As the main input nucleus of the basal ganglion, the striatum executes different functions, including motivation, reward and attention. The functions of the striatum highly rely on its subregions that receive projections from various cortical areas and the distribution of striatonigral neurons that express D1 dopamine (DA) receptors  and striatopallidal neurons that express D2 DA receptors (or D2 MSNs). Using bacterial artificial chromosome (BAC) transgenic mice, several studies have recently been performed on the spatial distribution of D1 and D2 MSNs. However, these studies mainly focused on enumeration of either D1-enhanced fluorescent protein (eGFP) or D2-eGFP in mice. In the present work, we used Drd1a-tdTamato and Drd2-eGFP double BAC transgenic mice to evaluate the spatial pattern of D1 MSNs (red fluorescence) and D2 MSNs (green fluorescence) along the rostro-caudal axis of the dorsal striatum. The dorsal striatum was divided into three subregions: rostral caudoputamen (CPr), intermediate CP (CPi), and caudal CP (CPc) across the rostral–caudal extent of the striatum. The results demonstrate that D1 and D2 MSNs were intermingled with each other in most of these regions. The cell density of D1 MSNs was slightly higher than D2 MSNs through CPr, CPi, and CPc, though it did not reach significance. However, in CPi, the ratio of D1/D2 in the ventromedial CPi group was significantly higher than those in dorsolateral, dorsomedial, and ventrolateral CPi. There was similar proportion of cells that co-expressed D1 and D2 receptors. Moreover, we demonstrated a pathway-specific activation pattern of D1 MSNs and D2 MSNs in a manic like mouse model induced by D-Amphetamine by utilizing this double transgenic mice and c-fos immunoreactivity. Our results may provide a morphological basis for the function or pathophysiology of striatonigral and striatopallidal neurons with diverse cortical inputs to the dorsal striatum. The striatum is a cardinal point of the basal ganglia (BG) that plays an important role in action selection, motor planning and other functions related to evaluation and selection . The results demonstrated a pathway-specific activation of striatal D1 MSNs and D2 MSNs with the cortico-striatal projectome in hyperactivities induced by psychostimulant.The generation of D1-tdTomato and D2-eGFP transgenic mice had previously been described  was dissolved in saline and administered in a volume of 10 ml/kg. One dose of amphetamine (2 mg/kg body weight) or vehicle was administered by intraperitoneal (i.p.) injection 30 min before placing the mouse into the testing chamber.The animals were allowed to habituate the experimental room at least 12 h before the tests. Mice were received either saline or D-AMPH challenge and placed in the center of a open field chamber (40 cm × 40 cm) for habituating 5 min. Then the locomotor activity was recorded for 30 min. Locomotor activity was analyzed by using the SMART Video Tracking System (Panlab S.L.U.). The chamber was divided into an outer zone  and a square center zone (20 cm × 20 cm). Total distance traveled was used for measuring locomotor activity, and distance traveled in the central zone was used for evaluating anxiety  cutting solution containing (in mM): 115 choline-chloride, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 8 MgCl2, 26 NaHCO3, 10 D-(+)-glucose, 0.1 L-ascorbic acid, and 0.4 sodium pyruvate (with osmolarity of 300–305 mOsm). The brains were then rapidly removed and placed in ice-cold cutting solution for slice preparation. The coronal slices (300 μm) were prepared using a slicer  and then incubated in a holding chamber at 32°C with carbogenated cutting solution for 15–20 min. The slices were then transferred to artificial cerebral spinal fluid (ACSF) containing (mM): 119 NaCl, 2.3 KCl, 1.0 NaH2PO4, 26 NaHCO3, 11 D-(+)-glucose, 1.3 MgCl2, 2.5 CaCl2  at room temperature for at least 1 h.Mixed gender D1-tdTomato and D2-eGFP mice at 5–8 weeks-old were used for whole-cell electrophysiology procedures. Acute coronal striatal slices were prepared as previous reported . The perfusion rate was 2.0 ml/min. The fluorescently labeled D1 or D2 MSNs were visualized and identified with a microscope equipped with GFP or red fluorescent protein (RFP) filter . Whole-cell patch clamp recordings were performed with infrared-differential interference contrast (IR-DIC) visualized guide. Recording pipettes  were pulled in a horizontal pipette puller  with a tip resistance of 3–5 MΩ. Patch pipettes were filled with a solution containing (in mM): 128 potassium gluconate, 10 Hepes, 10 phosphocreatine sodium salt, 1.1 EGTA, 5 ATP magnesium salt, and 0.4 GTP sodium salt. pH was adjusted to 7.3 with KOH, and osmolarity was adjusted to 300–305 mOsm with sucrose. Cells with series resistance more than 20 MΩ at any time during the recordings were discarded. Neurons with resting membrane potentials more negative than −60 mV and action potentials with overshoot were selected for further experiments. Liquid junction potentials were not corrected. An axon 200A amplifier  was used to record membrane potentials. Signals were low-pass filtered at 5 kHz and sampled at 20 kHz with a Digidata 1322A and Clampex 9.0 (Molecular Devices), and data were stored on a computer for subsequent off-line analysis.Some MSNs were labeled by adding 0.5% neurobiotin 350  to the internal solution. During the recording, simple diffusion of the dye from the pipette into the cell was sufficient to obtain complete labeling. Immediately after the recording, the slices were fixed by immersion in 4% paraformaldehyde (PFA), 0.1 M phosphate buffer  at 4°C. The slices were then cryoprotected by infiltration with 30% sucrose in 0.1 M PB overnight at 4°C, and were incubated at 4°C for 12 h with streptavidin-Alexa Fluor 350 . The slices were examined under a BX-51 microscope using FV 1200 software . We obtained the labeled cells from 10 to 20 serial optical sections that were 2 μm apart. Using a 3D reconstruction software , morphology reconstructions were performed from the sets of confocal images.Mice were deeply anesthetized with an overdose of sodium pentobarbital  and perfused with 100 ml of 0.01 M phosphate buffered saline , followed by 200 ml of 4% PFA in 0.1 M PB. The brains were dissected and post-fixed for 4 h in the same fixative at 4°C, and transferred to 30% (w/v) sucrose for 48 h at 4°C. After that, the brain blocks were cut into 30 μm thick coronal sections on a cryostat. All sections containing dorsal striatum were serially collected into 24 dishes containing 0.01 M PBS, with avoidance from light. Each dish contained a set of serial sections that were stored at 4°C for subsequent immunohistochemical staining.Coronal striatal sections were washed three times with 0.01 M PBS, blocked with 3% bovine serum albumin (BSA) in 0.01 M PBS at room temperature for 2 h, and then used for immunofluorescent staining. Slices were first incubated at 4°C for 48 h with rabbit anti-D1 receptor antibody , goat anti-D2 receptor antibody  and rabbit anti-cFos antibody , respectively. The antibody diluent was prepared with 0.01 M PBS containing 0.3% (v/v) TritonX-100, 0.05% (w/v) NaN3, 0.25% (w/v) λ-carrageenan, and 3% BSA. The sections were rinsed three times with 0.01 M PBS and incubated at room temperature for 2 h with Alexa Fluor 647-conjugated goat anti-rabbit IgG  and Alexa Fluor 647-conjugated donkey anti-goat IgG , respectively. After three times washing, sections were incubated with Hoechst 33342  for 15 min at room temperature to stain nuclei. All of the sections were displayed in order with an interval of 180 μm on glass slides. The stained sections were observed and captured under a confocal laser scanning  and fluorescence  microscopes.After sections containing striatum were taken, the panorama of striatal image was stitched manually by xuvstitch v1.8.0 software  or two-way ANOVA. The significance levels for all tests were set at *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.All data were transferred to Prism 6.0  for analysis and graphing. Data are presented as mean ± SEM, and the “n = 14; D2: −81.0 ± 1.19 mV, n = 12). With the depolarization current step stimulation, the delayed first action potential was manifest on these neurons  from Bregma +1.54 to +0.64, intermediate CP (CPi) from Bregma +0.46 to −0.08, caudal CP (CPc) from Bregma −0.26 to −0.98 based on Hintiryan et al.  recent w2; D2, 236.2 ± 60.88 cells/mm2; n = 6 slices, p > 0.05, unpaired t-test; Figure n = 6 slices, p > 0.05, one-way ANOVA; Figure n = 6 slices, p > 0.05, one-way ANOVA; Figure Since striatum from the rostral to caudal received different cortical inputs, it is necessary to perform the quantitative analysis in serial striatal parts. So we first examined the cell density of D1 MSNs and D2 MSNs in CPr. According to a previous study , dorsomedial (CPi.dm), ventrolateral (CPi.vl) and ventromedial (CPi.vm). The definition of the four areas is shown in Figure 2; D2, 168.4 ± 10.86 cells/mm2; n = 5 slices, p > 0.05, unpaired t-test; Figure 2; CPc.i, 184.0 ± 11.14 cells/mm2; CPc.v, 202.8 ± 5.86 cells/mm2; Density of D2 MSNs: CPc.d, 152.8 ± 11.01 cells/mm2; CPc.i, 185.2 ± 17.58 cells/mm2; CPc.v, 167.0 ± 5.83 cells/mm2; n = 5 slices, p > 0.05, one-way ANOVA). This finding was also supported by Figure n = 5 slices, p > 0.05, one-way ANOVA; Figure Next, we analyzed the spatial distribution of D1 and D2 MSNs in CPc. We separated the CPc into three areas, namely dorsal (CPc.d), intermediate (CPc.i), and ventral (CPc.v). Figure p < 0.0001, unpaired t test, Figure p = 0.02; D1: Saline, n = 8 slices; D1:D-AMPH, n = 7 slices, p < 0.0001; D1:D-AMPH vs. D2:D-AMPH, p = 0.03, Figure n = 8 slices; D1: D-AMPH, n = 8 slices, p < 0.0001; D2: Saline vs. D2: D-AMPH, p = 0.0001, Figure n = 8 slices; D1:D-AMPH, n = 8 slices, p < 0.0001; D2:Saline vs. D2:D-AMPH, p = 0.03, Figure n = 8 slices; D1:D-AMPH, n = 8 slices, p = 0.0002; D2:Saline vs. D2:D-AMPH, p = 0.0002, Figure Previous research suggested that D-AMPH could induce acute manic like behavior and trigger the expression of immediate early genes (IEGs) in the striatum (Uslaner et al., In this study, we explored the spatial distribution of D1 MSNs and D2 MSNs across the rostral-caudal axis of the striatum using D1-tdTomato and D2-eGFP double transgenic mice. Using these double transgenic mice, two MSN populations were visualized simultaneously, which minimized the experimental variability of traditional immunohistochemical staining. Moreover, we utilized these double transgenic mice and c-fos immunoreactivity to demonstrate a pathway-specific activation pattern of D1 MSNs and D2 MSNs in a manic like mouse model, which provided a potential method to study activation pattern of D1/D2 MSNs balance in striatal related disease.As the major input nucleus of the BG, the striatum is well known direct and indirect pathways of BG normal function and related diseases. D1 and D2 MSNs critically define these two pathways based on close attention to their cytoarchitecture, spatial distribution, function and distinct modulation. Previous results suggested that D1 and D2 MSNs were intermingled with each other and without clear lamina organization through the whole striatum (Lanca et al., The whole striatal regions are organized into distinct input-output sub-networks with functionally diverse D1 and D2 principal neurons located in a unique manner in the striatum Gerfen, . We diviThe balance of the D1 MSNs and the D2 MSNs are important for the normal function of the striatum (Yin et al., The striatum can also be divided into striatal micro-zones, including striosomes and matrix according to differential expression of neurotransmitter-related molecules (Graybiel, In this study, we used a double labeling transgenic mouse line to simultaneously visualize D1 and D2 MSNs, and found intermingled distribution patterns across the rostral-caudal extent of the striatum, and that the density of D1 MSNs was slightly higher than that of D2 MSNs. Besides, we found a specific spatial pattern of D1 MSNs and D2 MSNs activation in a manic like behavior, which provided an evidence for the unique function of the striatum.WW and SW conceived the project and designed the experiments. KR and BG performed the experiments, and analyzed the data. CD, HY and TS analyzed the data. WW and SW prepared the manuscript based upon the draft by KR, and BG, XL and ZB revised it critically for important intellectual content. All authors approved the final version of the manuscript submitted for publication, all persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.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.


Furanosteroids, represented by wortmannin, viridin, and demethoxyviridin, are a special group of fungal-derived, highly oxygenated steroids featured by an extra furan ring. They are well-known nanomolar-potency inhibitors of phosphatidylinositol 3-kinase and widely used in biological studies. Despite their importance, the biosyntheses of these molecules are poorly understood. Here, we report the identification of the biosynthetic gene cluster for demethoxyviridin, consisting of 19 genes, and among them 15 biosynthetic genes, including six cytochrome P450 monooxygenase genes, are deleted. As a result, 14 biosynthetic intermediates are isolated, and the biosynthetic pathway for demethoxyviridin is elucidated. Notably, the pregnane side-chain cleavage requires three enzymes: flavin-dependent Baeyer-Villiger monooxygenase, esterase, and dehydrogenase, in sharp contrast to the single cytochrome P450-mediated process in mammalian cells. Structure–activity analyses of these obtained biosynthetic intermediates reveal that the 3-keto group, the C1β–OH, and the aromatic ring C are important for the inhibition of phosphatidylinositol 3-kinase. Demethoxyviridin is a fungal steroid that inhibits a phosphatidylinositol 3-kinase, an enzyme contributing to tumor progression. Here, the authors elucidate the biosynthetic route that leads to the formation of demethoxyviridin in fungi. They are some of the most widely distributed small molecules in nature and serve a myriad of biological functions. Sterols are the most important form of steroids, with a hydroxyl group at C3 and a skeleton derived from cholestane, among which cholesterol in animals, sitosterol in plants, and ergosterol in fungi are well-known molecules, as they are the essential components of the cellular membranes in these eukaryotic organisms2. In addition, sterols are important precursors for many biologically important molecules, such as the steroid hormones from animals and the cardenolides from plants, by extensive carbon degradation4. In fungi, the oxidative removal of carbons from sterol precursors also produces active molecules, such as wortmannin, viridin, and demethoxyviridin (1), which are called furanosteroids because all of these molecules contain an extra furan ring fused between C4 and C6 of the steroidal framework , among which wortmannin has been developed as a commercial PI3K inhibitor widely used in various biological studies10. Notably, a semisynthetic analog of wortmannin, PX-866, was tested in a phase II clinical trial for treating cancers11. The intriguing structures and excellent biological activities of furanosteroids have thus led to extensive efforts toward their total chemical synthesis over the past 20 years, and the stereoselective synthesis of wortmannin and (–)-viridin was finally achieved in 201713. However, as compared with the progress in chemical synthesis, the biosynthesis of these important molecules in fungi is poorly understood.Steroids are modified triterpenoids containing the tetracyclic system of lanosterol, but lacking the three methyl groups at C4 and C14. Further modifications in the side chain lead to different sub-classes of steroids bearing C13C-labeled mevalonate or acetate revealed that viridin, demethoxyvridin, and wortmannin are formed from two farnesyl units in a steroid-like manner, and do not originate from a diterpenoid16. A subsequent study using 14C-labeled lanosterol confirmed that it is converted to viridin via a normal steroidal pathway17. To gain further insight into the biosynthesis of demethoxyviridin from lanosterol, Hanson et al. identified a group of ergosterol side-chain-derived C6 and C7 alcohols sharing a common origin with demethoxyviridin. This suggested that the removal of the sterol side chain during demethoxyviridin biosynthesis occurs between C20 and C22, in a pathway analogous to the sterol side-chain cleavage during the biosynthesis of steroid hormones in mammalian cells19. Despite these advances, the genetic basis for the biosynthesis of furanosteroids has remained elusive. Recently, a transcriptional comparison of Trichoderma virens IMI 304061, a high producer of viridin, and its mutant strain deficient in secondary metabolite production identified a four-gene cluster predicted to be responsible for the biosynthesis of viridin;20 however, it was soon realized that this gene cluster is involved in the biosynthesis of volatile terpene compounds, rather than viridin21. During our manuscript submission, Bansal et al. reported a biosynthetic gene cluster for viridin, but they did not provide substantial evidence for the biosynthetic pathway of viridin22.Early isotope labeling experiments with 24, we identified the endolichenic fungus Nodulisporium sp. (no. 65-12-7-1), which can produce large amounts of demethoxyviridin (1) and several analogs26. These findings provided a good chance to elucidate its biosynthesis.In our previous explorations for bioactive secondary metabolites from fungi1, by the combinational use of a transcriptome comparison analysis, CRISPR-Cas9-based gene disruption, an Aspergillus oryzae NSAR1 heterologous gene expression system, and an in vitro enzymatic assay. Our study sets the stage to uncover the biosyntheses of other furanosteroids and expands the chemical diversity of pharmaceutically important furanosteroids by engineered biosyntheses.Here, we report the identification of the gene cluster and the biosynthetic pathway for 27, the lanosterol-derived triterpenes/steroids, including ergosterol and ganoderic acid, do not conform to this rule29. This could be the reason why Kenerley et al. failed to find the biosynthetic gene cluster of viridin, when using terpene cyclase as the starting point21. Therefore, a different strategy based on other features of furanosteroids should be adopted. Since demethoxyviridin possesses a highly oxygenated structure, we inferred that there should be multiple cytochrome P450 monooxygenase (CYP) genes in its biosynthetic gene cluster. Thus, the CYP gene clusters in the genome could be potential targets. To identify the biosynthetic gene cluster of demethoxyviridin, we sequenced the whole genome of Nodulisporium sp. (no. 65-17-2-1), and identified a total of 103 CYP genes in the genome. Analyses of the relative localizations of these genes in the genome revealed 12 CYP clusters (clusters I–XII) containing two or more CYP genes  in the cluster, to generate the mutant strain ΔvidA-JN1001. As compared with the parent strain JN1001, in which 1 and its 3-keto reduced derivative demethoxyviridiol (2) were abundantly produced, the ΔvidA-JN1001 mutant strain lost the ability to produce 1 and 2, but instead produced three additional compounds, 5 (2.6 mg L−1), 6 (3.4 mg L−1), and 7 (2.1 mg L−1)  were individually disrupted. As shown in Supplementary Fig. g3262 , g3264 , and g3265 (a transporter protein), and the downstream g3285 (a heterokaryon incompatibility protein) had no effects on the production of 1 and 2, whereas the disruption of either g3266 (vidA) or g3284 (vidS) abolished their production and led to the production of different compounds  to g3284 (vidS), including 15 biosynthetic genes for the structural assembly of demethoxyviridin, two transporter genes, and two Bet v1-like genes   Fig. . This rends Fig. . These rvid cluster and decipher the biosynthetic pathway of demethoxyviridin, 15 biosynthetic genes, including the six CYP genes, were disrupted and the resulting mutant strains were subjected to a metabolite analysis  and 4 (10.5 mg L−1)  Fig. . Both 3 ely Fig. . These rvidA and vidD genes encode two other CYPs in the cluster. When vidA was disrupted, the resulting mutant strain ΔvidA-JN1001 failed to produce compounds 1 and 2, but accumulated three other compounds, 5 (2.6 mg L−1), 6 (3.4 mg L−1), and 7 (2.1 mg L−1)  and 8 (4.5 mg L−1)  Fig. . These c−1) Fig. . Structu L−1), 6 .4 mg L−1oup Fig. . Similar−1) Fig. . After s001 Fig. , suggest L−1), 6 .4 mg L−1C12 Fig. . These rvidH gene encodes an NAD(P)-binding dehydrogenase. The disruption of vidH resulted in the production of two distinct compounds, 7 (5.5 mg L−1) and 8 (3.5 mg L−1)  . The disruption of vidF thus led to the production of three additional compounds, 10 (2.2 mg L−1), 11 (3.2 mg L−1), and 12 (1.7 mg L−1)  Fig. . As compn 6 Fig. , suggest−1) Fig. , which wf 8 Fig. , indicat1) and 8 .5 mg L−1−1) Fig. . Their s001 Fig. , suggest1) and 8 .5 mg L−1vidS-JN1001 produced a single metabolite, 13 (12.6 mg L−1)   Fig. . The strtal Fig. . These r−1) Fig. . An HRES 13 Fig. . These rvidJ and vidM genes encode a 6-hydroxy-d-nicotine oxidase and a C-3 sterol dehydrogenase, respectively. The disruption of vidJ predominantly produced the 3-OH derivative 2  and the two transporter genes (vidB and vidL), only five biosynthetic genes in the gene cluster remained to be characterized, including three CYP genes , one short chain oxidoreductase gene (vidO), and one hypothetical gene (vidN). The deletion of these genes abolished 1 and 2, but did not yield stable intermediates . After JA1 was cultured in the induction medium for 5 days, the mycelia extract was analyzed by HPLC. As a result, three additional compounds, 13 (1.1 mg L−1), 14 (1.4 mg L−1), and 15 (1.7 mg L−1), were detected in the transformant JA1  or vidH (JA3) were performed, to verify their functions. As a result, JA2 converted 9 into 8, while the control strain did not , VidP (esterase), and VidH (dehydrogenase), during the demethoxyviridin biosynthesis, which is distinct from the single CYP-catalyzed process during the biosynthesis of steroid hormones in mammalian cells  by fungi has been well characterized, and it is mediated by a BVFMO, esterase, and dehydrogenase. However, the biochemical basis for this process is still not clear35. Since the cleavage between C17 and C20 by VidF, VidP, and VidH during the biosynthesis of demethoxyviridin is quite similar to that reported in the cleavage of progesterone35, we hypothesized that the side-chain cleavages of demethoxyviridin and progesterone are likely to be mediated by homologous enzymes. To verify this hypothesis, vidF, vidH, and vidP were simultaneously transformed into A. oryzae NSAR1 to construct the three-gene co-expression strain, JA4. As expected, incubating JA4 with progesterone (20) successfully led to the production of four additional compounds, 21−24  showed the most potent inhibitory activity against PI3K with an IC50 value of 6.6 nM, which is much better than that of 2 (IC50 = 1074 nM), suggesting that the 3-keto group is critical for the activity. This conclusion is also supported by the comparison of the activities between 3 and 4, 7 and 8, and 10 and 11  abolishes the activity  as a positive control , and deciphered its biosynthetic pathway. Notably, we found that an unusual pregnane side-chain cleavage process occurs during demethoxyviridin biosynthesis, which is distinct from the biosyntheses of structure-related steroid hormones in mammalian cells. Moreover, 14 demethoxyviridin intermediates were obtained by gene deletion experiments, providing insights into the structure–activity relationships of furanosteroids for PI3K inhibition.Furanosteroids are a group of remarkable, highly oxygenated steroidal metabolites derived from fungi, and are nanomolar-potency inhibitors of PI3K. Here, we reported the identification of the biosynthetic gene cluster for the furanosteroid demethoxyviridin 41, suggesting that the cleavage of the C20−C22 bond, to form the 20-keto, might be a key step in the divergence of furanosteroid biosynthesis from ergosterol biosynthesis. Based on these facts, we have proposed a reasonable early stage biosynthetic route for demethoxyviridin  were analytical grade from Fine Chemical Co., Ltd. . The compound standards  were purchased from J&K Scientific Co., Ltd. .Methanol (MeOH) was purchased from Yuwang Industrial Co., Ltd. . Acetonitrile (MeCN) was obtained from Oceanpak Alexative Chemical Co., Ltd. . Ethyl acetate (EtOAc) and chloroform . PCR was performed using KOD FX DNA polymerase, KOD Plus DNA polymerase , and Taq DNA polymerase . T4 DNA polymerase and an In-Fusion® HD Cloning Kit were used, as recommended by the manufacturer . DNA restriction enzymes and other DNA modification reagents were purchased from Thermo Fisher Scientific  and TOYOBO Co., Ltd. .d6: δH 2.50/δC 39.5; CD3OD: δH 3.30/δC 49.0; CDCl3: δH 7.26/δC 77.0) as internal standards.UV data were obtained with a JASCO V-550 UV/vis spectrometer . IR data were acquired with a JASCO FT/IR-480 plus spectrometer . Optical rotations were determined on a JASCO P1020 digital polarimeter . ECD spectra were recorded on a JASCO J-810 spectrophotometer , using MeOH as the solvent. The melting points were measured on an X-5 micromelting point apparatus  without corrections. The ESIMS spectra were recorded using a Bruker amaZon SL mass spectrometer  and the HRESIMS spectra were performed obtained with a Waters Synapt G2 mass spectrometer . The 1D and 2D NMR spectra were recorded on Bruker AV 300, Bruker AV 400, and Bruker AV 600 spectrometers , using the following solvent signals  . The preparative HPLC was performed on a Shimadzu LC-6-AD liquid chromatography system  with an SPD-20A detector, using a YMC-Pack ODS-A column  . Medium pressure liquid chromatography (MPLC) was performed with a UV preparative detector, a dual pump gradient system, and a Dr. Flash II fraction collector system .Column chromatography (CC) was performed on ODS resin . Analytical HPLC was conducted with a Dionex HPLC system equipped with an Ultimate 3000 pump, an Ultimate 3000 DAD, an Ultimate 3000 column compartment, an Ultimate 3000 autosampler , and an Alltech (Grace) 2000ES evaporative light scattering detector (ELSD) , using a Phenomenex Gemini CNodulisporium sp. (no. 65-12-7-1), a high producer of demethoxyviridin, has been deposited in GenBank as specimen KC894854. The strain was maintained on potato dextrose agar (PDA), and cultivated at 28 °C, 180 rpm in maltose medium  for 3 days, and was used as the source for whole-genome sequencing. The total RNA for RT-PCR and RNA-Seq was extracted from Nodulisporium sp. (no. 65-12-7-1), grown on maltose medium (viridins producing) and Czapek medium , at 28 °C, 180 rpm, for 2 days.1 and other metabolites, the Cas9-expressing strain (JN1001)30 and gene deletion strains were grown in 10 mL maltose medium at 28 °C, 180 rpm, for 2 days. The cells were then transferred into 500 mL Erlenmeyer flasks containing 100 mL maltose medium and the culture was continued at 28 °C on a rotary shaker at 180 rpm, for 2 days.For the production of Aspergillus oryzae NSAR1 34 was used as the host for heterologous gene expression. The A. oryzae NSAR1 transformants were cultured in 10 mL DPY medium  at 28 °C, 180 rpm for 2 days. The cells were then transferred into Czapek-Dox (CD) medium  to induce the expression of exogenous genes under the amyB promoter, and the cultures were shaken for 5 more days.E. coli DH5α was used for cloning, while E. coli BL21-Codon Plus (DE3) was used for expression. These strains were grown in Luria−Bertani (LB) medium supplemented with appropriate antibiotics.Nodulisporium sp. (no. 65-12-7-1) was performed by Sangon Biotech Co., Ltd.  with an Illumina HiSeq 2500 system. The SOAPdenovo version 2.04 (http://soap.genomics.org.cn/soapdenovo.html) sequence assembly system was used to produce 381 contigs, covering ~36.9 Mb. Gene prediction was then performed with AUGUSTUS , and manually revised according to the coding sequences obtained from the transcriptome and the homologous genes found in the NCBI database.The whole-genome sequencing of TM II 1st Strand cDNA Synthesis Kit (TaKaRa). In order to suppress the interference from the genomic DNA, all of the primers used for RT-PCR flanked an intron, and are listed in Supplementary Table Mycelia were collected from the maltose medium and the Czapek medium as described above, and ground in liquid nitrogen with a mortar. RNA was then isolated using an RNeasy Plant Mini Kit (QIAGEN), according to the manufacturer’s protocol. To eliminate the genomic DNA, RNA samples were treated with RNase-free DNase I (TaKaRa). The first strand cDNA was synthesized with the PrimeScript30. The genes  and the vid genes of the cluster, except for two transporter genes (vidB and vidL) and two Bet v1-like genes (vidC and vidI), were deleted.The gene disruption in JN1001 was performed by the CRISPR/Cas9 system established by our laboratoryeGFP, using the primers listed in Supplementary Table TM Express Large Scale RNA Production System .For the preparation of the in vitro transcriptional gRNA, the gRNA cassettes containing the T7 promoter, the protospacer sequence, and the synthetic gRNA scaffold for targeting genes were PCR amplified from the plasmid pUCm-gRNAscaffold-neo marker gene cassette was amplified from the plasmid pBSKII-PtrPC-neo-TtrPC, using the primers PtrpC-XbaІ-F/TtrpC-HindШ-R listed in Supplementary Table The linear 4)2SO4, 50 mM maleic acid, pH 5.5 at 30 °C for 3 h. The resulting protoplasts were then separated from mycelia by filtration and washed with TF solution 2 , and diluted to a concentration of 2 × 107 cells mL−1. Then, the linear neo marker gene cassette and the in vitro transcriptional gRNA were added to the 200 μL protoplasts solution, and incubated on ice for 30 min, which was blended with 1.35 mL solution 3  and incubated at the room temperature for 20 min. The resulting solution was then diluted with 10 mL solution 2 and centrifuged at 420 × g for 10 min. The precipitates were suspended with 200 μL solution 2 and added on the M medium 2SO4, 0.05% KCl, 0.05% NaCl, 0.1% KH2PO4, 0.05% MgSO4–7H2O, 0.002% FeSO4–7H2O, 2% glucose and 1.2 M sorbitol as well as 200 μg mL−1 G418, pH 5.5) with 1.5% agar, and covered with the upper M medium containing 0.8% agar. The plates were incubated at 30 °C for 5–7 days. All of the mutants used in this report are listed in Supplementary Table For transformation of JN1001, the strain was inoculated into 10 mL DPY medium and cultivated at 28 °C, 180 rpm, for 2 days. The cell culture was then transferred into 100 mL DPY medium and cultivated for 24 h. The mycelia were collected and digested using 1% Yatalase (Takara) in 0.6 M (NHvidF gene was amplified from the Nodulisporium sp. (no. 65-12-7-1) cDNA, with the primers listed in Supplementary Table NdeI and NotI cleavage to yield the E. coli expression vector pET-28a (+)-vidF. For the expression of the VidF protein, E. coli BL21-Codon Plus (DE3) was transformed with the pET-28a (+)-vidF plasmid. The transformant was incubated in LB medium supplemented with 50 mg L−1 kanamycin sulfate, at 37 °C/200 rpm for 16 h. Gene expression was induced by the addition of 0.1 mM IPTG when the OD600 reached 0.6, followed by further incubation with shaking at 200 rpm at 18 °C for 16 h. The cells were harvested by centrifugation, resuspended in lysis buffer , and lysed on ice by sonication. After centrifugation, the supernatant was applied to a Ni-NTA affinity column, which was washed with 30 column volumes of wash buffer . The His-tagged protein was eluted with 5 column volumes of elution buffer . The purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)  was performed in a 100 μL reaction mixture, containing 50 mM Tris-HCl (pH 8.0), 125 μM NADPH, 0.25 mM of 11, 1 mM FAD, and 2 μM of VidF. The reaction was performed at 25 °C for 24 h, with shaking at 150 rpm. The mixture was then extracted with EtOAc twice. After evaporation, the extract was analyzed by HPLC.The enzymatic reaction of VidF with 3-dihydrovirone , each gene was amplified from Nodulisporium sp. (no. 65-12-7-1) genomic DNA, with the primers listed in Supplementary Table To construct the fungal expression vectors for each gene  or the plasmid harboring two genes (vidH and vidP), DNA fragments including the amyB promoter and terminator were amplified from pTAex3-vidE, pTAex3-vidN, pTAex3-vidO, pTAex3-vidH, and pTAex3-vidP, with the primers listed in Supplementary Table XbaI-digested pAdeA vector, using the In-Fusion® HD Cloning Kit, to construct the expression plasmids listed in Supplementary Table For the construction of the plasmid harboring three genes , the plasmid pTAex3-vidF, together with pAdeA-vidH-vidP, was used for the transformation. All of the transformants used in this work are listed in Supplementary Table Transformation of A. oryzae NSAR1 transformant strain  was inoculated into 10 mL DPY medium and cultured for 2 days, and then fermentation was performed in 50 mL CD medium for the expression of exogenous genes. After cultivation for 1 day, 1.0 mg of substrate, dissolved in 20 μL DMSO, was added to the culture, which was further incubated for 4 days. The culture was extracted with an equal volume of EtOAc and evaporated to dryness. The dried extract was dissolved in MeOH for the HPLC analysis.The 2O system , at a flow rate of 1 mL min−1.Products from the mutants, the transformants, and the in vitro reaction mixtures were analyzed by HPLC, eluted with a MeOH−H2O, or subjected to silica-gel column chromatography using a CHCl3–MeOH gradient, and further purification by preparative HPLC to afford the main metabolites. The detailed purification procedures for each compound are described in the Supplementary Methods.For compound isolation, 1−3 L of the culture media were extracted with EtOAc thrice, and then the crude extract was subjected to MPLC by ODS column chromatography, eluted with a gradient of MeOH–HTM Assay kit  according to the manufacturer’s protocol. Briefly, the PI3K gamma kinase was incubated with 10 μM sample (tested compounds) in the reaction buffer containing 10 μM PIP2 in the wells of a 384-well opaque black plate  at room temperature. Kinase reactions were initiated by adding 10 μM ATP and incubating the plate at room temperature for 1 h. The reactions were quenched by adding 5 μL of Stop Solution (containing EDTA and biotin-PIP3), and then the detection buffer  PH domain, and streptavidin–APC) was added to each well. Following 2 h incubation in the dark, the HTRF signal was measured with a 2104 EnVision® Multilabel Reader  in the time-resolved fluorescence mode, set for excitation at 320 nm and dual emission detection at 615 nm (Eu) and 665 nm (APC). The emission ratio (ER) of 665 nm emission signal to 615 nm emission signal was then calculated and the inhibition ratio of a certain sample on PI3K was calculated according to the following formula: PI3K inhibition (%) = (ERsample − ER0%)/(ER100% − ER0%) × 100%. ER0% means 0% inhibition, which was calculated from the reaction of PI3K, PIP2, and ATP in the absence of sample. ER100% means 100% inhibition, which was calculated from the reaction of PIP2 and ATP without PI3K. BEZ235 was used as a positive control. For active inhibitors (relative inhibitory activity ≥50%), the IC50 values were estimated using the “Sigmoidal dose-response (variable slope)” equation in the GraphPad Prism® Version 5.0 software, with the maximum and minimum of the curve constrained to 100 and 0, respectively.The in vitro PI3K gamma kinase assay was carried out using a PI3-Kinase (human) HTRF2 atmosphere at 37 °C. The cytotoxicity assay was performed using the CCK-8  method in 96-well microplates, with cisplatin (DDP)  as the positive control. After a treatment with 40 μM of each compound (Dissolved with DMSO) for 48 h, the CCK-8 solution (10 μL) was added to each well and incubated for 2 h at 37 °C. The absorbance (Ab) of samples was detected at 450 nm using a microtiter plate reader . The cell viability of each sample was calculated by the following formula: [(Absample − Abblank)/(AbDMSO − Abblank)] × 100%, and the IC50 values were calculated using the “Sigmoidal dose-response (variable slope)” equation in the GraphPad Prism® Version 5.0 software.The human breast cancer MCF-7 cell line (ATCC HTB-22) was used in the cytotoxicity assay. The cell line was cultured in DMEM , supplemented with 10% fetal bovine serum , in a 5% CO1: white amorphous powder; HRESIMS (positive) m/z 323.0923 [M+H]+ , see Supplementary Fig. 26  m/z 325.1071 [M+H]+ , see Supplementary Fig. 45  m/z 307.0966 [M+H]+ , see Supplementary Fig. 46  m/z 309.1129 [M+H]+ , see Supplementary Fig. λmax (log ε) 204 (4.36), 248 (4.56), 316 (4.26) nm; ECD  λmax (Δε): 215 (+10.47), 225 (+11.43), 258 (–7.49), 302 (+3.17), 329 (+1.75), 354 (–4.56); IR (KBr) νmax 3514, 2947, 2866, 1689, 1662, 1585, 1439, 1057, 968 cm−1; NMR spectra, see Supplementary Figs. 4 was identified as -3-hydroxy-11b-methyl-1,2,3,7,8,11b-hexahydrocyclopentaphenanthrofuran-6,9-dione  m/z 325.1446 [M+H]+ , see Supplementary Fig. λmax (log ε) 205 (3.81), 221 (3.66), 296 (3.59), 342 (3.68) nm; ECD  λmax (Δε): 219 (+0.85), 240 (–10.92), 287 (–5.23), 340 (+7.67); IR (KBr) νmax 3410, 2962, 2877, 1720, 1651, 1616, 1454, 1377, 1053 cm−1; NMR spectra see Supplementary Figs. 5 was identified as -3-hydroxy-9a,11b-dimethyl-1,2,3,6b,7,8,9a,11b-octahydrocyclopentaphenanthrofuran-6,9-dione  m/z 327.1593 [M+H]+ , see Supplementary Fig. λmax (log ε) 205 (3.75), 270 (3.67), 312 (3.45) nm; ECD  λmax (Δε): 209 (+1.66), 225 (–7.00), 244 (+1.23), 267 (–4.46), 302 (+4.93), 345 (–1.47); IR (KBr) νmax 3487, 2947, 2873, 1728, 1658, 1616, 1377, 1041 cm−1; NMR spectra see Supplementary Figs. 6 was identified as -3-hydroxy-9a,11b-dimethyl-1,2,3,6b,7,8,9a,10,11,11b-decahydrocyclopentaph-enanthrofuran-6,9-dione  m/z 327.1602 [M+H]+ , see Supplementary Fig. λmax (log ε) 206 (4.05), 229 (3.84), 264 (3.77), 300 (3.63) nm; ECD  λmax (Δε): 219 (–7.77), 263 (–4.32), 321 (+2.28); IR (KBr) νmax 3433, 2939, 2877, 1693, 1658, 1531, 1354, 1103 cm−1; NMR spectra see Supplementary Figs. 7 was identified as -9-hydroxy-9a,11b-dimethyl-1,2,6b,7,8,9,9a,10,11,11b-decahydrocyclopentaphenanthrofuran-3,6-dione  m/z 329.1753 [M+H]+ , see Supplementary Fig. λmax (log ε) 205 (3.87), 263 (3.75), 310 (3.62) nm; ECD  λmax (Δε): 223 (–7.73), 244 (+1.28), 267 (–5.22), 306 (+2.17); IR (KBr) νmax 3406, 2947, 2873, 1651, 1616, 1454, 1346, 1053 cm−1; NMR spectra see Supplementary Figs. 19; and 8 was identified as -3,9-dihydroxy-9a,11b-dimethyl-2,3,6b,7,8,9,9a,10,11,11b-decahydrocyclopentaphenanthrofuran-6(1H)-one  m/z 371.1865 [M+H]+ , see Supplementary Fig. λmax (log ε) 205 (3.73), 218 (3.55), 264 (3.70), 310 (3.60) nm; ECD  λmax (Δε): 223 (–7.02), 242 (+2.03), 267 (–5.83), 306 (+1.61), 347 (–0.59); IR (KBr) νmax 3487, 3425, 2935, 2870, 1716, 1658, 1620, 1377, 1277, 1057 cm−1; NMR spectra see Supplementary Figs. 9 was identified as -3-hydroxy-9a,11b-dimethyl-6-oxo-1,2,3,6,6b,7,8,9,9a,10,11,11b-dodecahydrocyclopentaphenanthrofuran-9-yl acetate  m/z 353.1758 [M+H]+ , see Supplementary Fig. 46  m/z 355.1915 [M+H]+ , see Supplementary Fig. 47  m/z 363.2545 [M+H]+ , see Supplementary Fig. 41  m/z 373.2019 [M+H]+ , see Supplementary Fig. λmax (log ε) 204 (3.90), 216 (3.86), 255 (3.96) nm; ECD  λmax (Δε): 218 (–5.16), 238 (+3.36), 287 (+3.69), 356 (–0.43); IR (KBr) νmax 3375, 3197, 2943, 2870, 1693, 1577, 1454, 1354, 1057, 987 cm−1; NMR spectra see Supplementary Figs. 13 was identified as -9-acetyl-3,5a-dihydroxy-9a,11b-dimethyl-2,3,4,5a,6b,7,8,9,9a,10,11,11b-dodecahydrocyclopentaphenanthrofuran-6(1H)-one  m/z 397.1999 [M+Na]+ , see Supplementary Fig. λmax (log ε) 207 (4.02) nm; ECD  λmax (Δε): 220 (+2.96), 286 (+7.11); IR (KBr) νmax 3529, 3356, 3232, 2935, 2858, 1689, 1450, 1362, 1065, 991 cm−1; NMR spectra see Supplementary Figs. 14 was identified as 1--3,5a,6-trihydroxy-9a,11b-dimethyl-1,2,3,4,5a,6,6b,7,8,9,9a,10,11,11b-tetradecahydrocyclopentaphenanthrofuran-9-yl)ethan-1-one  m/z 395.1821 [M+Na]+ , see Supplementary Fig. λmax (log ε) 207 (4.10), 247 (3.96) nm; ECD  λmax (Δε): 205 (+17.86), 225 (–7.30), 258 (+6.89), 279 (+5.46), 325 (–1.16); IR (KBr) νmax 3515, 3315, 2969, 2931, 2882, 2839, 1677, 1446, 1383, 1365, 1232, 1212, 1093, 1050, 998 cm−1; NMR spectra see Supplementary Figs. 15 was identified as -9-acetyl-5a,6-dihydroxy-9a,11b-dimethyl-1,4,5a,6,6b,7,8,9,9a,10,11,11b-dodecahydrocyclopentaphenanthrofuran-3(2H)-one (Supplementary Note Compound ε): 205 +.86, 225 21: white amorphous powder; HRESIMS (positive) m/z 331.2281 [M+H]+ , see Supplementary Fig. 48  m/z 303.1976 [M+H]+ , see Supplementary Fig. 49 (Supplementary Note Compound vid gene have been deposited at GenBank under accession MG886384. The crystallographic data of small-molecule compounds related to this manuscript have been deposited at the Cambridge Crystallographic Data Center: CCDC 1821761−1821763. All relevant data are available from the corresponding authors on request.The sequence data for the Supplementary InformationPeer Review File


Paeonia suffruticosa Andrews (MC) and P. lactiflora Pall (PL). Paeonol can reduce cerebral infarction volume and improve neurological deficits through antioxidative and anti-inflammatory effects. However, the anti-inflammatory pathway of paeonol remains unclear. This study investigated the relationship between anti-inflammatory responses of paeonol and signaling pathways of TLR2 and TLR4 in cerebral infarct. We established the cerebral ischemia-reperfusion model in Sprague Dawley rats by occluding right middle cerebral artery for 60 min, followed by reperfusion for 24 h. The neurological deficit score was examined, and the brains of the rats were removed for cerebral infarction volume and immunohistochemistry (IHC) analysis. The infarction volume and neurological deficits were lower in the paeonol group  than in the control group (without paeonol treatment). The IHC analysis revealed that the number of TLR2-, TLR4-, Iba1-, NF-κB- (P50-), and IL-1β-immunoreactive cells and TUNEL-positive cells was significantly lower in the paeonol group; however, the number of TNF-α-immunoreactive cells did not differ between the paeonol and control groups. The paeonol reveals some neuroprotective effects in the model of ischemia, which could be due to the reduction of many proinflammatory receptors/mediators, although the mechanisms are not clear.Paeonol is a phenolic compound derived from Stroke generally causes a permanent disability and is the second leading cause of death worldwide. Moreover, approximately 70% of patients experience ischemic stroke. The only approved medicine for ischemic stroke is recombinant tissue plasminogen activator (rt-PA) , 2. HoweκB) and the triggering translocation of nuclear factor-κB (NF-κB) into the nucleus, which induces the production of proinflammatory cytokines such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) . Further, the sections were incubated with secondary antibodies  for 1 h at room temperature. Each of the abovementioned steps was followed by three 3-min rinses in 0.01% Tween 20/PBS. At the end of the procedure, the sections were covered with coverslip using a mounting medium  containing DAPI to counterstain DNA in the nuclei and dried overnight. The confocal images were captured using a laser-scanning confocal microscope .Data are expressed as mean ± standard deviation. Data from all the experimental groups were compared using one-way ANOVA followed by post hoc Scheffe's test. A probability value of less than 0.05 was considered statistically significant.2, and pCO2), and blood sugar levels did not significantly differ among sham, control, and paeonol groups at 10 before and 60 min after occluding the blood flow of the right MCA and at 10 min after the reperfusion (all p > 0.05, The physiological parameters including rectal temperature, heart rate, arterial blood pressure, arterial blood gas (pH, pOp < 0.001, p < 0.001; The control and paeonol groups exhibited varying cerebral infarction volume percentages after undergoing occlusion of the right MCA for 60 min, followed by reperfusion for 24 h . The cerp < 0.001, p < 0.001; The rats in the control and paeonol groups developed different severities of neurological deficits after undergoing occlusion of the right MCA for 60 min, followed by reperfusion for 24 h. The modified neurological severity scores in the sham, control, and paeonol groups were 0.00 ± 0.00, 10.58 ± 0.67, and 6.57 ± 0.97, respectively. The modified neurological severity scores were higher in the control and paeonol groups than in the sham group (both 2 (counts/1 mm2; All immunoreactive cells were counted within the dotted line area of 1 mmp < 0.001 and p < 0.05, respectively; Figures p < 0.01; Figures The number of TLR2-immunoreactive cells in the sham, control, and paeonol groups was 70.8 ± 13.2, 301.2 ± 63.3, and 172.5 ± 65.9, respectively. The number of TLR2-immunoreactive cells was significantly higher in the control and paeonol groups than in the sham group (p < 0.001 and p < 0.05, respectively; Figures p < 0.001; Figures The number of TLR4-immunoreactive cells in the sham, control, and paeonol groups was 35.2 ± 7.0, 586.7 ± 128.7, and 197.3 ± 86.3, respectively. The number of TLR4-immunoreactive cells was significantly higher in the control and paeonol groups than in the sham group (p < 0.05; Figures p > 0.05; Figures The number of Iba1-immunoreactive cells in the sham, control, and paeonol groups was 202.3 ± 66.4, 402.0 ± 78.5, and 259.5 ± 66.0, respectively. The number of Iba1-immunoreactive cells was significantly lower in the paeonol group than in the control group (κB-immunoreactive cells in the sham, control, and paeonol groups was 2.7 ± 2.6, 86.0 ± 67.7, and 15.2 ± 11.7, respectively. The number of NF-κB-immunoreactive cells was significantly lower in the paeonol group than in the control group (p < 0.05; Figures p > 0.05; Figures The number of NF-β-immunoreactive cells in the sham, control, and paeonol groups was 3.7 ± 2.7, 40.2 ± 7.8, and 7.8 ± 4.5, respectively. The number of IL-1β-immunoreactive cells was significantly lower in the paeonol group than in the control group (p < 0.001; Figures p > 0.05; Figures The number of IL-1α-immunoreactive cells in the sham, control, and paeonol groups was 17.0 ± 20.7, 249.7 ± 122.8, and 173.8 ± 40.7, respectively. The number of TNF-α-immunoreactive cells was significantly higher in the control and paeonol groups than in the sham group (p < 0.001 and p < 0.05, respectively; Figures p > 0.05; Figures The number of TNF-p < 0.001; Figures p > 0.05; Figures TUNEL assay was used to detect apoptotic cells 24 h after reperfusion. The number of TUNEL-positive cells in the sham, control, and paeonol groups was 6.5 ± 6.2, 473.7 ± 198.2, and 88.8 ± 97.7, respectively, in the ipsilateral hemisphere. No TUNEL-positive cells were observed in the contralateral hemisphere Figures . The numTo evaluate whether TLR2 and TLR4 are related to activating microglia after the ischemia-reperfusion injury, double immunofluorescence analysis was performed. The double immunofluorescence analysis of the brain sections, adjacent to the sections used for IHC analysis, revealed the colocalization of TLR2 staining with Iba1 in the cortical penumbra 24 h after reperfusion . By contκB-, and IL-1β-immunoreactive cell numbers; and TUNEL-positive cells in cerebral ischemia-reperfusion injured rats. These results are consistent with those of our previous study that paeonol pretreatment can reduce cerebral infarction volume and neurological deficits, scavenge superoxide anions, and inhibit microglial activation and IL-1β production [The results of the present study indicated that paeonol pretreatment can reduce cerebral infarction volume; neurological deficits; TLR2-, TLR4-, Iba1-, NF-oduction . Inflammoduction . Emerginoduction –23. The oduction –23. Moreoduction –28.κB and the translocation of NF-κB into the nucleus; this induces the production of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α [κB activity and proinflammatory cytokine suppression corresponding with decreased tissue damage [Microglia are resident macrophage-like cells in the central nervous system . During nd TNF-α , 20. Seve damage , 27, 28.β can be observed 2–6 h after cerebral ischemia and reaches a peak at 12–24 h [β is considered to be a neurotoxic mediator, which induces neuronal apoptosis and enhances chemokine expression in microglia and astrocytes [β production reduces infarction volume [κB-, IL-1β-, and TNF-α-immunoreactive cells and TUNEL-positive cells were observed in the control group, which is in accordance with the results of previous studies. In addition, paeonol pretreatment significantly reduced the number of Iba1-, TLR2-, TLR4-, NF-κB-, and IL-1β-immunoreactive cells and TUNEL-positive cells. Therefore, these results suggest that paeonol pretreatment can attenuate inflammatory responses through the suppression of TLR2 and TLR4 signaling pathways to ameliorate apoptosis within the penumbral area.IL-1 12–24 h , 32. IL-trocytes , 33. Morn volume . In the α levels did not significantly differ between the paeonol and control groups. TNF-α is another proinflammatory cytokine involved in cerebral ischemia-reperfusion injuries. TNF-α gradually increases from 1 to 3 h after cerebral ischemia and reaches a peak at 24–36 h [α can function both as a neurotoxic and as a neuroprotective mediator [α executes its neurotoxic function by promoting the production of oxygen free radicals and excitatory amino acids, which ultimately results in neuronal cell death. TNF-α deficiency and anti-TNF-α antibodies exert neuroprotective effects [α-knockout mice have a higher infarction volume [α promotes inflammatory responses and is involved in the suppression of inflammatory signal transduction; in other words, TNF-α may regulate both proinflammation and anti-inflammation after cerebral ischemia-reperfusion injuries [α-immunoreactive cells and lower infarction volume and neurological deficits were observed in the paeonol group. However, the present study was limited only to 24 h after reperfusion. Therefore, a long-term observational study is required to understand the role of TNF-α in ischemic stroke treated with paeonol.In our study, TNF- 24–36 h –37. Sevemediator , 39. TNF effects . By contn volume . It appeinjuries . In the TLR2 is mainly upregulated in lesion-associated microglia 24–72 h after cerebral ischemia  but is aκB expression levels in the future.Some limitations in the present study because the study used immunochemistry images alone are sometimes not enough to provide quantitative measurements. We will perform to complement these data with immunoblotting or ELISA, in particular, TLR2, TLR4, and NF-In conclusion, paeonol pretreatment ameliorated cerebral infarction and neurological deficits, probably by reducing many proinflammatory receptors and mediators in cerebral ischemia-reperfusion injured rats, although the mechanisms are not clear.


Branchinella kugenumaensis and Eulimnadia braueriana, and compared their hatching rate, maturation time, sex ratio, growth of body length, survivorship, clutch size, net reproductive rate R0, generation time TG, and intrinsic rate of natural increase r in relation to temperature . We found that E. braueriana had a significantly higher temperature-dependent fecundity and intrinsic population growth pattern (R0 and r). In contrast, B. kugenumaensis reproduced much slower than E. braueriana with much lower R0 (90–100 folds less) and r (about 10 folds less) at 15, 20 and 25°C and with a double as long TG at 20 and 25°C. In addition, E. braueriana increased its chance of hermaphroditic sexual reproductive mode at higher temperature because of a significantly delayed maturation of males from hermaphrodites. In contrast, B. kugenumaensis showed no significant change in reproductive mode with temperature. This is the first study indicating a significant differentiation in life history parameters of two sympatric branchiopods mediated by temperature.Temperature effects on organisms are of multiple scientific interests, such as for their life history performance and for the study of evolutionary strategies. We have cultured two sympatric branchiopod species from an ephemeral pond in northern Taiwan,  Calanus finmarchicus  grew faster with elevated respiration rate at higher temperature [Branchinecta lindahli Packard, 1883 matured faster with increasing temperatures along a latitudinal gradient [Busseola fusca  showed smaller clutch size at increased temperature [Gammarus pulex  died earlier with increasing temperature at an experimental range of 10–20°C [Temperature is a key factor for growth, development, reproduction, and survival of organisms –5. Varioperature . The fai 10–20°C .Anisops sardeus Herrich-Schaeffer, 1849 ate the larvae of Anopheles stephensi Liston, 1901 in a temperature-dependent manner. Its foraging activities increased at elevated temperatures between 20°C to 30°C [Scenedesmus obliquus (Turpin) Kützing, 1833 grew faster at higher temperatures (e.g. 35°C), however, formed smaller colonies (e.g. four-cell colony) especially under Daphnia filtrate condition [Anisops sardeus) as well as the colony formation of preys , affecting interspecific relationships and their niche.Temperature also affects animal behavior, interspecific relationships, and niche realization , 7. The  to 30°C . Algae aBranchinella kugenumaensis (Ishikawa 1895) and the clam shrimp Eulimnadia braueriana Ishikawa, 1895 coexisted for a long time. Siangtian Pond, a temporary wetland that is dry for most of the year and becomes water-filled following heavy rainfalls during typhoons [B. kugenumaensis exhibited the phenotypic plasticity on growth with presumably optimal resource [E. braueriana as it grew faster and died earlier at higher temperatures  with significantly different developmental time between males and hermaphrodites  [At Siangtian Pond of Yangmingshan National Park, Taipei, Taiwan , the fai itself) . TherefoR0), generation time (TG) and intrinsic rate of natural increase (r) of each resulting cohort. Finally, all above life history traits of these two species were compared and interpreted from an ecological as well as from an evolutionary perspective.We investigated the specific niche conditions that enable both branchiopods to co-exist in such a variable environment. Since temperature is an important factor in this habitat, we hypothesize that the performance of life history traits and different reproductive modes depend on temperature. To test this hypothesis, we cultured egg-containing sediments from Siangtian Pond in laboratory mesocosms at four different temperatures , recorded the earliest hatching time (EHT), earliest maturation time (EMT), maximum lifespan (MLS), sex ratio , and measured or estimated the growth (body length), survivorship, clutch size, net reproductive rate  is publicly owned. With the date of 2009/08/31 a valid field license number 20091403 was provided for the present study. In this area are no endangered nor protected species present nor involved in our study . Siangtian Pond is a seasonally temporary wetland and inundates after heavy rains mainly during typhoons. The pond can be sometimes 80 m in diameter with a depth of up to 5 m. The water level can fall for about 30 cm per day, keeping the ponds inundated for commonly only 2–3 weeks .2, 15 cm in height), which was covered with 2 L filtered water . The photoperiod was set at 14 hours light  and 10 hours dark [Collected substrate with dormant eggs was air dried indoors for more than two weeks. Then the substrate was gently stirred to become homogenous. In laboratory, four temperature trials  were conducted with 20 replicates each, making 80 cultures in total. Each culture contained 100 g of substrate mud with dormant eggs in a plastic container , earliest maturation time (EMT), maximum lifespan (MLS), sex ratio , body length, number of survivors, clutch size, net reproductive rate (R0), generation time (TG), and intrinsic rate of natural increase (r). Individuals were photographed and body lengths were measured using the software Image J [Life history traits of  Image J . The bodR0) was acquired by accumulating daily clutch size from the effective survival members  from EMT to MLS . The post hoc analysis for parametric testing was conducted using Tukey HSD and LSD. For nonparametric post hoc examination, Nemeny test was performed [The hatching rate  at different temperatures  was statistically compared by Chi-square test for independence. All parameters at the specific temperatures , including EHT, EMT, MLS, sex ratio, coefficients of body-length growth (b1), coefficients of survivorship (b2), erformed .B. kugenumaensis, p = 0.001 for E. braueriana, n = 20 for each temperature group). The hatching rate of B. kugenumaensis ranges 20–65%, significant higher (65% and 50%) at lower temperatures . However, the hatching rate of E. braueriana ranged 30–85% and showed highest (85% and 60%) at intermediate temperatures   .B. kugenumaensis hatched on day 4.7±0.7  at 15°C, on day 1.6±0.3 (n = 10) at 20°C, on day 1.2±0.2 (n = 9) at 25°C, and on day 1.0±0.0 (n = 4) at 30°C. The EHT of E. braueriana was on day 5.8±1.5 (n = 6) at 15°C, day 2.7±0.2 (n = 17) at 20°C, day 1.6±0.4 (n = 12) at 25°C, and day 1.7±0.4 (n = 6) at 30°C . Male B. kugenumaensis matured on day 16.0±1.2  at 15°C, day 10.8±1.1 (n = 10) at 20°C, and day 8.4±1.4 (n = 9) at 25°C. This fairy shrimp never matured under laboratory control at 30°C. The EMT of hermaphrodite E. braueriana was on day 18.3±4.7 (n = 6) at 15°C, day 11.9±1.3 (n = 17) at 20°C, day 7.5±0.7 (n = 12) at 25°C, and day 5.3±0.6 (n = 6) at 30°C  fairy shrimp with about equal sex ratio of 49.8–53.0%  at different temperatures  clam shrimp with biased sex ratio of 0.1–1.4% at four temperatures under laboratory control  as temperature increased  at 20°C than at 15°C (E. braueriana died earlier with significant larger coefficients (b2) at higher temperature, especially above 20°C  to 18.8 (post-EMT) in field from E. braueriana, making the B. kugenumaensis, a3 = 13.214 and b3 = 2.1036 for E. braueriana  of B. kugenumaensis was higher under the middle range of temperatures (20 and 25°C), and was estimated as 0.1±0.1  at 15°C, 0.5±0.3 (n = 10) at 20°C, 0.4±0.2 (n = 9) at 25°C, 0.0±0.0 (n = 4) at 30°C (R0) of E. braueriana was generally much higher than of B. kugenumaensis , and significantly increased at higher temperatures, i.e. 19.8±7.4 (n = 6) at 15°C, 11.8±2.1 (n = 17) at 20°C, 73.4±28.1 (n = 12) at 25°C, 297.0±98.5 (n = 6) at 30°C   .G) of B. kugenumaensis was 27.0±0.0 , 29.8±1.7 (n = 10), and 27.0±0.0 (n = 9) for the groups reared at 15°C, 20°C, and 25°C, respectively, with no significant difference among these three groups  of E. braueriana from the four temperature treatments were 20.3±5.3 (n = 6) at 15°C, 14.0±1.5 (n = 17) at 20°C, 11.0±0.8 (n = 12) at 25°C, 10.6±1.2 (n = 6) at 30°C (G of B. kugenumaensis was significantly higher than that of E. braueriana at intermediate temperatures (i.e. 20 and 25°C)  of B. kugenumaensis was -0.04±0.00  at 15°C, -0.01±0.01 (n = 10) at 20°C, 0.00±0.01 (n = 9) at 25°C, not available at 30°C (r) of E. braueriana was 0.17±0.05 (n = 6) at 15°C, 0.21±0.05 (n = 17) at 20°C, 0.32±0.05 (n = 12) at 25°C, 0.50±0.06 (n = 6) at 30°C  was significantly higher than that at lower temperatures, namely 15 or 20°C . The r-values of B. kugenumaensis were significantly lower than those of E. braueriana at all three temperature groups in laboratory cultures .The intrinsic rate of natural increase , developed faster as temperature increased from 5°C to 15°C [Acheta domesticus, grew faster with more oxygen consumption and earlier metamorphosis from larval instar to adult at 28°C, compared to 25°C [Eotetranychus carpini (Oudemans), matured faster with higher egg production at elevated temperatures from 15°C to 30°C. Its intrinsic rate of natural increase  “r” showed maximum levels at 26°C, which was considered as the optimal temperature for this species [Temperature is a key factor for development and population growth of organisms. We found that several life history traits of both perature , 15, the to 15°C . The hou to 25°C . The yel species .B. kugenumaensis and E. braueriana grew faster and died earlier with increasing temperature. A possible explanation for a shortened life expectancy at elevated temperature could be the reduced oxygen availability (or hypoxic conditions) [Palaemon peringueyi  and the copepod Pseudodiaptomus hessei  increase their respiratory rate with a temperature-increasing manner [Artemia franciscana Kelloggs, 1906, showed a higher heart beat rate at elevated temperature (32°C) than at lower temperature (24°C) [Triops longicaudatus  was reported to use an allosteric control of hemoglobin oligomerization for oxygen saturation [in situ in the fat cells of epipodites from thoracopods of the water flea Daphnia magna Straus, 1820 [D. magna showed an elevated heart beat rate, thoracopod movement rate, and the production of high-oxygen-affinity hemoglobin [The temperature effect on life history traits might result from phenotypic plasticity in physiological change. We found that both ditions) –21. Animg manner , 23. To g manner –29. The e 24°C) . It incre (24°C) . The tadturation , 26. Hem°C . It iGadus morhua Linnaeus, 1758 lowered their swimming speed upon hypoxia [Pseudocrenilabrus multicolor  even increased its gill surface at low-oxygen conditions [P. multicolor had enlarged brain and head for a better response in regards to predation or self-protection. While at extreme environmental conditions like hypoxia, this cichlid fish invested more energy in gill filament enlargement to improve oxygen transport for a better aerobic physiology and individual survival.Phenotypic plasticity upon hypoxia was also demonstrated in fish. Juveniles of the Atlantic cod  hypoxia . The gilnditions , 32. Thinditions , 32. At D. magna exhibited a higher sex ratio with male bias at lower temperature (20°C), while more females developed at higher temperature (30°C) [Bicyclus anynana  produced fewer but larger eggs at lower temperature (20°C), while more but smaller eggs at higher temperature (27°C) [B. kugenumaensis and E. braueriana did not show the temperature-dependent sex ratio variations, they demonstrated the reproductive flexibility on clutch size and EMT (earliest maturation time). Our clam shrimp E. braueriana even exhibited the switch of reproduction mode from gonochorism  to hermaphroditism (self-fertilization within a hermaphrodite) as temperature elevated. At higher temperature, E. braueriana produced much more hermaphroditic eggs that provide faster population growth since all emerging individuals could provide offspring themselves [E. braueriana might maintain its population at high temperature with hermaphroditism through the trade-off by offspring fitness reduction.Besides the effects on physiology and survival, temperature also influenced reproduction with respect to sex ratio, clutch size, and presumably the biological fitness of offspring . The wate (30°C) . The bute (27°C) , 36. In emselves . Howeveremselves , 39. TheE. braueriana was conducted mainly when male matured significantly behind hermaphrodite at high temperature. Male E. braueriana development was denoted by claspers modified from the first two enlarged thoracopods [D. magna epipodites of thoracopods [E. braueriana developed slowly due to hypoxic conditions at high temperature. The sexual reproduction of E. braueriana with combination of gonochorism and hermaphroditism at lower temperature, therefore, might be traded-off by hermaphroditism alone at higher temperature when clasper development is much slower and offspring fitness is presumably reduced from less genetic diversity.Hermaphroditism of racopods , 41. Theracopods  suggesteR0). Population growth is best reflected by intrinsic rate of natural increase (r). The clam shrimp, E. braueriana, showed significant differences in both R0 and r at higher temperatures as 25 and 30°C, indicating its optimal temperature at 25–30°C. However, the other sympatric branchiopod in Siangtian Pond, B. kugenumaensis, showed highest R0 and r at lower range (15–25°C). These two sympatric branchiopod species, therefore, demonstrated a temperature-adapted niche-differentiation on individual reproduction and population growth. It is in accordance with the studies in Austria [Eulimnadia sp. belongs to a group of warm-adapted branchiopods preferring 20–40°C [In this study, we showed differences in temperature adaptation for life history traits between two sympatric branchiopods, especially their reproductive modes and population growth. Individual productivity refers to net reproductive rate  [Lepidurus apus lubbocki Brauer, 1873 [Eulimnadia species are androdioecious animals composed of few males and majority of hermaphrodites [G) at 20°C and 25°C. The clam shrimp E. braueriana showed only half TG of the fairy shrimp B. kugenumaensis. This implies a faster evolutionary rate [E. braueriana than the dioecious B. kugenumaensis. Androdioecious breeding system contributes to a tactic reproductive strategy in response to dynamic and unpredictable environment, adding the androdioecious E. braueriana with more competitive strength in natural niche. Therefore, the coexistence of dioecious (B. kugenumaensis) and androdioecious (E. braueriana) breeding system might provide a notation of evolutionary balance from niche differentiation between these two branchiopod populations.Androdioecy is an evolutionarily transitional breeding system between monoecy and dioecy , 47. Mons, 1761) , which hs, 1761) , 50. Dios, 1761) , e.g. Leer, 1873 . Many Euhrodites . They rehrodites , 47. Howhrodites –53. In tary rate  of the aB. kugenumaensis and E. braueriana under different temperature regimes in laboratory. Our result implies a temperature-adapted niche-differentiation and reproductive strategies between these two sympatric branchiopods. In addition, comparing with field observation (Huang’s unpublished data), these two species grew larger , matured faster , and reproduced more  in field than in laboratory. Therefore, some other factors in addition to temperature (e.g. dissolved oxygen) might be involved.This study presented the contrasting life history characteristics and population growth of B. kugenumaensis and the androdioecious clam shrimp E. braueriana from Siangtian Pond in northern Taiwan were studied here. This pond shows slight seasonal water temperature variations. In laboratory cultures at 15–30°C, temperature-differentiation has its significant effect on fecundity and population growth. B. kugenumaensis showed optimal net reproductive rate (R0) and intrinsic rate of natural increase (r) at lower temperatures (i.e. 15–25°C) and E. braueriana conversely at higher temperatures (i.e. 25–30°C). We suggest that this temperature variation in life history traits relates to their sexual reproduction, i.e. dioecious B. kugenumaensis reproduced only by gonochorism at lower temperatures (i.e. 15–25°C); while the androdioecious E. braueriana laid both gonochoric and hermaphroditic eggs at lower temperatures (i.e. 15–20°C), and showed a switch to hermaphroditic egg production as temperature elevated to 25–30°C. This is the first study demonstrating a potential niche-differentiation related to temperature, which affects the reproduction and life history performance of two sympatric wetland branchiopods. More direct (e.g. hemoglobin expression and oxygen-binding affinity) and indirect  factors in addition to temperature need further studies in future. This may also allow a better understanding of niche-differentiation mechanisms between these two sympatric branchiopods from an ephemeral wetland.Life history traits of the co-existing dioecious fairy shrimp S1 FileTable A. Inundation information of Siangtian Pond, Yangmingshan National Park, Taipei, Taiwan, from 2006 to 2012. The record of accumulated rainfall came from Central Weather Bureau of Taiwan. The water temperature was calculated as mean±SE from sampling days (n). Table B (forand). Constants (a1) and coefficients (b1) for the sigmoidal growth curves of body length at different temperatures. A, B. kugenumaensis. B, E. braueriana. Table C (forand). Constants (a2) and coefficients (b2) for the exponential survival curves at different temperatures. A, B. kugenumaensis. B, E. braueriana. Table D (forR). Net reproductive rates (0) at different temperatures. A, B. kugenumaensis. B, E. braueriana. Table E (for). Generation times (TG) at different temperatures. A, B. kugenumaensis. B, E. braueriana. Table F (forr) at different temperatures). Intrinsic rates of natural increase . Multiple comparisons for net reproductive rates  from r) from E. braueriana using Tukey HSD and LSD test as post hoc analysis . Figure A (forand Table H). Multiple comparisons for intrinsic rates of natural increase . Linear relationship of clutch sizes and body lengths forB. kugenumaensis (n = 241). B, E. braueriana (n = 602). Figure B Part A (forB. kugenumaensis). Daily clutch sizes at different temperatures from . X-axis: day. Y-axis: clutch size. Figure B Part B (forE. braueriana). Daily clutch sizes at different temperatures from . X-axis: day. Y-axis: clutch size. Figure C Field license. A, Number: 20091403. B, Date: 2009.08.31. C, Studying site: Siangtian Pond, Yangmingshan National Park. D, Studying goal: life history traits of branchiopods in Siangtian Pond. forand. Constan forand. Constanle D for. Net reple E for. Generatle F for. Intrinsre A for. Linear rt A for. Daily crt B for. Daily c(DOCX)Click here for additional data file.


The resulting clots can form under different flow conditions in the veins as well as the arteries. The excessive and undesirable formation of clots  in our circulatory system may lead to significant morbidity and mortality. Some of these pathologies are deep vein thrombosis and pulmonary embolism and atherothrombosis (thrombosis triggered by plaque rupture) in coronary arteries, to name a few. The process of clot formation and growth at a site on a blood vessel wall involves a number of simultaneous processes including: multiple chemical reactions in the coagulation cascade, species transport and platelet adhesion all of which are strongly influenced by the hydrodynamic forces. Numerical models for blood clotting normally focus on one of the processes under a specific flow condition. Here, we propose a general numerical model that encompass a wide range of hemodynamic conditions in the veins and arteries, with individual platelets and their adhesive dynamics included explicitly in the models. Further, we include the biochemistry of coagulation cascade, which is essential to modeling thrombus formation, and couple that to our platelet aggregation model. The simulation results—tested against three different experiments—demonstrate that the proposed model is effective in capturing the  Platelets transporting through the regions with values of ω > 1 will become activated. In this study, we assume a negligible activating effect for TxA2, and the threshold values of [IIa]thr = 1 nM  is not known, and thus, we assume a cubic function in the form of [TF-VIIa] = [TF-VIIa]0(1 − ([Ia]/[Ia]thr)3).To initiate and drive the coagulation, a spatially varied concentration level of subendothelium-bound TF-VIIa complex is prescribed at the site of injury. This results in a few reactions at the wall (represented by flux conditions) that form enzymes IXa and Xa that drive the TF pathway. The concentration of [TF-VIIa]Fogelson  (estimats−1) may stimulate multiple ligand-receptor interactions, depending on the exposed ECM proteins (but is not strongly dependent on GPIb-vWF binding). We assume that the overall effect of interactions between receptors and ligands is incorporated into the adhesive model of Eqs s−1) may invivo experiment of Begent and Born  = [Va][Xa]/KdW, respectively [Dj∂cj/∂n = Bj. Surface reactions Bj along with their kinetic constants are given in W is prescribed at the wall as a decreasing cubic function of fibrin concentration given the fact that fibrin deposition leads to fewer subendothelium binding sites available for the complex. The initial value of the concentration is considered to be in the physiologic range [TF-VIIa]0 ≈ 1 (nM) [As mentioned in the main text, we use the coagulation model from Anand etal. , where bectively . At the ≈ 1 (nM) .(TEX)Click here for additional data file.S1 Table(TEX)Click here for additional data file.S2 Tablek9 = 11 min−1, KM9 = 160 nM, h9 = 0.0162 nM−1min−1, k8 = 194.4 min−1, KM8 = 112,000 nM, h8 = 0.222 min−1, hC8 = 10.2 min−1, HC8M = 14.6 nM, k5 = 27.0 min−1, KM5 = 140.5 nM, h5 = 0.17 min−1, hC5 = 10.2 min−1, HC5M = 14.6 nM, k10 = 2391 min−1, KM10 = 160 nM, h10 = 0.347 nM−1min−1, hTFPI = 0.48 nM−1min−1, k2 = 1344 min−1, KM2 = 1060 nM, h2 = 0.714 nM−1min−1, k1 = 3540 min−1, KM1 = 3160 nM, h1 = 1500 min−1, HM1 = 250,000 nM, k11 = 0.0078 min−1, KM11 = 50 nM, kPC = 39 min−1, KPCM = 3190 nM, hPC = 6.6 × 10−7nM−1min−1, KdZ = 0.56 nM, KdW = 0.1 nM.Kinetic constants are given as follows: (TEX)Click here for additional data file.S3 Tablek7,9 = 32.4 min−1, KM7,9 = 24 nM, k7,10 = 103.0 min−1, KM7,10 = 240 nM.The kinetic constants are given as (TEX)Click here for additional data file.


All topsoils were analysed for pH, acidity, EC, water-dispersible clay, Na, Mg, K, Ca, P, S, C, N, NH4, NO3, B, Mn, Cu and Zn. Applications of ammonium sulphate (AS), but not superphosphate (SP), greatly constrained tree abundance relative to control plots. Differences between control plots and plots that had received maximal AS application were particularly marked (16.3 ± 5.7 versus 1.2 ± 0.8 trees per plot). Soil properties most affected by AS applications included pH (H2O) , pH (KCl) (5.5 ± 0.2 to 4.0 ± 0.1), acidity (0.7 ± 0.1 to 2.6 ± 0.3 cmol kg-1), acid saturation (8 ± 2 to 40 ± 5%), Mg (386 ± 25 to 143 ± 15 mg kg-1), Ca (1022 ± 180 to 322 ± 14 mg kg-1), Mn (314 ± 11 to 118 ± 9 mg kg-1), Cu (3.6 ± 0.3 to 2.3 ± 0.2 mg kg-1) and Zn (6.6 ± 0.4 to 3.7 ± 0.4 mg kg-1). Magnesium, B, Mn and Cu were identified using principal component analysis, boundary line analysis and Kruskal-Wallis rank sum tests as the nutrients most likely to be affecting tree abundance. The ratio Mn/Cu was most related to tree abundance across the experiment, supporting the hypothesis that competition between herbaceous and woody plants depends on the availability of anabolic relative to catabolic nutrients. These findings, based on more than six decades of experimentation, may have global significance for the theoretical understanding of changes in vegetation structure and thus the practical control of invasive woody plants.The causes of the worldwide problem of encroachment of woody plants into grassy vegetation are elusive. The effects of soil nutrients on competition between herbaceous and woody plants in various landscapes are particularly poorly understood. A long-term experiment of 60 plots in a South African savanna, comprising annual applications of ammonium sulphate (146–1166 kg ha The encroachment of woody plants into rangelands is a growing problem worldwide , 2, 3. LNotwithstanding the effects of factors such as water availability, fire frequency and intensity of herbivory, the availability of certain nutrient elements is likely to be one ultimate cause of the dominance of trees over herbaceous plants in a particular landscape or region , 10. TheA little-known agronomic approach is to use ratios of elements–such as Ca/Mg–to determine the amount of a particular nutrient required to maximise herbaceous or tree crop production . This is-1 yr-1; -1 yr-1; Vachellia karroo, an acacia known as one of the most important woody invaders of grasslands in South Africa (DOCX)Click here for additional data file.S2 TableVachellia karroo; V. ger. = Vachellia gerrardii; V. r. = Vachellia robusta). AS = ammonium sulphate; SP = superphosphate. Values are numbers of individual trees. Abbreviations in parentheses indicate the species with the maximum height for each plot (V. k. = (DOCX)Click here for additional data file.S3 TableAS = ammonium sulphate; SP = superphosphate. (DOCX)Click here for additional data file.S4 TableInter-quartile factors (IQFs) are presented for data-negative zones. All 91 nutrient ratios were analysed, but only those nutrient ratios with IQFs >1 are presented. NA values indicate instances where zones of no-data were not identified (i.e. fewer than four points delineating the boundary between zones). (DOCX)Click here for additional data file.S5 Table-1 except where indicated. AS = ammonium sulphate. Means and standard errors are presented. All soil properties are reported in mg kg(DOCX)Click here for additional data file.S6 Table-1 except where indicated. SP = superphosphate. Means and standard errors are presented. All soil properties are reported in mg kg(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table-1 except where indicated. AS = ammonium sulphate; SP = superphosphate. Means and standard errors are presented. All soil properties are reported in mg kg(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S1 FigA boundary line separates a data-negative zone from a data-positive zone in a scatter plot , which enables delineation of bands of constraint as well as potential for the y variable. (TIF)Click here for additional data file.S2 Figst quartile (Q1) for values smaller than the median, or the 3rd quartile (Q3) for values larger than the median. For 0<IQF<1, OD falls between Q1 and Q1–1.5 times the inter-quartile range (IQR) for values smaller than the median or Q3 and Q3 + 1.5 times IQR for values larger than the median. For IQF >1, OD is greater than Q1–1.5 times IQR for values smaller than the median or Q3 + 1.5 times IQR for values larger than the median. For -1<IQF<0, the observed data (OD) falls between the median and the 1(TIF)Click here for additional data file.S3 FigIn the top row, four categories of tree abundance  are shown, with data points depicted as grey circles, and means ± standard errors depicted with black circles and error bars. Asterisks show significant differences in tree abundance according to Kruskal-Wallis rank sum tests . Different letters designate significant differences between means (p<0.05). (TIFF)Click here for additional data file.S1 Raw Data(XLSX)Click here for additional data file.


Conus geographus, is composed of 13 amino acids and two disulfide bonds. It is the most toxic component of Conus geographus venom with estimated lethal doses of 0.029–0.038 mg/kg for humans. There is currently no reported analytical method for this toxin. In the present study, a sensitive detection method was developed to quantify GI in human plasma using a solid-phase extraction (SPE) column (polystyrene–divinyl benzene copolymer) combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) in the multiple reaction monitoring (MRM) mode. The plasma samples were treated with a protein precipitating solvent . GI in the solvent was efficiently extracted with an SPE column and was further separated by a Grace Alltima HP C18  column at a flow rate of 0.4 mL/min. Water (with 2% methanol) acetonitrile (with 0.1% acetic acid) was selected as the mobile phase combination used in a linear gradient system. α-Conotoxin GI was analyzed by an API 4000 triple quadrupole mass spectrometer. In the method validation, the linear calibration curve in the range of 2.0 to 300.0 ng/mL had correlation coefficients (r) above 0.996. The recovery was 57.6–66.8% for GI and the internal standard. The lower limit of quantification (LLOQ) was 2 ng/mL. The intra- and inter-batch precisions were below 6.31% and 8.61%, respectively, and the accuracies were all within acceptance. GI was stable in a bench-top autosampler through long-term storage and freeze/thaw cycles. Therefore, this method is specific, sensitive and reliable for quantitative analysis of α-conotoxin GI in human plasma.α-conotoxin GI, a short peptide toxin in the venom of  Conus is a hyperdiverse group of specialized predators, which use venom to subdue the prey and for self-defense. There are an estimated number of more than 700 species of cone snails around the world   (GI and MI[ΔR2] were separated on a CMI[ΔR2])  under thMI[ΔR2]) .Stock solution of GI or internal standard MI[ΔR2] was diluted with the diluent and injected in the peristaltic pump mode. The optimized parameters for the MRM analysis of analytes (GI) and internal standard (MI[ΔR2]) were 480.0/473.6 and 446.5/437.6, respectively. In addition, transition 480.0→626.6 was also used to avoid false positives in the GI analysis. The MS/MS full scan spectrogram is shown in No significant interfering peaks were observed in the chromatograms of the six plasma blanks at the retention times of GI and the IS (MI[ΔR2]) A. The rer) of six batches was 0.996~0.999, which met the acceptance criteria of no less than 0.99. A typical calibration curve is shown in n = 6, The response was linear over the tested concentration range of 2.0–300.0 ng/mL. The correlation coefficient (n = 7). The intra-and inter-batch precision and accuracy of the QC samples were also evaluated (n = 6). The inter-batch precision (RSD%) ranged from 7.02 to 8.61% with an accuracy (RE%) range of 1.27 to 1.50% (n = 7). These results indicate that the method is accurate, reliable and reproducible.The inter-batch precision and accuracy of calibration standards were evaluated for seven different runs. The precision (RSD%) ranged from 1.31 to 8.83%, while the accuracy (RE%) ranged from −4.28 to 5.21% , respectively, with a mean of 108.12%. The mean spike recovery of MI[ΔR2] was 89.36 ± 2.36% (n = 6). Therefore, a minimal matrix effect was observed for GI and MI[ΔR2]. The extraction recoveries of GI QC samples were 52.81 ± 2.53%, 61.07 ± 9.19% and 58.88 ± 3.90% (n = 6), respectively. The mean recoveries of GI and MI[ΔR2] were 57.59% and 66.76 ± 2.36% (n = 6), respectively. The results are summarized in The spike recoveries of GI at low, mid, and high levels  were 99.10 ± 6.83%, 111.96 ± 3.06% and 113.32 ± 7.66% , and the absolute recovery of GI in human blood was 42.32% ± 5.50% (n = 4). GI in human blood was stable for 3 h at room temperature. The mean ratio of GI samples at room temperature for 3 h to GI samples at 0 h was 103.54 ± 13.09% (n = 4).As shown in v/v) was used. α-Conotoxin GI and internal standard MI[ΔR2] were soluble upon addition of the mixed solvent to the plasma samples. This method is similar to the analyses of small compounds, but different from the analyses of longer 125-I-peptides, in which the analyte is also precipitated together with plasma proteins.Generally, a precipitating solvent is used to precipitate most proteins in plasma and keep the analytes in solution ,28,29. I18 reverse phase column, ion exchange column and magnetic beads  were well recovered from human plasma after protein precipitation with the mixed solvent of methanol/acetonitrile  and α-conotoxin MI mutant MI[ΔR2] (purity > 98%) were synthesized using a previously described method . Drug-frv/v) was prepared in deionized water. Stock solutions of GI (1.00 mg/mL) and internal standard MI[ΔR2] (1.00 mg/mL) were prepared in the diluent. The working solutions of GI were prepared at concentrations of 20.0, 40.0, 100.0, 500.0, 1000.0, 2400.0, 3000.0, 10,000.0 and 100,000.0 ng/mL by diluting aliquots of the stock solution with the diluent. The QC working solutions of GI were prepared at concentrations of 60.0, 400.0, 2250.0, 10,000.0 and 100,000.0 ng/mL by spiking aliquots of the QC stock solution (1.0 mg/mL) into the diluent. QC standards of 6.0, 40.0 and 225.0 ng/mL were prepared by diluting 30 µL of the corresponding QC working solutions into 300 µL drug-free human plasma. All stock solutions were stored at 4 °C before analysis. All working solutions were freshly prepared before daily use.A diluent of 0.2% formic acid (v/v). The solution was vortexed and centrifuged at 12,000 rpm for 5 min. A 0.750 mL aliquot of the supernatant in each sample was mixed with 2.250 mL water. The resulting solution was extracted on the SPE column. The SPE column was rinsed with methanol (0.500 mL) twice, and conditioned twice with pure water (0.500 mL) prior to use. The prepared sample (3.0 mL) was then transferred onto the SPE column, and washed by 0.50 mL of 10% and 40% methanol/water (v/v) sequentially. Finally, 0.15 mL of 70% methanol/water with 1% acetic acid was added, and the eluent was collected, diluted five times in water, and analyzed by LC-MS/MS.A 0.30 mL aliquot of human drug-free plasma sample was spiked with 30 μL of the working solution of GI or QC. Unknown samples or blank control samples were spiked with 30 μL of the diluent. The internal standard solution (30 μL) was then spiked into each sample and fully mixed, followed by the addition of 0.60 mL precipitating agent . Chromatographic separation was carried out on a Grace Alltima HP C18   column at ambient temperature. The LC elution conditions were as follows : mobile phase A, 2% methanol in water; mobile phase B, 0.1% formic acid in acetonitrile; gradient: 0 min, 100% A/0% B; 0.2 min, 100% A/0% B; 1.3 min, 50% A/50% B; 1.5 min, 10% A/90% B; 1.9 min, 10% A/90% B; 2.1 min, 100% A/0% B and 3 min, 100% A/0% B. The flow rate was 0.4 mL/min. All injection volumes were 10 μL.LC-MS/MS spectra were collected on an Agilent 1200 HPLC system  coupled to an API 4000 triple quadrupole mass spectrometer . The ion source was a Turbo ion spray source operating in ESIm/z):480.0→473.6, declustering potential (DP) = 40 V, collision energy (CE) = 19 eV. The optimized parameters for the MRM analysis of MI[ΔR2] with disulfide bridges were set as follows: transition (m/z):446.5→437.6, declustering potential (DP) = 50 V, collision energy (CE) = 20 eV.The mass spectrometer was operated in the positive ion multiple reaction monitoring (MRM) mode. The ion source parameters were set as follows: curtain gas (nitrogen) = 30 p.s.i., temperature = 450 °C, gas 1 (nitrogen) = 50 p.s.i., gas 2 (nitrogen) = 50 p.s.i., ion spray voltage = 5500 V. The optimized parameters for the MRM analysis of GI with disulfide bridges were set as follows: transition (x2. The correlation coefficient was reported. The lower limit of quantification (LLOQ) was chosen as the concentration of the lowest calibration standard if the analyte response at the LLOQ was at least ten times the response of the blank human plasma. Precision was evaluated as relative standard deviation (RSD%), while accuracy was calculated as relative error (RE%). The precision and accuracy of standard curves were determined by seven inter-batch plasma samples containing GI at the calibration curve standards, with six replicates for each standard. The inter-batch and intra-batch precision and accuracy were determined by analyzing QC samples at low, mid and high levels  for all of the six different runs. The acceptance criterion for RSD% was within ±15% (±20% for LLOQ samples) and the criterion for RE% was within ±15 RSD% (±20% for LLOQ samples). The matrix interference on GI was evaluated by the spike recovery of GI at three QC levels spiked post extraction into the plasma extract against the corresponding peak areas of GI in the diluent. Matrix interference was considered negligible if the spike recovery was between 85% and 115%. The extraction recoveries of GI at three QC levels were determined by comparing the peak areas of the pre-extraction spiked plasma samples and those of the post-extraction spiked plasma samples. The stability of the QC samples was evaluated after sample storage at room temperature for 5.25 h, at 4 °C in the autosampler (pretreated) for 55 h, at −20 °C after three freeze/thaw cycles, and at −20 °C for 14 days (long term). The stability of the GI stock solutions was evaluated after sample storage at room temperature for 6 h (short term) and at 2–8 °C for 19 days (long term). The stability of the MI[ΔR2] stock solutions was evaluated after sample storage at room temperature for 6 h (short term) and at 2–8 °C for 23 days (long term).The FDA guidelines for bioanalytical method validation were followed . For speThe blood samples were collected from four individuals, each with two duplicate samples. A 40 μL aliquot of the GI working standard solution (5 μg/mL) was diluted in 1960 μL human blood and mixed slightly, resulting in a final GI concentration 100 ng/mL. The blood samples were incubated at 37 °C for 10 min to test recoveries and were incubated at room temperature for 3 h to test stabilities. The samples used for the recovery test were centrifuged at 3000 rpm to separate plasma. A 0.30 mL aliquot of the plasma samples was then processed as described in the “sample preparation” section and prepared for LC-MS/MS analysis. The mixed plasma was used in the preparation of standard curves.x2. Ratios of analyte peak areas versus IS peak area were calculated for each point.Data were processed using the Analyst (v1.6) software . Calibration curves were constructed by least square linear regression analysis using a weighting factor of 1/