump to a 150 mm calcium chloride solution. After the immobilized beads were stirred for 1 h to harden, they were filtrated and used immediately or within the next 24 h.1.56 g alginate was added to 100 mL ddH1H-NMR : σ = 5.40 , 4.25 , 4.16 , 4.05 , 4.02 , 3.86–3.83 , 3.86 – 3.79 , 3.83–3.80 , 3.83–3.74 , 3.82–3.78 , 3.79–3.77 , 3.74–3.71 , 3.73–3.64 , 3.71–3.67 , 3.52 , 3.46–3.43  ppm.13C-NMR : σ = 103.7 (CFru2-2″), 103.2 (CFru1-2′), 92.4 (CHGlu-1), 81.1 , 76.5 , 74.4 (CHFru2-4″), 73.8 (CHFru1-4′), 72.5 (CHGlu-3), 72.3 (CHGlu-5), 71.1 (CHGlu-2), 69.1 (CHGlu-4), 62.2, , 60.8 , 60.0  ppm.1H-NMR : σ = 5.35 , 4.17 , 4.14 , 4.07 , 4.02 , 3.94 – 3.91 , 3.86–3.84 , 3.84–3.79 , 3.83–3.77 , 3.77–3.75 , 3.74–3.71 , 3.66–3.59 , 3.54–3.52 , 3.45–3.42  ppm.13C-NMR : σ = 103.7 , 92.1 (CHGlu-1), 81.1 (CHFru2-5″), 80.3 (CHFru1-5′), 76.4 (CHFru2-3″), 76.0 (CHFru1-3′), 74.6 (CHFru2-4″), 74.3 (CHFru1-4′), 72.5 (CHGlu-3), 72.4 (CHGlu-5), 71.0 (CHGlu-2), 69.2 (CHGlu-4), 62.9 , 62.6 , 61.1 , 60.2 , 59.7  ppm.1H-NMR : σ = 5.40 , 4.24 , 4.16 , 4.15 , 4.10 , 4.04 , 4.02 , 3.94–3.91 , 3.87–3.84 , 3.83–3.82 , 3.83–3.76 , 3.82–3.80 , 3.82–3.78 , 3.81 , 3 0.76–3.75 , 3.75–3.72 , 3.73 , 3.68–3.64 , 3.67–3.65 , 3.52 , 3.45  ppm.13C-NMR : σ = 103.9 (CFru3-2″′), 103.7 (CFru2-2″), 103.2 (CFru1-2′), 92.4 (CHGlu-1), 81.2 (CHFru2-5″), 81.1 (CHFru1-5′), 80.2 (CHFru3-5″′), 76.5 (CHFru2-3″), 76.4 (CHFru1-3′), 76.3 (CHFru3-3″′), 75.1 (CHFru3-4″′), 74.6 (CHFru2-4″), 73.7 (CHFru1-4′), 72.5 (CHGlu-3), 72.3 (CHGlu-5), 71.0 (CHGlu-2), 69.1 (CHGlu-4), 63.1 , 62.5 , 62.0 , 60.7 , 60.3 , 60.0 , 59.8  ppm.Supplementary Information


H-indol-3-yl)methyl]imidazothiadiazoles , the molecules are linked by a combination of N—H⋯N and C—H⋯π interactions to form chains when the 6-aryl substituent is phenyl or 4-fluorophenyl and a three-dimensional framework when the 6-aryl group is 4-bromophenyl.In the crystals of three new 6-aryl-2-(4-chlorobenzyl)-5-[(1 H-indol-3-yl)methyl]-6-phenylimidazothiadiazole, C26H19ClN4S, (I), 2-(4-chlorobenzyl)-6-(4-fluorophenyl)-5-[(1H-indol-3-yl)methyl]imidazothiadiazole, C26H18ClFN4S, (II), and 6-(4-bromophenyl)-2-(4-chlorobenzyl)-5-[(1H-indol-3-yl)methyl]imidazothiadiazole, C26H18BrClN4S, (III), have been prepared using a reductive condensation of indole with the corresponding 6-aryl-2-(4-chlorobenzyl)imidazothiadiazole-5-carbaldehydes , and their crystal structures have been determined. The asymmetric unit of compound (I) consists of two independent molecules and one of the molecules exhibits disorder of the 4-chlorobenzyl substituent with occupancies 0.6289 (17) and 0.3711 (17). Each type of molecule forms a C(8) chain motif built from N—H⋯N hydrogen bonds, which for the fully ordered molecule is reinforced by C—H⋯π interactions. In compound (II), the chlorobenzyl unit is again disordered, with occupancies 0.822 (6) and 0.178 (6), and the molecules form C(8) chains similar to those in (I), reinforced by C—H⋯π interactions involving only the major disorder component. The chlorobenzyl unit in compound (III) is also disordered with occupancies of 0.839 (5) and 0.161 (5). The molecules are linked by a combination of one N—H⋯N hydrogen bond and four C—H⋯π interactions, forming a three-dimensional framework.Three title compounds, namely, 2-(4-chlorobenzyl)-5-[(1 Despite the close similarity in the chemical constitution of compounds (I)–(III), no two of these compounds are isomorphous. None of the molecules exhibits any internal symmetry, so that all of them are conformationally chiral. The centrosymmetric space group for the compounds (I)et al., 1996Although compounds (I)The asymmetric unit of compound (I)b]thiadiazole ring system differs quite significantly between compounds (I)x1—Cx2—Cx27—Cx21/Cx31 x4—Cx5—Cx51—Cx53 and Cx5—Cx51—Cx53—Cx52  chain motif running along [010]  chain along [010]. These chains differ in that the second chain is reinforced by two C—H⋯π interactions, whereas in the first chain, only the minor disorder component takes part in such an interaction; in the major disorder component, the shortest intermolecular H⋯Cg distance exceeds 3.3 Å  .In the crystal of compound (I)0] Fig. 4. Similar1 screw axis, into a C(8) chain running along [010]. This hydrogen bond is augmented by the C—H⋯π interaction on Fig. 5. There aCg distances (Table 2C(8) chain along [010] s Table 2. The N—H0] Fig. 6. Two C—H0] Fig. 6, cooperaon Fig. 7. Finally1] Fig. 8. The comB) in Fig. 9b]thiadiazole  in Fig. 9b]thiadiazole-5-carbaldehyde imidazothiadiazol-2-yl]methyl}-1,2-benzoxazole , were prepared in a three-step sequence, as shown in Fig. 9A), using an established methodology , followed by Vilsmeier–Haack formylation to give the corresponding 5-carbaldehydes, (C), and finally reductive condensation with indole in the presence of triethylsilane and trifluoroacetic acid , but unfortunately no crystals of this compound have yet been obtained, only a viscous gum.The title compounds, CX = H: yield 58%, m.p. 493–495 K; HRMS found 455.0000. C26H1935ClN4S requires for (M + H)+ 455.1019. Compound (II)X = F: yield 48%, m.p. 483–485 K; HRMS found 473.0620, C26H1835ClFN4S requires for (M + H)+ 473. 0925. Compound (III)X = Br: yield 52%, m.p. 393–395 K; HRMS found 532.8687, C26H1879Br35ClN4S requires for (M + H)+ 533.0124. Crystals of (I)–(III) suitable for single-crystal X-ray diffraction were grown by slow evaporation in the presence of air of solutions in ethyl acetate at ambient temperature. 4-Chlorophenyl analogue (X = Cl): yield 48%, m.p. 503–505 K; HRMS found 488.914, C26H1835Cl2N4S requires for (M + H)+ 489.0629.Compound (I)2), and with Uiso(H) = 1.2Ueq(C). For the H atoms bonded to N atoms, the atomic coordinates were refined with Uiso(H) = 1.2Ueq(N), giving refined N—H distances of 0.83 (3)–0.99 (5) Å. On this basis, the refined occupancies of the disorder components were 0.6289 (17) and 0.3711 (17) for (I)Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019016050/is5527sup1.cifCrystal structure: contains datablock(s) global, I, II, III. DOI: 10.1107/S2056989019016050/is5527Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989019016050/is5527IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989019016050/is5527IIIsup5.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989019016050/is5527Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019016050/is5527IIsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019016050/is5527IIIsup7.cmlSupporting information file. DOI: 1968781, 1968780, 1968779CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


X (X = O or N) interactions. One of them is a polymorph of a known structure.The title compounds, which differ in the substituent at the 4-position of the pendant benzene ring, show different packing motifs mediated by weak C—H⋯ H-benzo[7]annulen-5-one] derivatives, viz. 2-(4-methoxybenzylidene)-1-benzosuberone, C19H18O2, (I), 2-(4-ethoxybenzylidene)-1-benzosuberone, C20H20O2, (II), 2-(4-benzylbenzylidene)-1-benzosuberone, C25H22O2, (III), 2-(4-chlorobenzylidene)-1-benzosuberone, C18H15ClO, (IV) and 2-(4-cyanobenzylidene)-1-benzosuberone, C19H15NO, (V), are described. The conformations of the benzosuberone fused six- plus seven-membered ring fragments are very similar in each case, but the dihedral angles between the fused benzene ring and the pendant benzene ring differ somewhat, with values of 23.79 (3) for (I), 24.60 (4) for (II), 33.72 (4) for (III), 29.93 (8) for (IV) and 21.81 (7)° for (V). Key features of the packing include pairwise C—H⋯O hydrogen bonds for (II) and (IV), and pairwise C—H⋯N hydrogen bonds for (V), which generate inversion dimers in each case. The packing for (I) and (III) feature C—H⋯O hydrogen bonds, which lead to [010] and [100] chains, respectively. Weak C—H⋯π interactions consolidate the structures and weak aromatic π–π stacking is seen in (II) [centroid–centroid separation = 3.8414 (7) Å] and (III) [3.9475 (7) Å]. A polymorph of (I) crystallized from a different solvent has been reported previously [Dimmock et al.  in the present study. The Hirshfeld surfaces and fingerprint plots for (I) and its polymorph are compared and structural features of the 2-benzylidene-1-benzosuberone family of phases are surveyed.The syntheses and crystal structures of five 2-benzylidene-1-benzosuberone [1-benzosuberone is 6,7,8,9-tetrahydro-5 al. 1999 J. Med.  The conformations of the benzosuberone fragments in (I)–(V) are almost identical, as shown by the overlay plot generated with QMOL The molecular structures of (I)–(V) are shown in Figs. 1–5The dihedral angles between the C6–C11 fused benzene ring and the C13–C18 pendant benzene ring are clustered in a ∼12° range, with values of 23.79 (3) for (I)et al., 1999For (I)et al., 2016et al., 1999i.e.P21/c; VENQUA was recrystallized from methanol solution rather than ethanol for (I)PLATON There are obviously no classical hydrogen bonds in these structures and, in each case, just one C—H group can be identified as the donor for a weak hydrogen bond with atom O1 as the acceptor in (I)–(IV) and atom N1 in (V)C(8) graph-set motif results for both structures with the methyne group  involved as the donor.The packing motifs for the extended structures of (I)meta to the 4-substituent) is the donor group and The packing motifs for (II)C(8) chains arising from C15—H15⋯O1 hydrogen bonds seen in (I)−3) is significantly greater than that of (I)−3), suggesting that the former might be the more stable polymorph if the ‘rational packing rule’  corresponding to a C3—H2A⋯π interaction (H⋯π = 2.69 Å) to the centroid of the C6–C11 benzene ring, but there are also probably spurious features close to H8 and H17 corresponding to a short H⋯H contact of 2.07 Å between these atoms, which possibly arose because the H atoms of the C19 methyl group in VENQUA were geometrically placed and not treated using a rotating-group model. Notwithstanding this, the fingerprint plots for (I)In order to gain further insight into these different packing motifs, the Hirshfeld surfaces and fingerprint plots for (I) al. 2019. The Hiret al., 2016et al. Compounds (I)–(V) were obtained from the reaction of 1-benzosuberone (1 mmol) with the appropriate 4-substituted benzaldehyde (1 mmol) in ethanol (5 ml) treated with an ethanolic solution of sodium hydroxide (30 mg in 5 ml ethanol). After stirring for 3–4 h at room temperature, each reaction mixture was cooled to 0 °C and the precipitated solid was recovered by filtration and rinsing with ice-cold ethanol. Recrystallization from ethanol solution at room temperature yielded colourless blocks [(I), (III)Uiso(H) = 1.2Ueq(C) or 1.5Ueq(methyl C). The methyl groups in (I)Crystal data, data collection and structure refinement details are summarized in Table 810.1107/S2056989019014245/eb2024sup1.cifCrystal structure: contains datablock(s) I, II, III, IV, V, global. DOI: 10.1107/S2056989019014245/eb2024Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989019014245/eb2024IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989019014245/eb2024IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2056989019014245/eb2024IVsup5.hklStructure factors: contains datablock(s) IV. DOI: 10.1107/S2056989019014245/eb2024Vsup6.hklStructure factors: contains datablock(s) V. DOI: 1960122, 1960121, 1960120, 1960119, 1960118, 1960118, 1960119, 1960120, 1960121, 1960122CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Context: The effects of icariin, a chief constituent of ﬂavonoids from Epimedium brevicornum Maxim (Berberidaceae), on the levels of HIF-1α, HSP-60 and HSP-70 remain unknown.Objective: To explore the effects of icariin on the levels of HSP-60, HIF-1α and HSP-70 neuron-specific enolase (NSE) and cell viability.Materials and methods: PC12 cells were treated with icariin  for 3 h (1 h before oxygen–glucose deprivation (OGD) plus 2 h OGD). HSP-60, HIF-1α, HSP-70 and NSE were measured using enzyme-linked immunosorbent assay (ELISA). Cell viability was determined by metabolic 3--2,5-diphenyltetrazolium bromide (MTT) assay.Results: After 2 h OGD, levels of HIF-1α, HSP-60, HSP-70 and NSE were increased significantly , and cell viability was significantly decreased (0.26 ± 0.03), while icariin  significantly reduced the contents of HIF-1α, HSP-60, HSP-70 and NSE , and improved cell viability  in OGD-treated PC12 cells.Discussion and conclusion: These results indicate that the protective mechanisms of icariin against OGD-induced injury may be related to down-regulating the expression of HIF-1α, HSP-60 and HSP-70. Epimedium brevicornum Maxim (Berberidaceae)   , 5% heat-inactivated foetal bovine serum and 5% horse serum .Icariin (purity 98.3% by HPLC) was purchased from Nanjing Zelang Pharmaceutical Technology Co., Ltd . Icariin was dissolved in DMSO to 102 polystyrene flasks  with full culture medium as described above. The cells were incubated under an atmosphere of 95% air and 5% CO2. Culture medium was replaced every 48 h.PC12 cells  were seeded in 25 cmin vitro as previously described  under normoxic conditions.PC12 cells were deprived of oxygen and glucose for 2 h to simulate ischaemic injury −7, 10−6 or 10−5 mol/L) or nimodipine (10 μmol/L) under normoxic conditions for 1 h before hypoxia. The full culture medium containing drug was discarded. The cells were rinsed once with PBS, and incubated with Earle’s balanced salt solution containing icariin  or nimodipine (10 μmol/L) for 2 h of hypoxia.PC12 cells were incubated with full culture medium containing icariin   was added to each well. Plates were incubated at 37 °C in normoxia for 4 h. Dimethyl sulfoxide  (150 μL) was added to each well for 10 min to dissolve the dark blue crystals and the absorbance was read at 570 nm on a microplate reader .PC12 cells were seeded into 96-well plastic plates with 0.1 mL at the density of 1 × 105 cells/mL. It contained six samples in normal control group, OGD-treated group and OGD plus drug-treated groups, respectively. Then, after growth for 48 h, the cells were treated with OGD and drug administration as described above. After OGD and drug administration, 100 μL per well of cell culture supernatant fluid was used for measuring HSP-60 , HIF-1α , HSP-70  and NSE  using ELISA according to the corresponding manufacturer’s instructions.PC12 cells were seeded into 6-well plastic plates with 1 mL at the density of 5 × 10t test. A value of p < 0.05 was considered significant. All statistical analyses were performed with version SPSS 19.0 statistical software.Measurement data were expressed as mean ± standard deviation (Mean ± SD). Statistical significance was determined by two independent samples p < 0.01). After treatment with icariin  or nimodipine (10 μmol/L), respectively, cell viability was dramatically increased (p < 0.01) ] was 0.62 ± 0.06 in normal control cells. It was significantly decreased in OGD-treated cells  .p < 0.01). Icariin treatment at concentrations range from 10−7 to 10−5 mol/L reduced HIF-1α levels significantly, compared to OGD-treated cells (p < 0.01)  were increased to fivefold compared with normal control cells  .−7 to 10−5 mol/L reduced HSP-60 and HSP-70 levels significantly, compared to OGD-treated cells (p < 0.01) , compared to normal control cells . Icariin treatment at concentrations range from 10 < 0.01) .−7 to 10−5 mol/L reduced NSE levels significantly, compared to OGD-treated cells (p < 0.01) , compared to normal control cells (309 ± 72 ng/L). Icariin treatment at concentrations range from 10 < 0.01) .Neuroprotective mechanisms of icariin include reducing oxidative stress, inhibiting proinflammatory role of microglia, regulating metabolism of monoamine neurotransmitter, increasing synthesis and release of neurotrophic factors and promoting angiogenesis , responsible for cellular survival under hypoxic conditions  are a family of highly conserved proteins during biological evolution. HSPs exist in all living organisms, from bacteria to humans (Giffard et al. NSE is a sensitive indicator of the degree of brain damage (Yoshinori et al. −7, 10−6 and 10−5 mol/L. The expression of the HSP-60, HIF-1α and HSP-70 was not coincident among different concentrations of icariin, which did not mean either higher or lower concentration generate a stronger effect. Icariin is not an agonist or inhibitor of HSP-60, so the experimental data did not reflects that when concentration of icariin and HSP-60 expression is high, HIF-1α expression is high, or when HSP-60 expression is low, HIF-1α expression is low. HIF-1α, extracellular HSP-60 and extracellular HSP-70 can stimulate inflammatory responses, which aggravate ischaemia injury. The present study demonstrated that icariin reduced HIF-1α, HSP-60, HSP-70 and NSE levels, and increased cell viability in OGD-injured PC12 cells. Our previous study showed that icariin decreased TNF-α, IL-1β and IL-6 levels in PC12 cells following OGD injury. Taken together, these results and the above listed studies suggest that icariin may attenuate OGD-induced injury in PC12 cells through inhibiting the expression of HIF-1α, HSP-60 and HSP-70, and subsequently reducing the levels of inflammatory cytokines.Our previous study has indicated that nimodipine, a calcium antagonist, can reduce intracellular free calcium concentration and increased cell viability in OGD/R-treated PC12 cells (Mo et al.


Their structures were determined by extensive NMR spectroscopic data, ECD and OR calculations, as well as X-ray diffraction. The isolated compounds exhibited potent phytotoxicity against seedling growth of amaranth and lettuce. Harziane diterpenes were rarely reported for their remarkably bioactivities, and it was the first report to study the phytotoxicity of harziane diterpenes, which provide a new application of such compounds in agriculture for future research.Two new harziane diterpene lactones, possessing a 6/5/7/5-fused carbocyclic core containing a lactone ring system, harzianelactones A and B ( Nowadays more than half of the pesticides used are herbicides2. With the increasing attention to food safety and environmental protection, it is desiderate to develop new types of bio-herbicides with high efficiency and low toxicity.Increasing concerns for the management of weeds have been caused by scientists, as they can bring out greater reduction in crop yields than plant diseases and pestsTrichoderma spp. are one of the most commonly disseminated fungi in nature, and are distributed around the world ranging from the tundra to the tropics. They have been widely used as biocontrol agents , and commercially marketed as biopesticides, due to their capacity to parasitize in other fungi and to compete with deleterious plant microorganisms3. However, there are few Trichoderma spp. products sold in the commercial market, and limited studies are focused on the phtotoxicity of compounds from Trichoderma spp4.5. Marine-derived Trichoderma spp. have been reported to represent a potential source for producing compounds with novel structures and remarkable bioactivities, such as trichodermamides A and B6, dithioaspergillazine A7, tandyukisins E and F8, as well as harzianone9. Therefore, it has huge potential to find new phytotoxic compounds from marine-derived Trichoderma spp.Marine fungi have gained more and more attention over the past decades, as the recognition that they are a quite diverse group and an excellent source of natural products, possessing prominent bioactivities, including antibacterial, antifungal, antiviral, anti-inflammatory, antitumor, and insecticidal13, a T. harzianum XS-20090075 strain attracted our attention because the finger-print for the extract of the fungal culture on HPLC showed abundant peaks with interesting UV absorption spectra at around 250 nm, and the fungal extracts showed obvious phytotoxicity. Further chemical examination on the EtOAc extract resulted in the discovery of two new harziane diterpene lactones, harzianelactones A and B (1 and 2), and five new harziane diterpenes, harzianones A–D (3–6), and harziane (7)  was obtained as a colorless oil with the molecular formula of C20H30O3 by HRESIMS, requiring six degrees of unsaturation. The 1H NMR spectrum , 3.80 , and 3.73 , four methyl singlets at δH 2.33 (s), 1.43 (s), 0.85(s), and 0.84 (s), as well as one methyl doublet at δH 1.15 . The 13C NMR (Table δC 171.3), one oxymethylene carbon (δC 78.4), one oxymethine carbon (δC 73.7), four methylenes , three methines , five methyl groups , and five nonprotonated carbons  including two quaternary olefinic ones. The aforementioned data corresponded to two degrees of unsaturation, and the remaining four degrees of unsaturation suggested the existence of four rings. The planar structure of 1 was elucidated on the basis of COSY and HMBC experiments , in addition to the HMBC correlations from H-12 to C-10, C-11, C-14, and C-19, and from H-15 to C-13 indicated the existence of a five-membered lactone ring D connected to ring C. Finally, the connection of ring B and ring C was confirmed by the HMBC correlations from H-7 and H-14 to C-1, from H-7 to C-5, and from H-15 to C-13. Therefore, the planar structure of 1 was determined.Harzianelactone A  was obta3-16 with H-2 and H-14 indicated a cis-relationship of them  data (see Supplementary Information). The predicted ECD for -1 was in agreement with the experimental result of 1 (λmax (Δε) 200 (−1.55), 239 (+7.03) nm) -1, and +38.8 for -1, respectively, and the experimental value was +34.0. Based on both of ECD and OR calculations, the absolute configuration of 1 was assigned as 2R, 4S, 5S, 6S, 13S, 14R.The absolute configuration of 20. The cyclization mechanism of these unique diterpenes was illuminated by studies of selectively 13C- and 2H-labeled synthetic mevalonolactone isotopologues21. Distinguishing 1 from classic harziane diterpenes such as harzianone and harziandione was the D ring, which is a product of a Baeyer-Villiger monooxygenase catalyzed oxidation of a 6/5/7/4 fused tetra-cyclic skeleton; only two such harziane diterpene lactones have been discovered22. Moreover, only one report has appeared on the absolute configuration of such a harziane diterpene lactone (harzianelactone) by comparison its optical rotation data with that of the classic harziane diterpene, harzianone17. This report is the first to determine the absolute configuration of a harziane diterpene lactone by comparison of calculated and observed ECD spectra.Harziane diterpenes are a unique class of terpenes, and only 16 such skeletons have been reported2) was also isolated as a colorless oil and was assigned the same molecular formula C20H30O3 as 1 by HRESIMS results. Extensive analysis of the 1D and 2D NMR data indicated that 2 was also a harziane diterpene lactone, possessing a 6/5/7/5-fused tetra-cyclic ring scaffold like 1. Compared to 1, the disappearance of the oxymethylene signals in the 1H  and 13C NMR (δC 78.4) spectra, and its replacement with ketomethylene signals at δH 2.46, 2.33, and δC 47.3, and the significant downfield shift of C-10, as well as the different UV absorption , in combination with biogenetic considerations, suggested that C-10 is connected to the O-atom of the ester carbonyl. Detailed analysis of the HMBC correlations from H-8 and H3-20 to C-10, from H-12 to C-11, from H-14 to C-12, and from H-15 to C-13 -2, and the experimental value was +18.9 (see Supplementary Information). Therefore, the absolute configuration of 2 was determined as 2R, 4S, 5S, 6S, 13S, 14R. This is the first report of the absolute configuration of harziane diterpene lactones with the acyloxy group connected to C-10, such as 2.Harzianelactone B  was also3) was obtained as a colorless oil. Its molecular formula of C20H30O2 (six degrees of unsaturation) was determined by HRESIMS and NMR data , and the downfield shift of C-2 to C-4 indicated that there was a hydroxy group anchored at one of these three carbons. The COSY correlations of H-2/H-3/H-4/H-5/H-18 and the HMBC correlations from H-4 to C-2, C-3, C-5, C-6 and C-18 suggested the hydroxy group was attached to C-4. Therefore, 3 has a 6/5/7/4-fused tetra-cyclic ring scaffold different from 1 and 2. Analysis of the NOESY spectrum allowed the relative configuration of 3 to be the same as those of 1 and 2. The positive first Cotton effect at 340 nm (Δε + 4.09) and the negative second one at 251 nm (Δε −3.11)  was afforded as a colorless oil, and had a molecular formula of C20H30O3 evidenced from its HRESIMS spectrum. Analysis of the NMR data demonstrated that the structure of 4 resemble that of 3. The 1H and 13C NMR spectra of 4 displayed signals of four methyl groups, while those of 3 revealed five ones. Compared to 3, the C-20 methyl group was replaced by an oxymethylene in 4, which was defined by the HMBC correlations from H-20 to C-8, C-9, and C-10. As expected, subsequent analyses of the coupling constants, NOESY correlations, and experimental ECD data  as that of 3.Harzianone B  was isolated as colorless crystal needles. The molecular formula, C20H30O2, was assigned to be the same as that of 3 by its HRESIMS. The 1H and 13C NMR spectra of 5 showed similar characteristic signals to 3  was obtained as a colorless oil. The NMR spectral features suggested that 6 was closely related to 3. The additional carbonyl group (δC 216.9) and the disappearance of the oxymethine group  in 6 indicated that the hydroxy group at C-4 in 3 was replaced by a carbonyl group in 6, which was confirmed by the HMBC correlations from H-2, H-3, H-5, and H-18 to C-4. The relative configuration was determined as the same as 1−5 through NOESY spectrum. Similar cotton effects observed for 6  to 3 and 4 in their ECD spectra .Harzianone D (7) was obtained as colorless crystals. Its molecular formula, C20H32O2, was deduced from its HRESIMS data with five indices of hydrogen deficiency, one fewer than those of 1−5. The 1H and 13C NMR data  in 3 was disappeared and one more oxymethine (δC 67.8) was emerged in 7. These evidences as well as the unsaturation degrees of these two compounds indicated that the ketone carbonyl group at C-11 in 3 was replaced by an oxymethine group in 7, which was confirmed by the HMBC correlations from H-11 to C-9, C-10, and C-13. The relative configuration of all chiral centers but C-11 was determined by the NOESY spectrum of 7 like 1−6, and the correlation of H-11 with H3-18 indicated they were cis-oriented. To clarify its absolute stereochemistry, 7 was recrystallized in a dichloromethane/methanol (20:1) mixture to yield crystals. The low-temperature X-ray diffraction (CuKα) of the single crystals -configuration.Harziane , compared to the positive control glyphosate. No compound was found to inhibit the root growth of lettuce at 200 ppm. It seemed that the isolated compounds caused weaker inhibition to lettuce than to amaranth, and have stronger toxicity on the growth of root growth than hypocotyl. Although there are three reports on the phytotoxicity of crude extracts of Trichoderma spp.24, no one had studied the phytotoxicity of compounds from Trichoderma spp. Thus, this is the first report of the phytotoxic compounds from Trichoderma spp., and the phytotoxicity of harziane diterpenes is also reported for the first time. None of the isolated compounds exhibited antibacterial activities.Harziane diterpenes have rarely been reported to have significant bioactivities. In the present study, compounds pm Table . CompounT. harzianum XS-20090075 resulted in the discovery of a series of harziane diterpenes (1–7). Compounds 1 and 2 represent a unique type of harziane diterpene lactone derived from harziane diterpenes though Baeyer-Villiger monooxygenase catalyzed oxidations. Harziane diterpenes have rarely been studied, and only 18 such compounds have been reported, including two harziane diterpene lactones. In this study, the structures of harziane diterpenes were determined by NMR spectroscopic data, ECD and OR calculations, together with X-ray diffraction. The phytotoxicity of compounds from Trichoderma sp. was evaluated for the first time, and the isolated compounds exhibited potent phytotoxicity towards amaranth and lettuce.In summary, the present chemical investigation on the soft coral-derived α radiation (Aglient Technologies). A 1525 separation module (Waters) equipped with a C18  column was used for semi-preparative HPLC. ODS , Sephadex LH-20 (Amersham Biosciences), and silica gel  were applied for column chromatography. TLC  was used in the compounds detection.Optical rotations were measured using a P-1020 polarimeter (JASCO). UV spectrua were obtained with a DU 640 spectrophotometer (Beckman). ECD spectra were acquired on a JASCO J-815-150S CD spectrometer. IR spectra were obtained via a Nicolet-Nexus-470 spectrometer. NMR spectra were recorded on an Agilent DD2 NMR spectrometer (500 MHz). ESIMS and HRESIMS spectra were obtained by a Q-TOF (Micromass) and a LTQ Orbitrap XL (Thermo Scientific) spectrometer, respectively. Single-crystal analysis were performed on a Gemini A Ultra system using Cu KT. harzianum by morphological characteristics and ITS sequence. A voucher specimen was deposited at School of Medicine and Pharmacy, Ocean University of China, PR China (KU866299).The fungal strain (XS-20090075) was isolated from the inner part of an unidentified soft coral, and was identified as 2O with 3% salinity. The culture medium was extracted by EtOAc and CH2Cl2−MeOH  for three times, and the solution was concentrated under reduced pressure to afford a residue. The residue was mixed with 1000 mL of H2O, and extracted with ethyl acetate to yield the crude extract (18.5 g). The extract was fractioned by silica gel column chromatography (CC) eluted with gradient EtOAc in petroleum ether (0%–100%), and then with MeOH/EtOAc (10%–50%) to yield six fractions (Fr. 1−Fr. 6). Fr. 1 was first repeatedly chromatographed on silica gel column by EtOAc/petroleum ether (10%), and then separated by ODS eluted with MeOH−H2O (30−80%) to afford Fr. 1-1−Fr. 1–5. Fr. 1–3 was further purified over semipreparative RP-HPLC  to yiled 2 (24.0 mg), 3 (94.3 mg), and 6 (4.2 mg). Fr. 2 was first separated by silica gel CC (EtOAc/petroleum ether = 20/80), and the eluent were combined, concentrated, and submitted to Sephadex LH-20 CC , followed by purification on HPLC with 55% MeOH−H2O to afford 1 (8.9 mg) and 5 (22.8 mg). Fr. 3 was chromatographed on silica gel CC (EtOAc/petroleum ether = 20%) and separated by ODS CC using 50% MeOH−H2O to obtain Fr. 3-1–Fr. 3-3. Fr.3-1 was futher purified on HPLC (65% MeOH−H2O) to give 4 (38.2 mg). Fr. 3-2 was purified by semipreparative RP-HPLC  to yield 7 (6.4 mg).The fungus XS-20090075 was fermented at room temperature for four weeks in 100 conical flask (1 L) containing 80 g rice and 120 mL Hα]20D + 33.8 ; UV (MeOH) λmax (log ε) 237 (3.81) nm; ECD  λmax (Δε) 200 (−1.55), 239 (+7.03) nm; IR (KBr) νmax 3425, 2933, 2362, 2340, 1738, 1653, 1029 cm–1; 1H and 13C NMR data, Tables m/z 319.2263 [M + H]+ .colorless oil; 20D + 18.9 ; UV (MeOH) λmax (log ε) 204 (3.62) nm; IR (KBr) νmax 3398, 2931, 2362, 2340, 1779, 1703, 1029 cm−1; 1H and 13C NMR data, Tables m/z 319.3 [M + H]+, 341.3 [M + Na]+, 637.4 [2 M + H]+, 659.5 [2 M + Na]+; HRESIMS m/z 319.2271 [M + H]+ .colorless oil; [α]20D + 72.1 ; UV (MeOH) λmax (log ε) 259 (3.98) nm; ECD  λmax (Δε) 251 (−3.11), 340 (+4.09) nm; IR (KBr) νmax 3400, 2932, 2361, 1735, 1669, 1441, 1260, 1150, 1027 cm−1; 1H and 13C NMR data, Tables m/z 303.2316 [M + H]+ .colorless oil; [α]20D + 32.2 ; UV (MeOH) λmax (log ε) 203 (3.49), 256 (3.64) nm; ECD  λmax (Δε) 248 (−6.98), 345 (+7.02) nm; IR (KBr) νmax 3425, 2935, 2363, 1722, 1689, 1029 cm−1; 1H and 13C NMR data, Tables m/z 319.2262 [M + H]+ .colorless oil; 20D + 14.7 ; UV (MeOH) λmax (log ε) 256 (3.58) nm; ECD  λmax (Δε) 254 (−6.21), 337 (+4.66) nm; IR (KBr) νmax 3398, 2932, 2363, 1737, 1659, 1444, 1382, 1020 cm−1; 1H and 13C NMR data, Tables m/z 303.2318 [M + H]+ .colorless crystals; mp 168−169 °C; [α]20D + 52.6 ; UV (MeOH) λmax (log ε) 255 (2.72) nm; ECD  λmax (Δε) 255 (−2.83), 338 (+3.02) nm; IR (KBr) νmax 2948, 2356, 1728, 1655, 1438, 1260, 1022 cm−1; 1H and 13C NMR data, Tables m/z 301.2161 [M + H]+ .colorless oil; [α]20D + 5.1 ; UV (MeOH) λmax (log ε) 206 (3.58) nm; IR (KBr) νmax 3404, 2929, 2362, 1653, 1382, 1033 cm−1; 1H and 13C NMR data, Tables m/z 287.2364 [M + H − H2O]+ .colorless crystals; mp 214−215 °C; .The Single-crystal X-ray diffraction data were recorded on a Xcalibur, Atlas, Gemini ultra diffractometer at 120 K. Crystallographic data for 20H30O2, Mr = 302.44, monoclinic, a = 7.13670 (10) Å, b = 13.7978 (3) Å, c = 8.4352(2) Å, α = 90.00°, β = 94.516 (2)°, γ = 90.00°, V = 828.04 (3) Å3, space group P21, Z = 2, Dx = 1.213 mg/m3, μ = 0.586 mm−1, and F (000) = 332. Crystal size: 0.42 × 0.25 × 0.13 mm3. Reflections collected/unique: 8011/2950 [R(int) = 0.0230]. The final indices were R1 = 0.0295, wR2 = 0.0733 (I > 2σ(I)). Flack parameter = 0.13 (19).C20H32O2, Mr = 304.46, monoclinic, a = 18.9468 (16) Å, b = 8.3433 (2) Å, c = 13.245 (4) Å, α = 90.00°, β = 124.739 (8)°, γ = 90.00°, V = 1720.6 (5) Å3, space group C2, Z = 4, Dx = 1.175 mg/m3, μ = 0.564 mm−1, and F (000) = 672. Crystal size: 0.21 × 0.20 × 0.19 mm3. Reflections collected/unique: 9303/3064 [R(int) = 0.0261]. The final indices were R1 = 0.0307, wR2 = 0.0748 (I > 2σ(I)). Flack parameter = 0.13 (19).CAmaranthus retroflexus L.) and lettuce (Lactuca sativa) was assayed by the method reported previously25. Glyphosate was used as the positive control.Phytotoxicity against seeding growth of amaranth , Staphyloccocus aureus (ATCC 27154), and Gram-negative Escherichia coli (ATCC 25922), Ralstonia solanacearum, Vibrio anguillarum (ATCC 19019), and V. Parahemolyticus (ATCC 17802) were used, and ciprofloxacin and streptomycin sulfate were used as positive controls.The antibacterial activity was evaluated by the conventional broth dilution assaySupplementary Information