Clusterin activated Akt, STAT3 and significantly reduced Bax activity; in addition to inducing phosphorylated STAT3 in Müller cells, which suggests it may indirectly acts on photoreceptors. Thus, clusterin treatment may interferes with mechanisms leading to rod death by suppressing cell death through activation of Akt and STAT3, followed by Bax suppression. Novel insights into the pathway of how clusterin promotes the rod cell survival suggest this treatment may be a potential therapeutic strategy to slow progression of vision loss in human RP.Retinitis Pigmentosa (RP) begins with the death of rod photoreceptors and is slowly followed by a gradual loss of cones and a rearrangement of the remaining retinal neurons. Clusterin is a chaperone protein that protects cells and is involved in various pathophysiological stresses, including retinal degeneration. Using a well-established transgenic rat model of RP (rhodopsin S334ter), we investigated the effects of clusterin on rod photoreceptor survival. To investigate the role of clusterin in S334ter-line3 retinas, Voronoi analysis and immunohistochemistry were used to evaluate the geometry of rod distribution. Additionally, immunoblot analysis, Bax activation, STAT3 and Akt phosphorylation were used to evaluate the pathway involved in rod cell protection. In this study, clusterin (10μg/ml) intravitreal treatment produced robust preservation of rod photoreceptors in S334ter-line3 retina. The mean number of rods in 1mm Retinitis Pigmentosa (RP) is the cause of inherited blindness that results from gene mutations –6. RegarRecently we discovered that retinal ganglion cells are still functional at later stage of S334ter rats. The recordings of RGCs at P60  showed that distinct RGC types are functionally intact. This result indicates that parallel processing of visual pathways remains largely intact in S334ter at P60 .Clusterin, a multifunctional heterodimeric glycoprotein (70–80 kDa), is constitutively synthesized by a variety of tissues and plays a role in several biological events such as programmed cell death, lipid transport, sperm maturation, complement regulation and barrier cytoprotection –32. Up-rIn the present study, we investigated the neuroprotective feature of clusterin in a transgenic rat model of RP. We show for the first time that clusterin intravitreal treatment produced a robust preservation of rod photoreceptors in S334ter-line3 retina and Bax activity was significantly reduced. In addition, clusterin treatment induced Akt and STAT3 phosphorylation.The Veterinary Authority of University of Southern California’s Institutional Animal Care and Use Committee approved all procedures. The homozygous S334ter-line-3 , a transgenic RP prototype with a rhodopsin mutation caused by truncated murine opsin gene at Serine residue 334 (S334ter-line-3). Female rats were obtained from Matthew LaVail, Ph.D.  and were mated with Long-Evans (LE) male rats to generate heterozygous offspring. The heterozygous S334ter line 3 was designated as RP in this study. For controls, age-matched SD rats  were used. Female or male SD rats and RP rats were euthanized at postnatal (P) days 15, 16, 17, 30, 45, 60, and P75 (number (n) = 9~12 and n = 6, respectively for each stage). For all experiments, animals were kept in cyclic 12-hour light/dark conditions with free access to food and water.Clusterin  was prepared in phosphate buffered saline (PBS). For adjustment of clusterin dosage, 2 μl of three concentrations  were administered by intravitreal injection with a Hamilton syringe (Sigma-Aldrich Corp) through the sclera on the temporal side of a P30 normal eye under anesthesia. Induction of anesthesia was done by intraperitoneal (IP) injection of ketamine  and xylazine  with similar procedures as our published protocols , 59, 60.Previous detailed protocols for tissue preparation were described , 60. BriThe detailed protocols for immunohistochemistry were performed as previously published , 60, 62.Cell death was tested using an In Situ Cell Detection kit  in line with the manufacturer’s recommendations. The vertical sections were incubated with TUNEL reactive mixture for 90 minutes in 37°C. The sections were then washed with 0.1M PB for 30 minutes and cover-slipped with Vectashield mounting medium.For nuclear layer staining, we embedded the eyecups in OCT embedding medium (n = 5). Eyecups were sectioned along the vertical meridian on a cryostat at a thickness of 10 μm. TOPRO-3  was incubated for 10 minutes and washed for 30 minutes with 0.1 M PB and cover-slipped with Vectashield mounting medium. The Zeiss LSM image browser software was used to measure the thickness of ONL. The measurements were taken within 2 mm from the optic nerve.2 area. At these locations we made serial optical sections using a confocal microscope. By following each rhodopsin-positive rod throughout the sections, we ensured that every rhodopsin positive cell in the selected region was counted. Using these images, the Voronoi domain of each cell was generated. The coefficient of clustering (CC) was also analyzed by the ratio between the global coefficient of variance and average local coefficient of variance in Voronoi domain sizes.Voronoi analysis was used to quantify the distribution of rod cells stained with rhodopsin. The detailed instructions for the Voronoi analysis were described in our previous study , 64. BriImmunoblot analysis was performed on whole retinal cell lysate as previously reported , 66. BriAll the statistics were expressed as mean ± standard error of the mean (SEM). Student’s t-test was used for comparison . One-way ANOVA, two-way ANOVA and Fisher's least significant difference procedure (LSD test) were used to examine the differences among the group of means. The tests were performed by MATLAB version 8.2.0  and graphs were generated by GraphPad Prism 6. The difference between the means of separate experimental groups was considered statistically significant at P < 0.05.in vitro [Increased clusterin expression is associated with retinal degenerative diseases –46 incluin vitro –70. The In P30 normal retina, clusterin immunohistochemical labeling was not observed. In contrast, clusterin expression was noted in some processes in the inner nuclear layer (INL) of RP retina . To idenThe induction of clusterin expression in pathological conditions is still controversial since some reports suggest that clusterin is involved in apoptosis, whereas other reports demonstrated that clusterin participates in a protective role. Thus, we investigated if an exogenous application of clusterin could affect the rod survival in RP retina, with the hypothesis that clusterin was an “innate defender.” Before application of clusterin in RP, we tested three dosages of clusterin in normal retinas. Either saline or 10, 20, and 50 μg/ml of clusterin was injected and we observed the effects after 3 days , 1 week Intravitreal injection of clusterin (10 μg/ml) at P15, when rod photoreceptor cells show peak cell death in RP , resulte2 sampling areas  and RP Clusterin (Lt) retinas showed higher numbers of 7,529±994 and 9,650±534 cells/mm2 at P30, respectively. The one-way ANOVA analysis showed significant differences in mean among the different groups , and RP Clusterin Multiple (Lt) retinas. The mean density of cells in saline—treated RP retinas at P45, P60, and P75 were 500±24/mm2, 18±2/mm2, and 7±1/mm2, respectively. The density from the clusterin—single treated RP retinas at P45, P60, and P75 were 7,094±391/mm2, 1,188±36/mm2, and 711±111/mm2, respectively. The density from the clusterin—multiple treated RP retinas showed higher number of 8,790±337/mm2, 3,988±31/mm2, and 2,181±298/mm2, respectively. The two-way ANOVA analysis showed significant differences between the mean of different groups of retinas and the different postnatal days  nuclear, (2) secretory, and (3) cytoplasmic. The nuclear clusterin binds poptosis . The secpoptosis , 105. Wepoptosis , 107. Inpoptosis –111. We poptosis  at diffeWhat are the possible mechanisms underlying rod survival with the secretory form of clusterin? In our study, we did not observe clusterin expression in photoreceptor cells in RP retinas  whereas In S334ter RP retina, Bax is highly expressed in the early stage of retinal development . Bax plaIn RP retina, we also observed up-regulation of pSTAT3 in Müller cells after clusterin treatment . In the In this study, we investigated if an exogenous application of clusterin could affect the rod survival in RP retina, with the hypothesis that clusterin was an “innate defender.” Our data showed neuroprotective features of clusterin in a transgenic rat model of RP. Our study clearly demonstrated that clusterin treatment promotes rod cell survival by activating both Akt and STAT3 while deactivating Bax. These effects are encouraging and so dramatic that clusterin may be a potential therapeutic agent in slowing the progression of vision loss in human RP.S1 FigDensitometry analysis of clusterin-α bands at P15, P30, and P60 in normal and RP retinas was shown by measuring the intensity relative to the control β-actin. Data represents mean ± SEM.(TIF)Click here for additional data file.S2 FigConfocal micrographs taken from vertical cryostat sections processed for TOPRO-3 staining in P21-, P30-, P45-RP Saline and P21-, P30-, P45-RP Clusterin (Lt) retinas. The thickness of the ONL for P21, P30, and P45 RP Saline retinas was 13.2 ± 0.2 μm, 10 ± 0.2 μm and 4.2 ± 0.15 μm, respectively. The thickness of the ONL for P21, P30, and P45 RP Clusterin (Lt) retinas was 14.5 ± 0.3 μm, 12 ± 0.3 μm and 4.3 ± 0.1 μm, respectively. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; P, postnatal; N, normal; RP, Retinitis Pigmentosa. Data represents mean ± SEM. Scale bar = 20 μm. Data represents mean ± SEM, *** P<0.001.(TIF)Click here for additional data file.S3 FigCleaved caspase-3 expression level was evaluated by immunoblot analysis in saline- and clusterin-treated RP retinas (A). Retinas were collected at 5 min, 1 hour, 6 hours, and 24 hours after injection at P15. Cleaved caspase-3 expression was significantly decreased at 24 hours after clusterin injection (+) compared to 24 hours of saline injection (-). Densitometry analysis of cleaved caspase-3 expression was shown by measuring the intensity relative to the control β-actin (B). Data represents mean ± SEM, *** P<0.001.(TIF)Click here for additional data file.S1 TableLegend: Intensity of immunoreactive bands of clusterin precursor in RP retinas compared to normal retinas.(DOCX)Click here for additional data file.S2 TableLegend: Intensity of immunoreactive bands of clusterin- α in RP retinas compared to normal retinas.(DOCX)Click here for additional data file.S3 Table2 sampling areas of whole-mount retinas (Legend: The rhodopsin-immunoreactive rods were counted from the 1 x 1 mm retinas .(DOCX)Click here for additional data file.S4 TableLegend: The coefficient of clustering was measured in all groups .(DOCX)Click here for additional data file.S5 TableLegend: Immunoblot analysis shows up-regulation of clusterin precursor and clusterin-α in both RP Saline (Rt) and RP Clusterin (Lt) retinas compared to RP Saline retinas. Beta actin was used as loading control to obtain relative clusterin precursor  and clus(DOCX)Click here for additional data file.S6 Table2 sampling areas of whole-mount retinas (Legend: The rhodopsin-immunoreactive rods were counted from the 1 x 1 mm retinas .(DOCX)Click here for additional data file.S7 TableLegend: Immunoblot analysis shows up-regulation of pAKT expression in RP Clusterin (Lt) retina compared to RP Saline retinas from 5 minutes after injection at P15. Beta actin was used as loading control to obtain relative pAKT expression .(DOCX)Click here for additional data file.S8 TableLegend: Immunoblot analysis shows up-regulation of pSTAT3 expression in RP Clusterin (Lt) retina compared to RP Saline retinas at 5minutes, 1 hour, and 6 hours after injection at P15. Beta actin was used as loading control to obtain relative pSTAT3 expression .(DOCX)Click here for additional data file.S9 TableLegend: Immunoblot analysis shows suppression of BAX at 24hours after clusterin injection at P15. Beta actin was used as loading control to obtain relative BAX expression .(DOCX)Click here for additional data file.


Polypropionates—motifs with alternating methyl and hydroxy groups—are important segments of many natural products possessing high bioactivity and therapeutic value. Synthetic access to these structures remains an area of intensive interest, focusing on the establishment of the contiguous stereocentres and a desire for operational simplicity. Here we report an efficient strategy for the stereoselective assembly of polypropionates with three or four stereocentres through a three-step relay process that include Diels–Alder reaction, silylenol ether hydrolysis and Baeyer–Villiger oxidation. The stereochemistry and functionality of the resulting polypropionates depend on the substitution pattern of the diene and dienophile substrates of the Diels–Alder cycloaddition. More importantly, the relay sequence is effectively performed in one pot, and the product could potentially undergo the same sequence for further elaboration. Finally, the C1–C9 segment of the macrolide etnangien is constructed with four of the six stereogenic centres established using the relay sequence. Polypropionates are present in many natural products possessing high bioactivity and therapeutic value. Here the authors show a strategy for the stereoselective assembly of polypropionates with three or four stereocentres through a process that includes a Diels–Alder reaction, silylenol ether hydrolysis and Baeyer–Villiger oxidation. They exhibit a wide range of biological activities, including antibiotic, antitumour, antifungal, cholesterol-lowering, antiparasitic and immunosuppressive properties2. In particular, several polyketide-containing natural products, such as the antibiotics erythromycin3 and monensin A4 and the antitumour discodermolide5 , for example, could have 16 possible diastereomers. Consequently, the preparation of polypropionates has been a longstanding interest in the synthesis community10.Polyketides are a large class of structurally diverse secondary metabolites isolated from fungi, bacteria and sponges11, but crotylation14, allenylation15, epoxide opening6, [2 + 2]-cycloaddition16 and many other methods6 are available. Although the application of these methods in iterative protocols allowed for more complex polypropionate subunits, additional step-consuming transformations are required for each subsequent C–C bond extensions. In addition, the stereochemical control of the newly created stereocentres over the course of the propionate chain elongation is often complicated. Attractive alternatives, especially considering atom and step economy, are multicomponent domino (or cascade) reactions19 that could create more than one C–C bond, and hence, multiple stereocentres in a single-pot operation. Nevertheless, only particular sets of stereoisomers could be achieved in these procedures. Thus, further development of convenient entries to several possible stereotriads, stereotetrads or stereopentads, as well as their related structures that exploit operationally simple strategies in library design and drug discovery of polypropionate-containing molecules are still highly desirable.Numerous synthetic approaches have been developed to address the architectural complexity of polypropionates. Forging a single C–C bond into adjacent methyl and hydroxy-bearing stereogenic centres could be accomplished by established aldol reactionsMotivated by the intensive interest towards the expedient synthesis of polypropionates, we herein devise an alternative stereoselective strategy from simple and readily available precursors. This approach involves sequential Diels–Alder cycloaddition of silylenol ethers and enones acting as the respective dienes and dienophiles, silylenol ether hydrolysis to afford a cyclic ketone and Baeyer–Villiger oxidation to enable double oxygen insertions with stereoretention at key positions. Ultimately, the relay sequence provides a suitable access into polypropionates with three or four contiguous stereocentres both in stepwise and one-pot manner. Using this procedure, the synthesis of the C1–C9 segment of the macrolide natural product etnangien is also successfully demonstrated.1, harbouring three to four stereocentres / = 86/14). Alternatively, use of triethylamine as base to permit the same reaction led to lower yield (82%) and diastereoselectivity for the desired -isomer (85/15). The cycloaddition of diene 8 and dienophile 9 is expected to produce the exo-adduct based on the methyl substitution at C1 of 8 and Cβ of 921  triflate, scandium(III) triflate, trimethylsilyl triflate, dimethylaluminum chloride and tin tetrachloride, also gave similar exo-preference, but only in moderate-to-good yields -isomer of 8 and the enone 9 was not observed in these cases. The desilylation of 10 could be achieved by tetrabutylammonium fluoride (TBAF), but acid hydrolysis offers better compatibility with Baeyer–Villiger oxidation in light of our desire for a one-pot operation. Consequently, treatment with trifluoroacetic acid (TFA) for 20 min delivered the corresponding dione, which was directly treated with meta-chloroperoxybenzoic acid (mCPBA) for the double oxygen insertion to afford the expected ε-lactone 11 carrying a polypropionate syn,syn,syn-stereotetrad in 75% yield . The structure of 11 was unambiguously confirmed by X-ray crystallographic analysis  as a single diastereomer, providing a possible point of access to another stereotetrad structure.To examine the proposed relay concept, we first applied the designed procedure to the prototypical enones ner Fig. . Deprotor yield 8% and diar yield 8% and diasis Fig. . Curious11 with catalytic sodium methoxide in methanol resulted in deacetylation and transesterification, supplying the γ-lactone 13 (96%). This cyclic system should offer more stability than the initial seven-membered ring structure and could serve as key intermediate with various potential for further manipulation. With the intent to explore the possibility of reiterating the tandem Diels–Alder/Baeyer–Villiger reaction process, we examined the elaboration of compound 13 to further generate an advanced enone structure. The operation started from the silyl group protection of the free hydroxy group (97%), followed by nucleophilic addition to the carbonyl using allylmagnesium chloride. Subsequent base-promoted double bond isomerization ultimately afforded the desired enone 14 (78% in two steps). This and similar compounds offer prospective applications for the assembly of longer and more complex polypropionate chains.Treatment of the lactone exo-adduct, formed in situ from compounds 8 and 9 by the catalytic assistance of boron trifluoride, was smoothly converted to the desired ε-lactone 11 in 63% yield upon successive treatment with TFA and mCPBA in the same vessel (entry 1). For cycloaddition with enone 15, we found that copper(II) triflate provided the best product yield and selectivity for the expected endo-adduct20 . Further application of the one-pot procedure to different diene and enone combinations all provided satisfactory results (entry 3–12). Curiously, when the phenyl-substituted 32 was employed as starting enone (entries 11 and 12), minor amounts of the separable corresponding γ-lactone (34 and 36), apparently resulting from acid-induced translactonization22, was acquired along with the expected ε-lactone (33 and 35). The structures of the duly formed lactones were confirmed by NMR spectroscopy as well as X-ray crystallographic analysis for compounds 16, 23, 29 and 34  of the commercially available (S)-3-phenylbutanoic acid (37) / = 70/30).To demonstrate the utility of the tandem one-pot Diels–Alder/Baeyer–Villiger relay process for the assembly of polypropionates leading to more complex molecular architectures, we targeted the stereohexad structure present at the C1–C9 subunit of the macrolide etnangien  Fig. . Nucleop39 in hand, the formation of the target ε-lactone through the crucial relay process was first performed in a stepwise fashion. Thus, the Diels–Alder cycloaddition of the diene 39 and enone 9 employing the boron trifluoride-catalysed condition produced two exo- and two endo-cycloadduct diastereoisomers (exo/endo = 84/16) of which the desired silylenol ether and exo-adduct 40 was obtained as the major diastereomer in 65% yield; the other diastereomers were collectively afforded in 25% yield . Again, an X-ray analysis supported the structure and relative configuration of the stereopentad motif of the crystalline 42 . This fragment possesses the synthetic versatility that would enable further elaboration towards etnangien and other more advanced molecules.With implicitly positioned terminal groups, te yield . SilylatmCPBA formed the lactone and ester functionalities. Using this procedure, a range of polypropionate stereoisomers with high stereochemical flexibility and substantial skeletal diversity was accessed. The amenability of our strategy for a one-pot operation offers a rapid and convenient method to the many possible polypropionate motifs as demonstrated herein. Importantly, the resulting products not only feature the polyketide subunits, but also render functionalities that could, in principle, be further transformed for use in subsequent C–C bond forming events or other elaborations to more complex structures. Further extension of this methodology to provide more comprehensive access to other biologically significant polypropionate motifs is ongoing and will be reported in due course.We have developed an efficient approach to stepwise and one-pot stereoselective synthesis of polypropionate structures through a three-step Diels–Alder cycloaddition/Baeyer–Villiger oxidation relay process. The highly substrate-controlled Diels–Alder reaction, facilitated by the Lewis acid catalysts boron trifluoride and copper(II) triflate, enabled the establishment of the necessary cyclohexene ring featuring the silylenol ether and three-to-four stereogenic centres. Following an acid hydrolysis to provide the aptly positioned dicarbonyl groups, Baeyer–Villiger oxidation with 9  in CH2Cl2 (2 mL) was added by syringe to a solution of BF3·OEt2  in CH2Cl2 (2 mL) at −78 °C under argon atmosphere. After stirring at −78 °C for 10 min, diene 9 / = 70/30, 0.600 g, 1.99 mmol) in CH2Cl2 (2.0 mL) was added dropwise, and the reaction was allowed to stir for 12 h at the same temperature. TFA  was next added in one portion, and the reaction was allowed to warm to 0 °C. After 20 min at 0 °C, the resulting mixture was poured into a solution of mCPBA  in CH2Cl2 (20 mL) at 0 °C. The reaction was warmed to ambient temperature and stirred for 24 h. The reaction mixture was slowly quenched with satd. Na2S2O3(aq) at 0 °C, and the crude organic portion was extracted with CH2Cl2. The combined organic layer was washed with satd. NaHCO3(aq) and brine, dried over MgSO4, filtered and concentrated under reduced pressure. Purification via flash column chromatography (n-hexane/ethyl acetate = 4/1) provided the ε-lactone 41  as a colourless solid. [α]23D = −7.28 ; m.p. 130–131 °C ; Infrared Spectrum (thin film): ν 2973, 1734, 1637, 1454, 1239, 1183, 1087, 1020, 767, 703 cm–1; 1H NMR : δ 7.28 , 7.21 , 7.12 , 4.70 , 4.67 , 3.28 , 2.99 , 2.44 , 2.10 , 1.90 , 1.56–1.54 , 1.38 , 1.14 , 1.01 ; 13C NMR : δ 173.6 (C), 168.9 (C), 143.1 (C), 128.9 (CH × 2), 127.2 (CH × 2), 127.2 (CH), 80.9 (CH), 77.7 (CH), 41.9 (CH), 37.4 (CH), 35.1 (CH2), 32.6 (CH), 21.1 (CH3), 20.2 (CH3), 16.9 (CH3), 10.1 (CH3); High Resolution Mass Spectrum (Electrospray Ionisation): m/z calcd for C18H24O4Na ([M + Na]+): 327.1572, found: 327.1580. An X-ray analysis of the crystal supported the structure of 41 and the relative configuration of its contiguous chiral centres , under deposition numbers CCDC 1511846, CCDC 1511847, CCDC 1511848, CCDC 1511849, CCDC 1511880, CCDC 1511850, CCDC 1556026 and CCDC 1556034, respectively. These data can be obtained free of charge from the CCDC via www.ccdc.cam.ac.uk/data_request/cif. All data that support the findings of this study are available from the corresponding author upon reasonable request.The X-ray crystallographic coordinates for compounds Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8


This dataset provides detail information on the analytical methods of organophosphate esters (OPEs) in sludge samples, including the sample preparation, ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) analysis, quality assurance and quality control (QA/QC). The concentration of target OPE compounds in collected samples of four individual treatment was provided, including aerobic composting combined with pig manure (T1), aerobic composting without pig manure (T2), anaerobic digestion combined with pig manure (T3), and anaerobic digestion without pig manure (T4). To investigate the variation of bacterial community compositions, principal components analysis (PCA) was provided based on the high-throughput sequencing. These data would be useful for clarifying the removal of OPEs under aerobic and anaerobic conditions. Besides, it also provides important information on the potential bacterial strains responsible for the biodegradation of OPEs in each treatment. Specifications TableValue of the data•The dataset provides the detail information on the analytical method, including sample preparation, UPLC-MS/MS analysis, and QA/AC.•The concentration of OPEs in each sampling would be useful to understand the removal rate and make a comparison among different treatments.•To understand the variation of bacterial community compositions, abundance, and diversity in sewage sludge with different conditions.•To identify the potential bacteria responsible for the degradation of OPEs in sewage sludge with different conditions.1Organophosphate esters (OPEs) are widely used as flame retardants and plasticizers in recent years 22.127 at 5 mg L−1 as surrogate before extraction. Additional diatomaceous earth was added to fill the remaining free space of the cell. Two pieces of cellulose filter were placed on the bottom and top of the extraction cell, respectively. After ASE procedure, the extract was evaporated to almost dryness by using a rotary evaporator. The extract was re-dissolved in 6 mL of ACN and diluted to 200 mL with ultrapure water. The solution was filtered by GF/C membrane  and then subjected to an Oasis HLB cartridge . The analytes were eluted by 8 mL of acetonitrile (ACN) and then concentrated to nearly dryness. The residue was redissolved in 1.5 mL ACN/water  and 5 μL of the solution was injected into UPLC-MS/MS for analysis.Briefly, the extraction procedure was performed on a Dionex ASE 350 system . Small amount of diatomaceous earth and 0.5 g of sample were loaded into a 33 mL capacity stainless steel cell. Each sample was spiked with 10 μL of TnBP-d2.2−1 was applied. The gradient was set as follows: 0 min (40% B), 0.5 min (40% B), 3 min (50% B), 4.5 min , 8.5 min (70% B), 9 min (100% B), 13.8 min (100% B), 13.9 min (40% B), 17 min (40% B).A UPLC system  equipped with a triple quadruple mass spectrometer  was used for the determination and identification of OPEs. The separation of analytes was performed on a Hypersil GOLD C18 . A binary mobile phase of an aqueous solution of 0.1% formic acid (A) and ACN containing 0.1% formic acid (B) at a flow rate of 0.3 mL minm/z, spray voltage 4500 V, sheath gas pressure 35 units, auxiliary gas pressure of 20 units, and capillary temperature 300 °C.For MS/MS analysis, the electrospray ionization (ESI) was run in the positive ion mode. The optimal conditions were set as follows: peak width resolution 0.7 2.3n = 3), procedural blanks (n = 3), spiked blanks (n = 3), spiked matrix (n = 3), and replicate samples (n = 7) were analyzed with extraction to control contamination. In each spiked sample, 50- and 100-ng mixture of OPEs were added. All samples were spiked with TnBP-d27 as surrogate. TCEP was not found in the blank; TnBP, TPhP, TCPP, and TBEP were detected at 2.95, 9.38, 3.90, and 2.00 g L−1 in the blank. The recoveries of standards in spiked samples were within 56–113% at two different spiked concentration levels. The matrix effect was evaluated by addition of standards into the pre-extracted samples were in the range of 83–121% at two different spiked concentration levels. Each batch of ten samples included one procedural blank to check potential contamination. All glassware was solvent rinsed and heated overnight at 400 °C before usage.Field blanks (


Zuotai  is one of the famous drugs containing mercury in Tibetan medicine. However, little is known about the chemical substance basis of its pharmacodynamics and the intrinsic link of different samples sources so far. Given this, energy dispersive spectrometry of X-ray (EDX), scanning electron microscopy (SEM), atomic force microscopy (AFM), and powder X-ray diffraction (XRD) were used to assay the elements, micromorphology, and phase composition of nine Zuotai samples from different regions, respectively; the XRD fingerprint features of Zuotai were analyzed by multivariate statistical analysis. EDX result shows that Zuotai contains Hg, S, O, Fe, Al, Cu, and other elements. SEM and AFM observations suggest that Zuotai is a kind of ancient nanodrug. Its particles are mainly in the range of 100–800 nm, which commonly further aggregate into 1–30 μm loosely amorphous particles. XRD test shows that β-HgS, S8, and α-HgS are its main phase compositions. XRD fingerprint analysis indicates that the similarity degrees of nine samples are very high, and the results of multivariate statistical analysis are broadly consistent with sample sources. The present research has revealed the physicochemical characteristics of Zuotai, and it would play a positive role in interpreting this mysterious Tibetan drug.  Zuotai , known as “King of Nectar's Essence,” is one of the most famous and important drugs in traditional Tibetan medicine [ Nengchi Eight Metals, Nengchi Eight Minerals, and other natural drugs through special processing technology by senior Tibetan medicine practitioners [ Zuotai generally is not used alone, but it is usually used as an adjuvant with other compound drugs [ Zuotai could enhance the efficacy and reduce the toxicity of other drugs [ Zuotai has already been used as a key component of Renqing series drugs in Tibetan medicine for 1300 years [Renqing Mangjue,” “Renqing Changjue,” “Rannasangpei,” “Qishiwei Songshi Wan,” “Shanhu Qishiwei Wan,” “Zuota Demazi,” “Zuozhu Daxi,” “Dangzuo,” and other preparations [Tibetan medicine is one of the world's existing traditional medicines having completely medical theory, with more than 2000 years' phylogeny –3. Zuotamedicine –7. It isitioners –6, 8–10.nd drugs . And theer drugs . Zuotai 00 years . By now,arations , 11, 12.arations , 6, 8. Zuotai containing mercury need urgently to be reevaluated from a modern medical science perspective. Surprisingly, its toxicity tests suggest that Zuotai has no apparently adverse effects on animal subjects under clinical equivalent dose and clinical medication cycle [ Zuotai not only can tranquilize and allay excitement, promote sleep, and produce antipyretic effect [+3 is an essential element for mammals with a role in maintaining proper carbohydrate and lipid metabolism; on the contrary, Cr+6 has carcinogenic toxicity and genotoxicity [ Zuotai for elucidating its pharmacological functions and mechanisms.As known, heavy metals could pose potentially serious hazard to human health. So, the biological safety and efficacy ofon cycle , 13–16. c effect , 18, butc effect . It is ctoxicity –25. So,  Zuotai are available. Yan [ Zuotai was mainly an inorganic mixture of HgS, sulfur, graphite, and other tiny organic molecules, and it was composed of nanoparticles, which further aggregated into microsized particles. However, there still lacks more information about the chemical structure, micromorphology, and XRD fingerprint features of Zuotai from large sample sizes. Therefore, in the present research, nine Zuotai samples were collected from four major Tibetan regions  in China. The element compositions, phase structures, and micromorphology of these samples are analyzed by energy dispersive spectrometry of X-ray (EDX), powder X-ray diffraction (XRD), scanning electron microscopy (SEM), and atomic force microscopy (AFM), respectively. Additionally, the powder XRD fingerprint features of Zuotai were analyzed by applying multivariate statistical analysis methods .Until now, only a few literatures about physicochemical characterization ofble. Yan , 27, Lanble. Yan , and Zhable. Yan  reported Zuotai samples were kindly provided from the four Tibetan medicine organizations of four major Tibetan regions in China, respectively. The sources of Zuotai samples were listed in Nine Zuotai samples were detected by energy dispersive spectrometry of X-ray . EDX is a kind of semiquantitative elemental analysis method. The working conditions of EDX are operating voltage of 20 kV, beam spot size of 55 nm, working distance of 8 mm, and hit rates of 2500 cps.The elements in nineα1) energy of 40 keV, current intensity of 30 mA, scanning speed of 2°/min, and 2θ angle scan range of 5°–80°. Powder X-ray diffraction data were acquired by X'Pert Data Collector and analyzed by X'Pert HighScore Plus using PDF2-2004 of the International Centre for Diffraction Data. Powder XRD spectra were drawn by Origin 8.0. The crystal structure graphs of phase compositions in Zuotai were acquired by Diamond 3.2 and FindIt 2009.The phase compositions of samples were characterized by powder X-ray diffraction . The working conditions of XRD are Cu target, wavelength of 0.15406 nm, K  and atomic force microscopy . The working conditions of SEM are LV mode, acceleration voltage of 30 kV, working distance of 8 mm, and working pressure of 1 MPa in electron gun chamber. The working conditions of AFM are ScanAsyst Auto Control, RTESPA-300 probe, scan size of 1 μm, scan rate of 1 Hz, sample/line of 256, peak force amplitude of 150 nm, and Z range of 17.9 μm.The micromorphologies ofThe powder XRD fingerprint features were studied with multivariate statistical analysis methods  by SPSS 22.0 statistical analysis software. Zuotai samples by EDX analysis. Besides, a small amount of other elements has also been found, such as ferrum (Fe), aluminum (Al), and cuprum (Cu). The results are shown in http://dx.doi.org/10.1155/2016/7010519). However, we should note that the oxygen (O) in Zuotai may be interfered with inevitably to a certain extent by the residual oxygen from air in the sample testing chamber of EDX. So, whether Zuotai samples contain such high level oxygen element needs further study.A large amount of mercury (Hg), sulfur (S), and oxygen (O) was found in nine Zuotai samples display a variety of micromorphologies, which are composed of a large number of irregular nano-microparticles by scanning electron microscopy (SEM) and atomic force microscopy (AFM), as shown in Figures μm loosely amorphous particles. There are a number of literature reports in both the basic science and pharmaceutics which scientifically define dimensions of nanoparticles ranging in size from 1 to 1000 nm [ Zuotai should be a kind of ancient nanodrug in traditional Tibetan medicine.Nine 1000 nm –34. Accoβ-HgS) and orthorhombic crystal elemental sulfur (S8) in nine Zuotai samples (β-HgS in Zuotai is F-43m (216) with cell parameters a = b = c = 5.8580 and α = β = γ = 90°. The spacing group of S8 in nine samples is Fddd (70): a = 10.4646, b = 12.8660, c = 24.4860, and α = β = γ = 90°. Additionally, except for #9 sample, all the other samples contain a small amount of hexagonal crystal mercuric sulfide (α-HgS)  with cell parameters a = b = 4.1495, c = 9.4970, and α = β = 90°,   γ = 120°. Besides, there are also many unknown weak diffraction signals in the nine samples.Powder XRD analysis found that the common major phase compositions are cubic crystal mercuric sulfide  and hexagonal mercuric sulfide (α-HgS) without elemental mercury. Both mercuric sulfides are typical covalent insoluble sulfides. The solubility product constant of β-HgS is 1.6 × 10−52; the solubility product constant of α-HgS is 4.0 × 10−53 [This result indicates that the chemical species of mercury in × 10−53 . So far, × 10−53 , 37.θ angles were acquired by overlaying the diffraction topology spectra of nine samples firstly and then averaging. According to these means, the powder XRD fingerprint (average) of Zuotai was drawn by Origin 8.0  was also drawn by Origin 8.0  of nine Zuotai samples were calculated with SPSS 22.0. The results show that the similarity degrees of the nine samples are very high  in The XRD fingerprints (average and median) ofI/I0) of 25 common characteristic peaks were considered as indicators; nine Zuotai samples were clustered with systematic cluster analysis [Dkl = (1/nknl)∑xi∈Gk∑xi∈Gldij) is used to measure the distance between both groups; the Mahalanobis distance method (dij = (xi − xj)′S−1(xi − xj)) is applied to measure the distance between samples, in which n refers to sample number, nk and nl refer to the kth sample and the lth sample, Gk and Gl refer to the kth group and the lth group, Dkl refers to the distance between group Gk and group Gl, dij refers to the distance between sample xi and sample xj, and S−1 refers to the inverse matrix of sample covariance matrix. The result of cluster analysis is shown in 25 common peaks (Supplementary Table 1) are found through analyzing all the diffraction peaks of nine samples in Jade 5.0. And the relative intensities , orthorhombic elemental sulfur (S8), and hexagonal mercuric sulfide (α-HgS). But #9 sample only contains cubic mercuric sulfide and orthorhombic sulfur, but not hexagonal mercuric sulfide.When the relative distance is 10, nine samples are clustered in four groups: #3, #4, and #5 samples clustering together, #1, #6, and #8 samples clustering together, #2 and #7 samples clustering together, and #9 sample as a group alone. When relative distance is 23, nine samples are clustered in two groups: #1, #2, #3, #4, #5, #6, #7, and #8 samples clustering together and #9 sample still as a single group. Only when relative distance is 25 can all of the samples be clustered together. This result is consistent with the phase composition differences of nineI) of 25 common peaks of nine Zuotai samples were analyzed with PCA by SPSS 22.0, and then the initial eigenvalues  is a kind of reducing dimensions method, in that data are projected from high-dimensional space onto low-dimensional space, namely, using a smaller number of new variables instead of a huge number of original variables with minimal information loss –41. In tenvalues  and load F1, F2, and F3, resp.) can well explain the inherent information of the original 25 characteristic peaks of nine Zuotai samples.In rij refers to the load of principal component, i = 1,2,…, n; j = 1,2,…, p; n is sample number; p is the indicator number of sample). According to this equation, standardized eigenvectors (shown in Supplementary Table 3) of the first three principal components  are calculated. The linear equations of the first three principal components  are obtained through multiplying the standardized eigenvectors with the corresponding normalized peak intensity (ZX). The equations of F1, F2, and F3 of Zuotai are as follows:And the variables coefficients of principal component linear equation, namely, standardized eigenvector, can be calculated from the load values of principal components. The standardized eigenvector equation of principal components is  Zuotai, the principal component scores of nine Zuotai samples were calculated, and the 3D space scatter diagram , a Tibetan medicine classic, in the AC 8th century, and was improved by U rgyan pa Rin chen dpal, a famous Tibetan medicine practitioner, in AC 13th century [ Zuotai survives and flourishes till today; this may have been because Zuotai has been used as a core adjuvant in many precious Tibetan medicine component preparations [ Zuotai has been collected in the Chinese Intangible Cultural Heritage List [Tibetan medicine has been playing an important role in the health, reproduction, and development of Tibetan people. effects –8. Zuotainto ash , 42–44. chnology , 11. The century , 45. Thiarations . Meanwhiage List . Zuotai particles are mainly in 100–800 nm range, and some are less than 100 nm, which commonly aggregate into about 1–30 μm loosely amorphous particles. This is consistent with the reports of Yan et al. [ Zuotai is a kind of ancient nanodrug. There are three main absorption ways of oral nanoparticles in the intestinal tract [μm). It is surprising to find that the scale of Zuotai particles coincides with modern nanodrug concept. This finding greatly arouses our interests in this ancient nanodrug. According to the above absorption ways of nanodrugs in the intestine, Zuotai particles may mainly be absorbed through the intestinal epithelial cells and the M cells of Peyer's patches.Nanometer is not only a concept of spatial scale, but also a new way of thinking. The present research found thatn et al. , Lan et n et al. , and Zhan et al. . In the n et al. . So, thial tract , 49: (1) Zuotai is the prerequisite of understanding its peculiarly biological effects. The present study suggests that Zuotai contains cubic α-HgS , orthorhombic S8 , and hexagonal α-HgS . Besides, there are still many unknown very weak X-ray diffraction signals, which are difficult to analyze. According to the processing technology of Zuotai, we can know that it is obtained through mixing the burned Nengchi Eight Eshes and the burned Nengchi Eight Mineral Ashes into the prepared mercury powder at 3.3% rate, respectively. The chemical components of Nengchi Eight Eshes and Nengchi Eight Mineral Ashes have been reported [ Zuotai can be deduced from the Nengchi Eight Eshes and the refined Nengchi Eight Mineral Ashes. Therefore, except for the main phase compositions above, Zuotai also should contain minor or trace quadratic AuPb2 , quadratic PbO , orthorhombic PbO , cubic Pb , monoclinic Ag2S , quadratic Cu1.98(Zn0.73Fe0.29)Sn0.99S4, hexagonal CuS , hexagonal SiO2 , hexagonal NaCu2S2 , rhomboidal Ca(CO3)2, orthorhombic Cu7S4 , monoclinic Cu7S4 , monoclinic CuO , hexagonal FeS , cubic PbS , cubic ZnS , rhomboidal CaCO3 , cubic Fe+2Fe2+3O4 , orthorhombic SnS , orthorhombic PbSO4 , hexagonal SnS2 , cubic KCl , monoclinic K3Si3O102 , orthorhombic Mg2SiO4 , monoclinic KMg3Si3AlO102 , triclinic (Na0.4Ca0.6)Al1.6Si2.4O8 (unknown spacing group), orthorhombic FeAs , monoclinic K2Ca(SO4)2·H2O , orthorhombic FeAs2 , quadratic Fe2As , rhombic-hexahedral CaCO3 , cubic Cu2S , and so on [ Zuotai may also contain some amount of FeC, ε-Fe2O3, (S4N3)Cl, C, and trace organics. As seen from the above, the chemical components and crystal structures of Zuotai are extremely complex, and this may be the substance basis of its peculiar curative effects.Accurate characterization for the chemical species of heavy metals inreported , 51 by snd so on , 47. Bes Fm-3m, 2, cubic Znd so on  reported Zuotai. The chemical species of mercury in Zuotai are insoluble cubic mercuric sulfide (β-HgS) and minor hexagonal mercuric sulfide (α-HgS). And orthorhombic sulfur (S8) is also the main phase composition in Zuotai. Moreover, Zuotai is a kind of ancient nanodrug in traditional Tibetan medicine. Its particles are mainly in the range from 100 nm to 800 nm and further aggregate into 1–30 μm loosely amorphous particles. Besides, the intrinsic relationship among samples from different sources is revealed through multivariate statistical analysis, according to the established powder XRD fingerprint of Zuotai. As an important drug containing heavy metals in Tibetan medicine, Zuotai has many mysteries, which are worth exploring. Science always has its historical limitation, and if the knowledge of contemporary science cannot explain it, we cannot abandon it blindly, but it needs to be handed down, waiting for the science of the future to explain it.In summary, the present research suggests that mercury (Hg) and sulfur (S) are the major elements, and ferrum (Fe), aluminum (Al), and cuprum (Cu) are the minor metal elements in0) of 25 Common XRD Peaks in Nine Zuotai Samples, Load Matrix of Principal Components, Standardized Eigenvector Matrix, and The Lattice Distances (Å) of Nine Zuotai Samples XRD Peaks). These materials could provide further experimental data details for this research paper, and could provide more reference for some interested researchers.This Supplementary Materials are composed by one supplementary figure (EDX Spectrum of Nine Zuotai Samples) and four supplementary tables (Peaks Position (2θ/°) and Relative Intensity (I /I


Let β are positive numbers. In α-modulatIn order to prove the theorems, we also need some lemmas.Van der Corput lemma ([p.334). Letφandϕbe real valued smooth functions on the intervalandIffor alland (1) is monotonic onor (2) then we havet lemma .Ifandthen, for anyis bounded fromto(1) Ifandthen, for anyis bounded fromto(2) (1) It suffices to show thatondition , we haveLetorIfandthenis bounded fromtoBy checking the following proof, we can only consider the operatorCase 1. If Case 2. If milar to , we getombining  and (3.3ing with , we getLetorIfandthenis bounded fromtoAs the proof of Theorem Ifandthenis bounded fromtoWhen proof in , when \dideas in  and follIn Theorems sults in . Unlike sults in , we use sults in . Unfortuentclass1pt{minimaα-modulation space cannot be obtained by interpolation between modulation spaces In , the autγ in M such that We can extend this result to the well-curved ved. See , 18 for ved. See  (Proposiideas in , combiniLetbe well-curved. Ifandthenis bounded fromtoα-modulation spaces, combining the Fourier transform and Van der Corput lemma, we obtained the strongly singular integrals along homogeneous curves are bounded on the α-modulation spaces for all In this paper, using the equivalent discrete definition of sults in . Our metsults in .


Our results demonstrated that this combination therapy significantly decreased disease severity, inflammatory cell infiltration, secondary demyelination, and axonal loss, thus reducing neural death. In conclusion, our study suggests a possible treatment that can relieve progressive blindness and paralysis in an animal model of NMO through improvement of the inflammatory milieu.Neuromyelitis optica (NMO) is an autoimmune inflammatory demyelinating disease that mainly affects the spinal cord and optic nerve, causing blindness and paralysis in some individuals. Moreover, NMO may cause secondary complement-dependent cytotoxicity (CDC), leading to oligodendrocyte and neuronal damage. In this study, a rodent NMO model, showing typical NMO pathogenesis, was induced with NMO-IgG from patient serum and human complement. We then tested whether the combination of C16, an  Neuromyelitis optica (NMO) is an autoimmune inflammatory disease that selectively targets the optic nerves and spinal cord, leading to blindness and paralysis . Since NWorldwide, the prevalence of NMO is far lower than that of MS, but NMO is more common in Asian populations . NMO hasαvβ3, interfering with leukocyte transmigration across the endothelial cell layer in vitro [αvβ3 could decrease monocyte binding to intercellular adhesion molecule-1 (ICAM-1) and block monocyte migration across the endothelium [in vivo [The synthetic C16 peptide (KAFDITYVRLKF), representing a functional laminin domain, selectively binds to the integrin in vitro , 13. Pre[in vivo –16. In tThe study protocols were approved by the Ethics Committee of Zhejiang University. All animal procedures used in this study were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.Ninety Lewis rats  were used in this study. To induce NMO, serum was obtained from two patients with an established diagnosis of NMO and strong AQP4 autoantibody serum positivity. Human NMO immunoglobulin G was purified as described previously . AQP4-Abμg NMO-IgG and 50 μL (5 μg/μL) human complement  for 3 days (1 μL/hr). Meanwhile, the vertebrae were carefully separated using two fine tweezers to expose the lumbar spinal cord (L4–L5); the same amount of NMO-IgG and human complement was infused for 3 days intrathecally with Alzet 1003D minipumps and catheters.The rats were anesthetized by 1% Nembutal . For intraventricular injection, the coordinates of the injections were as follows: anteroposterior (AP) −0.7 mm; mediolateral (ML) −1.7 mm from the bregma; and depth, 5 mm from the skull surface . An osmon = 30) and C16 + Ang1- (C + A-) treated (n = 30). Non-NMO animals (n = 30), receiving normal serum with human complement, were also included as a control group.NMO rats were randomized into two groups: vehicle-treated  solution (4 mg/mL) was prepared as described previously , 14. Briμg/mL. The mixture of 0.5 mL of Ang1 (400 μg) and 0.5 mL of C16 (2 mg) was injected intravenously via the tail vein every day for 1 week. The first dose was given immediately after NMO induction. Because scrambled C16 acts as an αvβ3 and α5β1 antagonist, reverse C16 has some binding activities for endothelial cells in culture [Ang1 protein  was dissolved in distilled water to a final concentration of 800  culture , and the culture , 19, we Disease severity was assessed on a daily basis by two observers using a scale ranging from 0 to 10: 0, normal; 1, reduced tone of the tail; 2, limp tail, impaired righting; 3, absent righting; 4, gait ataxia; 5, mild paraparesis of the hindlimb; 6, moderate paraparesis; 7, severe paraparesis or paraplegia; 8, tetraparesis; 9, moribund; and 10, death [Cortical somatosensory-evoked potentials and cortical motor-evoked potentials were recorded at 1 and 8 weeks postinjection (pi), as described previously , 14–16.n = 10/group at each time point) after NMO-IgG injection. The spinal cords, brains, and eyes, with the optic nerve cut at 1 mm behind the eye globe, were collected. The tissues were fixed in 4% paraformaldehyde overnight at 4°C and were sequentially cryoprotected in 15% and 30% sucrose in PBS, embedded in Tissue-Tek optimal cutting temperature compound , and made into cryosections (20 μm thickness). The sections were then mounted on 0.02% poly-L-lysine-coated slides. The remains of the CNS tissue were fixed in 5% glutaraldehyde solution and examined by transmission electron microscopy.To prepare cryosections, rats were anesthetized with sodium pentobarbital and transcardially perfused with 4% paraformaldehyde at 3 days, 1 week, and 8 weeks ; 3, moderate cellular infiltration in the parenchyma (11–100/section); and 4, serious cellular infiltration in the parenchyma (100/section) , 20.The sections were stained with cresyl violet (Nissl staining) to observe the neuronal structure, as described previously , 20. NeuDemyelination was evaluated by Luxol fast blue staining and scored using a 6-point scale system: 0, normal white matter; 1, rare foci; 2, a few areas of demyelination; 3, confluent perivascular or subpial demyelination; 4, massive perivascular and subpial demyelination involving one half of the spinal cord with the presence of cellular infiltration in the CNS parenchyma; and 5, extensive perivascular and subpial demyelination involving the whole cord section with the presence of cellular infiltration in the CNS parenchyma , 20.Axonal loss was estimated by Bielschowsky silver staining using the following 4-point scale: 0, no axonal loss; 1, a few foci of superficial axonal loss involving less than 25% of the tissues; 2, foci of deep axonal loss that encompassed over 25% of the tissue; and 3, diffused and widespread axonal loss , 20.Five transverse sections from each animal were selected at random, and digital photomicrographs were obtained at 200x magnification in three visual fields per section. Quantification of the histological results was done by investigators blinded to the treatments.The sections were preincubated with PBS containing 0.1% Triton X-100 (PBST) for 10 min at room temperature, then blocked with 10% normal goat serum in PBST (blocking solution) for 1 h, followed by incubation in blocking solution overnight at 4°C with one of the following antibodies: mouse anti-neurofilament M , glial fibrillary acidic protein , myelin basic protein , and aquaporin-4 , followed by the appropriate fluorescent secondary antibody . The sections were mounted with Antifade Gel/Mount aqueous mounting media . All control sections were incubated in PBS without primary antibodies. Five transverse sections from each animal were randomly selected, and digital photomicrographs were obtained at 200x magnification in three visual fields per section. Areas that exhibited immunoreactivity were analyzed with NIH ImageJ software.2O2 in 0.005 Tris-HCl, the sections were counterstained with hematoxylin. Controls without adding primary antibody were used to further confirm the specificity of the immunohistochemical labeling. Five transverse sections from each animal were selected at random, and images were photographed under 200x magnification in three visual fields per section. Caspase-3-immunoreactive cells were counted on sections of the retina as well as anterior horns of each spinal cord.Rabbit anti-activated caspase-3  and CD45  were incubated overnight at 4°C. Sections were incubated with secondary biotinylated goat anti-rabbit IgG antibody  for 1 h at 37°C followed by avidin-biotin peroxidase complex . After incubation for 5 min with 0.02% 3,3′-diaminobenzidine and 0.003% Hn = 3/group). Briefly, the brain surface was exposed by drilling the parietal bone using a stereotactic apparatus. A total of 2.1 mL of 5% FluoroGold  was injected bilaterally at 5.5 mm caudal to the bregma, 1.2 mm lateral to the midline, and 4.5 mm in depth from the skull surface. To count the RGCs, isolated retinas were fixed in 4% paraformaldehyde and then mounted on a glass slide with the RGC layers facing up. FluoroGold-positive RGCs were identified under a fluorescence microscope using an ultraviolet light filter set (377/407 nm). RGCs were counted in 12 areas of 0.072 mm2 each  by an investigator blinded to the treatment conditions [RGCs were retrospectively labeled by bilateral stereotactic injection of FluoroGold into the superior colliculus 5 days before treatment with the NMO-IgG and human complement , whole-cell lysates were prepared from the lumbar spinal cord segments and brain tissue. The procedures used for Western blotting were as described previously [At 3 days, 1 week and 8 weeks postimmunization .Data were analyzed using SPSS version 13.0. Values are presented as the mean ± standard deviation (SD). Comparisons between two groups were performed using unpaired Student's Functional scoring demonstrated that the onset of disease symptoms  was observed in the vehicle-treated rats at 3 days pi, and the symptoms peaked at 1 week pi .In the vehicle-treated NMO group, significant neuronal loss Figures  was alsoThe expression of activated caspase-3, responsible for the execution of apoptosis, was upregulated in neurons of the spinal cord anterior horn and retina in the vehicle-treated NMO rats, compared with the control group Figures . This efμg of NMO-IgG and 0.5 μL of human complement was used to induce a rodent NMO model in Sprague-Dawley rats [μg of AQP4-IgG and 3 μL of 20% human complement in 8 μL of PBS intracerebrally into the brain of mice [Asavapanumas et al. employed the recombinant monoclonal NMO antibody rAb-53 (referred to as NMO-IgG), which was derived from a clonally expanded plasma blast population from cerebrospinal fluid of an NMO patient, and human complement to produce a mouse model of NMO . Their r of mice , and Zha of mice . Moreove of mice . Ratelad of mice . Further of mice  attempte of mice , 4–8. FuMost patients are seropositive for NMO-IgG, which specifically interacts with AQP4 , 23. AQPPrevious studies have clearly demonstrated that NMO-IgG is pathogenic in inducing or exacerbating the disease . It is b+ (leukocyte common antigen) leukocytes in the CNS of vehicle-treated NMO rats. Since extravasated inflammatory cells can activate a series of noxious factors and destroy the microenvironment in the CNS, they ultimately cause secondary injury. Because the oligodendrocytes are highly vulnerable to the inflammatory milieu, inflammation may lead to demyelination, which is probably secondary to oligodendrocyte apoptosis due to loss of trophic support from astrocytes. Extensive demyelination leads to the denudement of axons and destruction of normal axons. Ultimately, extensive axonal injury causes clinical symptoms such as sensory/motor impairment and paralysis [Following the acute lesion, the secondary lesions lead to further pathological changes. It has been shown that the interaction between NMO-IgG and cells of the neurovascular unit (astrocytes and the brain endothelium) alters BBB permeability and granulocyte recruitment , 28. Thearalysis , 29. In αvβ3 is critical for leukocyte-endothelium interactions and transmigration of leukocytes across the endothelium [αvβ3 is also an important receptor that regulates macrophage differentiation and responses to external signals. Furthermore, αvβ3 activation can maintain chronic inflammatory processes in pathological conditions [αvβ3 may also directly inhibit macrophage-related inflammation.The integrin othelium . C16, asothelium , 31, 32.nditions . Thus, tα4 integrin-mediated adhesion of immune cells to the BBB [ανβ3 and α5β1. Moreover, C16 also works as an ανβ3 agonist and promotes angiogenesis [A case series study of five patients by Kleiter et al. has shown that Natalizumab is not beneficial for NMO, although it is an effective treatment for relapsing-remitting multiple sclerosis (MS) . This re the BBB . In thisogenesis , 34, 35.Ang1, as an antileakage factor, modulates the BBB during acute injury and inflammation, can reduce vascular permeability, and promotes vessel survival , 36. In κB and the adhesion molecules intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin, which are required for the migration of inflammatory cells [In addition, by securing paracellular junctions, Ang1 also effectively limits the progression of the inflammatory response by inhibiting the expression of the inflammatory cytokine nuclear factor-ry cells . Our datStudies in an MS model have shown that C16 effectively suppresses inflammatory cell infiltration , 20, 21 Although the pathogenesis of MS and NMO as well as the expression patterns of APQ4 and GFAP in these two diseases is quite different , 37, the


Background. Water channel proteins, also called aquaporins, are integral membrane proteins from major intrinsic protein (MIP) family and involved in several pathways including not only water transport but also cell signaling, reproduction, and photosynthesis. The full cDNA and protein sequences of aquaporin in Chorispora bungeana Fisch. & C.A. Mey (C. bungeana) are still unknown. Results. In this study, PCR and rapid amplification of cDNA ends approaches were used to clone the full cDNA of LRB7 (GenBank accession number: EU636988) of C. bungeana. Sequence analysis indicated that it was 1235 bp, which had two introns and encoded a protein of 250 amino acids. Structure analysis revealed that the protein had two conserved NPA motifs, one of which is MIP signature sequence (SGxHxNPAVT), six membrane helix regions, and additional membrane-embedded domains. Phylogenetic analysis suggested that the protein was from TIP2 subgroup. Surprisingly, semiquantitative RT-PCR experiment and western blot analysis showed that LRB7 and TIP2 were only detectable in roots, unlike Arabidopsis and Raphanus. Connecting with our previous studies, LRB7 was supported to associate with chilling-tolerance in C. bungeana. Conclusion. This is the first time to characterize the full sequences of LRB7 gene and water channel protein in C. bungeana. Our findings contribute to understanding the water transports in plants under low temperatures.  Arabidopsis thaliana called γ-tonoplast intrinsic protein  [Water channel proteins (aquaporins) are integral membrane proteins from a large family of major intrinsic protein (MIP) . The disP, TIP1) , hundredP, TIP1) –7. In plP, TIP1) , tonoplaP, TIP1) , nodulinP, TIP1) , small bP, TIP1) , and X iP, TIP1) . The strP, TIP1) , 14. AquP, TIP1) , 16.Chorispora bungeana Fisch. and C.A. Mey.  is an alpine perennial herb belonging to the Brassicaceae family. It grows in the freezing and thawing tundra in the borders of glaciers, subalpine meadow, and gravel. Some studies of this model plant have investigated mainly cold-tolerance [ Arabidopsis and Raphanus, aquaporins have been characterized with high expression in young shoot, rosette, and flower tissues and other tissues [ LRB7 cDNA sequence encoding an aquaporin in the root of C. bungeana. In addition, the encoding aquaporin was characterized as δ-TIP, also called TIP2 [ C. bungeana.olerance –19; howe tissues , 21. In led TIP2 . We furt C. bungeana were grown in the source area of Urumqi River in Tianshan Mountains, Xinjiang, China , and transplanted to the experimental field of botanical garden at Lanzhou University. After one year, leaves, stems, and young roots were collected from three individual plants during their growth period (from June to September) and stored in liquid nitrogen.The wild species ofµL of chloroform : isoamyl alcohol (24 : 1), mixed by inversion, and centrifuged at 13000 RPM for 1 min. The supernatant (contains the DNA) was next moved to a clean tube and added 50 µL of 7.5 M ammonium acetate followed by 500 µL of ice-cold absolute ethanol. To precipitate the DNA, invert the tubes slowly several times and incubate the tubes at −20°C for 1 hr. After precipitation, samples were centrifuged at 7000 RPM for 1-2 min to concentrate the DNA. The pellet was then washed by 20 mL of wash buffer (70% ethanol and 10 mM ammonium acetate): following an initial gentle swirling of the tube to break up the pellet, samples were left at room temperature for 20 min. The DNAs were then centrifuged at 13000 RPM in for 5 min, air-dry the pellets (~30 min), were resuspended in 1 mL TE , and were incubated in RNase A solution  for 30 min at 37°C. The solution was made 2.5 M in ammonium acetate and 2.5 volumes of cold ethanol were added to precipitate DNA. The DNA pellet was then air-dried again on a desktop overnight and resuspended in 1 mL TE. The quality of DNA was evaluated and controlled by NanoDrop 2000 (Thermo Scientific).Genomic DNA was isolated according to the method described by Doyle and Dickson . Young rµg) was added in as carriers, followed by 1 mL of 100% isopropanol. Next, the samples were incubated at room temperature for 10 min and centrifuged at 12,000 ×g for 10 min at 4°C. The RNA pellet was washed by using 2 mL of 75% ethanol, vortex briefly, and centrifugation at 7500 ×g for 5 min at 4°C. The RNA pellet was air-dried, suspended in RNase-free water, water-bathed at 57°C for 10 min, and stored at −80°C. RNA quality was evaluated by Agilent 2100 Bioanalyzer. Young roots, stems, and leaves were used for RNA isolation separately.Total RNA was isolated using Trizol® reagent (Invitrogen) according to the manufacture's instruction. Briefly, a total of 200 mg plant samples  were incubated in 2 mL Trizol reagent at room temperature for 5 min. After transferring the mixture into clean tubes and adding 0.4 mL of chloroform, the samples were homogenized by shaking the tubes for 15 sec, incubated for 3 min, and centrifuged at 12,000 ×g for 15 min at 4°C. The aqueous phase was transferred into new tubes. RNase-free glycogen (5 µg) was used for cDNA synthesis with One Step RNA PCR Kit (Takara Bio) and AMV Reverse Transcriptase XL (Takara Bio), according to the protocols. The reaction mixture contained 1.25 U of Ex Taq DNA polymerase (Takara Bio), 2.5 μL of 10x buffer (100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2), 2.5 mM of each dNTP, and 0.4 μM of each primer. PCR amplifications were performed at 94°C for 5 min followed by 30 cycles of amplification  and a final elongation at 72°C for 5 min.To confirm the full sequence of LRB7 gene, PCR amplification was performed using primers shown in ™ RACE Ready cDNA Kit (Invitrogen) strictly following the manufacturer's instructions. The amplified fragments were then transferred into the pMD18-T vector (Takara Bio), nucleotide sequences were determined with CEQ 2000XLDNA Analyzer (Beckman Coulter), and the data were analyzed and visualized by using Sequencer (Genes Codes Corporation). Three independent clones of each amplification product were sequenced to avoid errors caused by PCR. The primers used for this experiment can be found in The reactions of Rapid Amplification of cDNA Ends (RACE) were performed using GeneRacer C. bungeana, semiquantitative RT-PCR experiments were conducted using the RNA samples isolated from roots, stems, and leaves, respectively. Briefly, total RNA (2 µg) was used for cDNA synthesis, as described. Primers P7/P8 and P9/P10 ( actin gene (GenBank accession number AY825362) and LRB7 gene determined in this study. PCR amplifications were performed following the process: 25 cycles (94°C for 5 min), 30 cycles , and a final elongation at 72°C for 10 min. The PCR products were separated and purified using 1% agarose gels and stained with ethidium bromide and analyzed by Gene Tools software (Gene Company Ltd.).To analyze the tissue specificity of LRB7 at transcriptional level ind P9/P10  were des E. coli BL21 (DE3) pLysS. The transformed E. coli BL21 (DE3) pLysS was cultured in lysogeny broth (LB) medium for overnight. Then, 0.4 mL of liquid bacteria was cultured in 20 mL LB medium until the optical density (OD) reached 0.6 at 600 nm. By adding 24 µL of 20% isopropyl-β-D-thiogalactoside (20% IPTG), fusion proteins (GST-LRB7) were induced for 6 hr from 1 liter of liquid transformed bacteria, obtained by boiling for 12 min, separated by 12% SDS-PAGE gels, and purified by using GSTrap 4B  following the protocols. Next, purified GST-LRB7 proteins (100 µg) and Complete Freund's Adjuvant  were used to simultaneously immunize two Japanese adult male rabbits every two weeks. Blood serum of the rabbits was harvested and polyclonal antibody was purified by Protein A Sepharose (CL-4B) following standard protocol (Invitrogen) and purified by using affiliation column with bound GST to remove the anti-GST antibody. The purified anti-LRB7 was stored at −80°C in a buffer containing 1% BSA, 50% glycerol, and 0.02% sodium azide for further use.pGEX-4T-3-LRB7 plasmid was built by combining LRB7 coding regions and PGEX-4T-3 plasmid and introduced intoµg) were separated by 12% SDS-PAGE gels and then blotted onto PVDF membrane (EMD Millipore) following the standard protocol. Primary antibody (anti-LRB7) was used to detect TIP, followed by secondary antibody . The signals were detected and visualized by using ECL Western Blotting system (Amersham Bioscience).Plant samples (200 mg) of roots, stems, and leaves were lysed by using Radio Immunoprecipitation Assay (RIPA lysis buffer) following protocol (Beyotime Institute of Biotechnology) and homogenized in the ice-cold RIPA lysis buffer ). After the precipitate was discarded, crude membrane fraction was collected by centrifugation at 12,000 RPM for 20 min at 4°C. Protein samples (30 http://www.mbio.ncsu.edu/bioedit/bioedit.html). The phylogenetic tree was constructed based on the genetic distance of protein sequence by ClustalX2 [http://www.megasoftware.net/). The transmembrane helices of TIP2 were predicted using an online software TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/). The membrane-spanning regions and its orientation were predicted using TMpred (http://www.ch.embnet.org/software/TMPRED_form.html).The deduced protein of LRB7 gene was aligned with TIPs from other species using BioEdit 7.2.5 , Nicotiana tabacum (GenBank accession number S45406), Lycopersicon esculentum (GenBank accession number LEU95008), and Daucus carota (GenBank accession number AB000506) were used to design PCR primers P1, P2, and P4  is consistent with the LRB7 cDNA sequence characterized by the 5′ and 3′ RACE experiments.To characterize the full cDNA sequence of LRB7 in, and P4 . Using P3 primer , obtaine3 primer  for 5′ a5 and P6 , which w C. bungeana was identified to contain an open reading frame (ORF) of 753 bp, 5′-untranslated region , 3′-untranslated region , and 24 bp poly(A) tail  tail . By comp(A) tail . C. bungeana. LRB7 was predicted to encode a protein of 250 amino acids, shown in  Arabidopsis thaliana, Raphanus sativus, Pyrus communis, Triticum aestivum, Nicotiana glauca, Populus trichocarpa, Tamarix albiflonum, Oryza sativa, and Saccharum officinarum showed that the deduced protein had two conserved characteristic motifs of asparagines-praline-alanine (NPA)  together with the N- and C-terminals towards the cytoplasmic side were predicted to strongly support that the protein product of LRB7 was from MIP (Then we characterized the protein product of LRB7 inne (NPA) . The firfrom MIP  family. C. bungeana was from TIP subgroup . We showed the phylogenetic tree analysis of LRB7 encoded protein with TIP sequences from other species  from R. sativus, and PIP1 (EU585602) from S. officinarum, respectively.We next used ClustalX2 to analyze the conservation between the protein encoded by LRB7 and other MIP sequences. In total, we obtained 119 MIP sequences of 13 other species from NCBI database . Phyloge species . These rwas TIP2 –29. In a C. bungeana by semiquantitative RT-PCR. The actin gene was used as internal control and primers for actin and LRB7 can be found in  actin gene product; however, the expected fragments (600 bp) for LRB7 suggested that it was expressed only in roots of C. bungeana.We next examined the expression of LRB7 gene in different tissues of C. bungeana to measure the abundance of TIP2. To obtain the antibody (anti-LRB7) for western blot, we first confirmed the fusion protein GST-LRB7 in the cell lysates from E. coli BL21 (DE3) pLysS transformed with PGEX-4T-3-LRB7. In  E. coli BL21 (DE3) pLysS transformed by PGEX-4T-3-LRB7 were cultured at 2, 3, 4, 5, and 6 hr. Compared with CK1 and CK2, a protein band of approximately 45 kDa was found in these five lanes revealing that fusion protein GST-LRB7 was expressed successfully. The expression of GST-LRB7 was increased by induction time and reached the maximum at 4 hr. After antibody (anti-LRB7) was purified from the serum of male adult rabbits immunized by GST-LRB7 proteins, it was used for western blot analysis in leaves, stems, and roots of C. bungeana, respectively. The antiserum against protein encoded by LRB7 specially recognized 25 kD band only in roots, but not in stems or leaves  family and have been characterized in essentially all living organisms , 31. The of MIPs . As show Arabidopsis and Raphanus the genes encoding TIP2s are expressed highly in young shoot, rosette, and flower tissues [ C. bungeana. Our findings fill the gaps of water channel protein identification of C. bungeana and are important to understand the mechanism of flowing water through roots to other tissues in C. bungeana. It may help understand the potential functions of aquaporins in chilling-tolerance because C. bungeana is an ideal material for investigating the chilling adaptation mechanism of the plant that can survive under frequent temperature fluctuations and freezing temperatures but does not possess special morphological characteristics for preventing chilling damage [TIPs and other aquaporins can be detected in many cellular types of plants. For example, in tissues , 21. Alt tissues , this isg damage .2+ distribution is a response of C. bungeana in adapting to the alpine subnival environment and the accumulation of Ca2+ can play an important role in active cold-hardiness [ C. bungeana to sudden freezing [The mechanism of chilling-tolerance is tightly linked with redox balance of the cellular redox molecule. In early chilling, the redox transition of ubiquinone can not only ensure the fluency of electron transfer in mitochondria but also facilitate the regulation of the whole-cell redox states leading to adaptation of cellular regulations. Caardiness . A synerfreezing . C. bungeana by more functional experiments.In rice, a gene called RWC1 encoding 290-residue protein (a water channel protein) is experimented to involve in chilling-tolerance . It is sSupplementary Figure S1. Phylogenetic analysis of the protein encoded by LRB7 in Chorispora bungeana and MIPs from other species. It revealed the protein encoded by LRB7 was from TIP2 subfamily because it was clustered with TIP2 proteins encoded by A. Thaliana (NP_193465.1 and NP_199556.1) and A. lyrata (XP_002870110.1 and XP_002863344). LRB7 encoded protein and TIP2 proteins encoded by other species were colored in light green.


Based on our previous work, a practical, scalable synthesis of alkaloid (1) was developed and reaction could be readily scaled up to more than 100 g. In addition, twenty-six analogues of alkaloid (1) by replacement of tryptamine moiety with different aromatic and aliphatic amines were also prepared. The bioassay results showed that most of these derivatives displayed potent algicidal activity against three harmful algae S. obliqnus, M. aeruginosa, and C. pyrenoidosa with IC50 values in the range of 1.5–5.0 μg/mL. Amongst them, compounds (10) and its hydrochloric salt (10S) were found to reveal powerful growth inhibitory activity against harmful cyanobacterial algae with IC50 values as low as 0.08 μg/mL, comparable to those of commercial algicide CuSO4 and herbicide Diuron.Currently, harmful algal blooms are being one of ever-increasing global environmental problems. Much attention has been paid to the use of natural products as the selective algaecides due to their low toxicity, high selectivity and eco-friendly properties. In the present study, the thiazole alkaloid ( To control HABs efficiently, extensive strategies including physical methods (e.g. clays and flocculants)6, chemical methods 10, and biological methods 13 have been developed to date. Despite this significant progress, unfortunately, all these strategies are shown to be either too expensive to implement, or nonspecific to harmful algae. As a result, development of novel selective and eco-friendly algicidal agents against harmful algae is highly desirable.Over the past decades, harmful algal blooms (HABs) have been becoming an increasing global environmental problem. Virtually, they do not only cause seriously public health problems, but also substantially economic lossesOscillatoria perornata17. Amongst them, anthraquinone-59 was shown to have the highest algicidal activity against musty-odor O. perornata (LC50 = 2.06 mg/L). The cyanobacterial alkaloid nostocarboline was also found by Gademann and co-workers to reveal potent algicidal activity with IC50 values of 2.1 μM against Microcystis aeruginosa, 5.8 μM against Synechococcus obliqnus, and 29.1 μM against Kirchneriella contorta, respectively19. Additionally, Mizuno et al. reported that natural stilbene analogues exhibited fully inhibitory activity against O. perorata at a concentration as low as 10 μM20.Recently, the effectiveness of natural products (NPs) as the powerful algicidal agents against marine and freshwater harmful algae has received more and more interests from academic and industrial communities due to their intrinsically low toxicity, high selectivity and eco-friendly properties. Schrader and co-workers discovered that 9,10-anthraquinones, produced or released during lignin decomposition act via photosynthetic electron transport, display selectively algicidal activity against cyanobacterium 22. Generally, in addition to killing algal cells directly by contact action, bacteria attack algae by releasing secondary metabolites to inhibit the growth of harmful algae27, namely allelopathy28. These natural secondary metabolites, termed alleochemicals, show extremely low toxicity to mammalian animal, high selectivity, readily degradable, and environment-friendly properties. To date, several types of natural secondary metabolites with algicidal activity including pigments22, diketopiperazines31, norharmalane32, and proteins34, have been isolated and identified from bacteria. The thiazole alkaloids ethyl)-2-acetylthiazole-4-carboxamide ), was originally isolated from Thermoactino-myces strain TM-64 in 197635. As the congeners of this class of alkaloids, bacillamides A–C, were recently isolated from marine Bacillus endophyticus collected during termination of a toxic algal bloom37. It was found that bacillamide A show potent antibiotic activity against dinoflagellates and raphidophytes. All these alkaloids highlight a dipeptide skeleton incorporating a central thiazole motif and a C-terminus tryptamide. As an only exception, neobacillamide A, firstly isolated from marine bacterium B. vallismortis C8938, has a unique C-terminus phenethylamide unit in its molecular scaffold. Due to their limited availability from natural source, the algicidal activity of this class of secondary metabolites has not been explored.On the other hand, the use of algicidal bacteria turns out to be a versatile alternative approach to control the bloom of harmful algae in marine and freshwater environments40. In particular, M. aeruginosa and S. obliqnus were shown to be the most frequently reported HABs species in the freshwater bodies. Therefore, controlling cyanobacteria blooms is becoming one of the most important aspects in the comprehensive management of water source. As parts of our ongoing research on natural alleochemicals, we herein report the algicidal potential of bacillamide alkaloids against three typically harmful cyanobacterial algae, S. obliqnus, M. aeruginosa, and Chlorella pyrenoidosa. In addition, a mini synthetic library consisting of twenty-six analogues of bacillamide alkaloids were also prepared and evaluated for their algicidal activity. Meanwhile, considering the low solubility of these compounds in water, their water-soluble hydrochloric salts were also subject to evaluation for selective algicidal activity against harmful freshwater algae.Nowadays, cyanobacterial blooms are becoming a worldwide aquatic environmental problem, which is tightly coupled with increasing water eutrophication and climate change1H and 13C nuclear magnetic resonanance (NMR) spectra are recorded in CDCl3 or d6-DMSO solution on a BRUKER (400 MHz) instrument  using tetramethylsilane as an internal standard, and chemical shift values (δ) are reported in parts per million. High resolution mass spectrometer (HRMS) data are obtained on a high resolution ESI-FTICR mass spectrometry (Ionspec 7.0 T). All reagents are commercially available and purified according to the standard methods prior to use.Melting point are determined using an X-4 digital melting point apparatus and thermometer uncorrected. 1) was synthesized using L-alanine (2) as the starting material according to a slightly modified procedure of our previous work  of alkaloid (1) in which tryptamine moiety was taken place by substituted aromatic and aliphatic amines were also prepared.The thiazole alkaloid  was added slowly to a cooled (0 °C) and stirred solution of L-alanine (2)  in 2 M aqueous NaOH solution (600 mL). The reaction mixture was warmed to room temperature gradually over 2 h and stirred at room temperature until the starting materials was consumed completely, and at that time the pH value of the solution was adjusted to 2 with concentrated hydrochloric acid. The mixture was extracted with EtOAc (600 mL × 3) and the combined organic layers were dried over anhydrous sodium sulfate. After removal of solvent in vacuum, the resulted residue was redissolved in 1,4-dioxane (1200 mL). To this solution was added NH4HCO3 , pyridine (55 mL) and (Boc)2O  in turn. The reaction mixture was then stirred at room temperature for another 4 h. The solvent was removed under reduced pressure and water (1000 mL) was then added. The mass was re-extracted with EtOAc (600 mL × 3) and the combined organic layers were dried over anhydrous Na2SO4 followed by removal of solvent in vacuum. The obtained residue was triturated with acetone (100 mL) to give the pure amide (3) as a white solid (211 g) in 92% yield41, m. p. 120–122 °C, 1H NMR  δ: 7.77 , 7.46 , 5.19 , 4.53 , 1.52 , 1.48 .In a 2.5 L three-neck flask equipped with mechanical agitator, (Boc)2S5 , Ethyl bromopyruvate , and anhydrous Na2SO4 (284 g) was added slowly to a cooled (0 °C) and stirred solution of (S)-tert-butyl (1-amino -1-oxopropan-2-yl)-carbamate (3)  in acetone (500 mL) under Ar2. The reaction mixture was stirred at room temperature overnight and the solid substances were then filtered off from the solution. After removal of the solvent under reduced pressure, the obtained residue was purified by flash column chromatography  on silica gel to give compound (4) as a yellow oil liquid in 85% yield (127 g).45 1H NMR  δ: 8.10 , 5.31 , 5.13 , 4.43 , 1.64 , 1.46 , 1.41 .In a 2 L three-neck flask equipped with mechanical agitator, P4)  was dissolved in a solution of THF/MeOH/H2O , and then LiOH  was added in one portion. The reaction mixture was stirred at room temperature for another 2 h and the solid substances were filtered off from the solution. The filtrate was concentrated under reduced pressure and neutralized to pH 7 with diluted hydrochloric acid. The mixture was extracted with ethyl acetate (100 mL × 3) and the combined organic phase was dried over anhydrous Na2SO4 followed by removal of the solvent in vacuum. The obtained residue was purified by flash column chromatography  on silica gel to give compound (5) as a white solid (8.8 g) in 87% yield, m. p. 101–103 °C41. 1H NMR  δ: 8.05 , 4.45 , 4.36 , 1.95 , 1.50 , 1.35 .In a 250 mL three-necked flask equipped with mechanical agitator, the ester (5)  in dry dichloromethane (25 mL) was added iso-butyl chloroformate  and N-methylmorpholine (NMM) . The resulted mixture was vigorously stirred at room temperature for 1 h. Tryptamine  and another part of NMM  in dichloromethane (10 mL) was added to the aforementioned solution and the reaction mixture was stirred for another 2 h. Water (10 mL) was added and organic phase separated off dried over anhydrous MgSO4. The solvent was evaporated under reduced pressure to give a crude product (6), which was used directly in the next step reaction without further purification.To a solution of acid (6) (1 mmol) was dissolved in EtOAc (5 mL), then H2O (1 mL) and concentrated hydrochloric acid (1 mL) was added. The reaction mixture was stirred at room temperature for 12 h until the reaction is finished monitored by TLC. The reaction solution was neutralized to pH 8–9 with saturated K2CO3 solution. The organic phase was separated off and aqueous phase was extracted with with EtOAc (2 × 20 mL). The combined organic layer was dried over anhydrous sodium sulfate followed by removal of the solvent under reduced pressure. The obtained residue was subjected to purification by flash column chromatography  on silica gel to give alkaloid (1) as a light brown oil (252 mg) in 80% yield. [α]D20=+ 0.015 . 1H NMR  δ: 1.49 , 1.76 , 3.09 , 3.78 , 4.28 , 7.03 , 7.12 , 7.19 , 7.36 , 7.55 , 7.66 , 7.99 , 8.77 ; 13C NMR  δ: 24.7, 25.5, 39.9, 49.6, 111.4, 112.7, 118.8, 119.3, 122.0, 122.3, 122.7, 127.4, 136.6, 149.9, 161.4, 178.5. HRMS(ESI-TOF) calcd. for C16H19N4OS+ [M+H]+ 315.1274, found: 315.1281.In 25 ml sealing tube, the crude product (7–32) were prepared readily in 80–94% overall yields from the carboxylic acid (5) through using substituted aromatic or aliphatic amines as the starting materials (See the Supporting Information for the detailed characterized data).Following the similar procedure, amides  and its amide analogues (7–32), as well as their hydrochloric salts (7S–27S), was evaluated against three typical Cyanobacterial algae, S. obliqnus, M. aeruginosa, and C. Pyrenoidosa at different concentrations. After three algae species were inoculated, the variation of algae density over 7 days were recorded continuously. It was found that the best period of inoculation is 4th day in the growth cycle of three algae species. The wavelength at 650 nm was determined as ideal incident wavelength for the measurement of algae density. Three Cyanobacterial algae S. obliqnus, M. aeruginosa, and C. pyrenoidosa were offered by Freshwater algae culture collection of the institute of Hydrobiology (FACHB-collection). Each culture for three species was maintained separately in continuous, steady-state growth microplates, which were placed in a growth chamber held at 28 ± 1 °C and illuminated continuously under fluorescent lights at a photon flux density of 28 μE·m−1s−1. Absorbance values of each wells were measured by a Packard model Spectra Count microplate photometer.According to the method established by Schrader 1) and its analogues (7–32), as well as their hydrochloric salts (7S–27S), were dissolved separately in DMSO to provide stock solution concentrations. Initially, each stock solution was aseptically pipetted into the bottom of separate wells (20 μL per well) in a 96-well microplate and the final test concentrations for each sample were 0, 0.78, 1.56, 3.12, 6.25, 12.5, and 25 μg/mL. Wells of control samples contained only unialgal culture. The solution of CuSO4 and Diuron at the same concentration were chose as the reference sample. Each experiment was performed at least three times. Through measuring the area values under the individual growth curves, the inhibitory rate (%) for the individual tested concentration was calculated according to the following equation (Ii: inhibitory rate of biomass growth by toxicant concentration i (%)Ac: average area under the growth curve by control sampleAi: average area under the growth curve by test concentration iAlkaloid  and its 50 were determined for each group of plates, and standard deviation were calculated and reported as the mean ± SD.Mean values of 96-hour IC50 values of all compounds against three freshwater algae species were calculated using SPSS v19.0, and the algicidal activities of alkaloid (1) and its amide analogues (7–32), as well as the corresponding hydrochloric salts (7S–27S) are listed in Table The ICogues 7–3, as well10) as a representative sample. Herbicidal activity of compound (10) against two terrestrial plants, Brassica campestris and Echinochloa crusgalli, and two aquatic plants, Ceratophyllum demersum L and Hydrilla verticillata, was evaluated at a concentration of 500 mg/L using a previously reported method43. In addition, toxicity of compound (10) toward fish Barchydanio rerio var was also tested at the concentrations of 200 mg/L and 100 mg/L, respectively. The preliminary results were reported in Table Toxicity of this class of thiazole amides towards non-target organisms, such as higher plants and fishes, was evaluated preliminarily using compound (1) and its amide analogues were prepared from L-alanine in five linear steps. Generally, the thiazole ester (4) is the key intermediate for synthesis of bacillamide-type alkaloid. To the best of our knowledge, although there are several approaches to synthesis this compound49, none of them are practical for scale-up preparation. In our previous work41, we developed a procedure through utilizing N-Boc-alanine as the starting materials. Firstly, it was converted into N-Boc-amino acid amide with excellent yield using the (Boc)2O-pyridine system. Subsequent thionation using Lawesson’s reagent afforded the desired N-Boc-amino acid thioamide, which was then reacted with ethyl 3-bromopyruvate to afford an intermediate followed by treatment with trifluoroacetic anhydride and 2,6-lutidine to give the thiazole ester (4). Unfortunately, careful purification by column chromatography is necessary for each step in the synthetic sequence. In present study, this synthetic route was modified for the convenience of scale-up synthesis. Therefore, a practical one-pot synthetic methodology for the key intermediate thiazole ester (4) was developed. Without Lawesson’s reagent, N-Boc-L-amino acid amide (3) directly reacted with ethyl 3-bromopyruvate in the presence of P2S5 to provide compound (4) in 85% yield, and isolation and purification of the related intermediate is not necessary. On the basis, reaction could be readily scaled up to more than 100 g.As illustrated in Fig. 4) was then successfully hydrolyzed to the carboxylic acid (5) in 87% yield using LiOH in a mixture solvents of THF/MeOH/H2O. Finally, treatment of acid (5) with iso-butyl chloroformate and subsequent tryptamine in the presence of NMM followed by the removal of the N-Boc protected group provided smoothly alkaloid (1) in an overall yield of 80%. In a similar manner, a series of the thiazole amide analogues (7–32) were prepared in 80–94% yields by coupling of the carboxylic acid (5) with substituted aromatic and aliphatic amines.The ester 50, most of synthetic analogues exhibit obviously higher algicidal activity with IC50 values of less than 5.0 μg/mL. For free amine derivatives, compounds (7), (10), (12), (15), (19), (21), and (22) show excellent algicidal activity against S. obliqnus with IC50 values of less than 1.0 μg/mL. In particular, compounds (10) and (12) display highest algicidal activity against S.obliqnus with an IC50 value as low as 0.08 μg/mL, respectively, while natural alkaloid (1) only show algicidal activity with an IC50 value of 14.33 μg/mL under the same conditions. Notably, related to commercial algicide CuSO4 and herbicide Diuron, compound (10) displays comparably inhibitory potential toward all three harmful algae with IC50 values of 0.08, 1.51, and 1.52 μg/mL, respectively. From data outlined in Table 10S), (13S), and (27S) appear to be the promising algicidal agents which show remarkable algicidal efficacy against all three algae species with IC50 value of less than 1.0 μg/mL.As shown in Table , (10), 1, (15), (29–32) , almost all these compounds display obviously higher algicidal activity than alkaloid (1) under the experimental conditions, suggesting that it is thiazole amide motif, not tryptamine, that is the active pharmacophore for their algicidal activity. Generally, structural modification using substituted anilines instead of tryptamine further increases the algicidal activity of this class of thiazole amide derivatives. The different substitution modes in the phenyl ring of aniline also influence their algicidal activity greatly. For Cyanobacterial algae S. obliqnus, the thiazole amides derived from 4-bromo and 4-OMe substituted anilines, compounds (10) and (12)  and herbicide Diuron (IC50 0.79 μg/mL). In addition, compound (10) also exhibits comparable algicidal activity to the commercial control samples against algae M. aeruginosa (IC50 1.51 μg/mL). While for algae C. pyrenoidosa, a 4-chloro or 4-bromo substituent group , 1.52 μg/mL for compound (10)). Generally, the thiazole amides in hydrochloric salt form show higher algicidal activity than the corresponding free amines against Cyanobacterial algae M. aeruginosa, and C. pyrenoidosa. For instance, the hydrochloric salts of compounds (13) (R = 4-trifluoromethylphenyl), (20) (R = 2-bromophenyl) and (27) (R = 4-acetylphenyl) reveal the obviously enhanced algicidal activity against all three Cyanobacterial algae than the relative free amines  and its hydrochloric acid salt (10S) show a comparable algicidal activity relative to commercial algicide CuSO4 and herbicide Diuron.In order to deduce the structure-activity relationship (SAR), several alkyl-substituted thiazole amide analogues  and its hydrochloric acid salt (10S) were subject to further survey for the optimal inhibition time and concentration  and its hydrochloric salt (10S) can completely inhibit the growth of M. aeruginosa in 4–5 days at a concentration of 3.13 μg/mL or above. Therefore, the lowest complete inhibition concentration for thiazole amide (10) and its salts (10S) is determined to be 3.13 μg/mL against M. aeruginosa. The variation of growth inhibitory ratios of compounds (10) and (10S) against M. aeruginosa at the lowest complete inhibition concentration is illustrated in Fig. 10), the growth inhibitory ratios after 96 and 120 hours increase gradually to 79% and 92%, respectively. Under the same experimental conditions, growth inhibitory ratios for the salt (10S) are 82% and 88%, respectively.Currently, ion Figs  and 4. F10) as a representative compound (Table 10) is still none-toxic towards both terrestrial plants, B. campestris and E. crusgalli, and aquatic plants, C. demersum L and H. verticillata. Additionally, compound (10) is also shown to be safe towards fish Barchydanio rerio var at a concentration of 100 and 200 mg/L, respectively.Finally, toxicity tests of this class of thiazole amide derivatives on non-target organisms were investigated preliminarily using free thiazole amide (nd Table . The res1). On the basis of the strategy, a mini compound library comprised 47 thiazole amide analogues derived from aryl- and alkylamines was prepared and investigated their algicidal activity against three harmful cyanobacterial algae, S. obliqnus, M. aeruginosa, and C. pyrenoidosa. The biological evaluation results showed that some amide analogues, such as compounds (10), (10S), (13S), and (27S), exhibit comparable algicidal activity relative to those of commercial algicide CuSO4 and herbicides Diuron. The preliminary results on toxic tests to non-target organisms also demonstrated that these amides are safety to terrestrial, aquatic plants, and fish. All the above-mentioned results suggest this class of thiazole amide analogues to be a novel class of algaecides against harmful Cyanobacterial algae. The studies on detailed algicidal mechanism of these thiazole amides, as well as extensive toxicity tests toward non-target organisms, are being pursued in our laboratory.In summary, we have achieved a practical and scalable synthesis of bacillamide alkaloid (Supplementary Information


Scientific Reports6: Article number: 30750; 10.1038/srep30750 published online: 08012016; updated: 06262017.This Article contained errors.Figure 1f incorrectly showed curves for the values N = 233, 279, 335, 402, 482, and 579.The correct The Results section contained a typographical error under the subheading “The role of network size”.μ ⪆ 0.2”.“Most of the algorithms can well uncover the communities when now reads:μ ⪅ 0.2”.“Most of the algorithms can well uncover the communities when The Supplementary Information file originally published with this Article contained errors in the f panels of Supplementary Figures 1, 2, 3, 4, and 5, where these panels incorrectly showed curves for the values N = 233, 279, 335, 402, 482, and 579.These errors have now been corrected in the PDF and HTML versions of this Article.