These newly synthesized compounds were characterized by 1H-NMR, 13C-NMR, and FT-IR spectroscopy, and gel permission chromatography (GPC). Three novel oligo-pyrazole thin films were prepared using oligo-pyrazole 4 with these respective values of thickness: 20, 21, and 24 μm. The optical properties of the films, including the absorbance, transmittance, and optical band gap, were determined using UV-vis spectroscopy. The Eg values of the films were found to be 1.426, 1.537, and 1.648 eV for the 20, 21, and 24 μm thick organic films, respectively. Atomic force microscopy (AFM) was used to examine the surface morphology and properties of the organic films. In the AFM images, a few black regions were observed and several yellow regions appeared over a large area, and the surface of the oligo-pyrazole films had an extremely low roughness value. The as-synthesized oligo-pyrazole has great potential in optoelectronic applications according to the optical properties of the as-prepared films.Pyrazole-3,4-dicarboxylic acid The online version of this article (10.1007/s00396-018-4342-7) contains supplementary material, which is available to authorized users. Conjugated polymers are important target molecules for synthetic chemists due to their use in solar cells, organic photovoltaics, organic light emitting diodes, and thin film transistors, as well as their industrial applications –3. The sPoly-pyrazoles are an important class of heterocyclic polymers, which have become more popular than before due to their wide range of properties such as film formation and high thermal stability , 10. PolOne of the most common applications of polymer materials is in thin film devices. Thin films that are made of these polymers have several advantages when used as optical coatings, coatings in electronics, and decorative protective coatings, thanks to the fundamental characteristics they have. For instance, optical, mechanical, and electrical properties are significantly increased when polymer materials are used as a thin film , 18. In This study aimed to synthesize different oligo-pyrazole-based thin films that are newly developed and to investigate their optical properties such as the optical band gap and transmittance. Furthermore, the study determined the surface morphology of the new thin film (21 μm) that had different thickness levels using atomic force microscopy (AFM).1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on a Bruker DRX-400 high-performance digital FT-NMR spectrometer. NMR spectra were obtained in solutions of deuterated chloroform. Molecular weight and PDI of the synthesized oligo-pyrazole were determined by gel permeation chromatography (GPC) using Agilent 1100 Series, equipped with refractive index detector. Optical measurements of thin films at different thicknesses were carried out with a Shimadzu model UV-1800 spectrophotometer in the wavelength range of 1100–190 nm at room temperature. An Ambios Q-Scope AFM device was used to study the surface structures of the films.All reagents and solvents were purchased from Merck, Sigma and Aldrich companies. These materials were used without purification. Infrared spectra were recorded on a Shimadzu IR-470 spectrophotometer. 1, procedure existed in literature was followed  337.3562; observed 337.3565.Compound −1) 3062 (aromatic C-H), 2928 , 1720, 1700 , 1H NMR  δ (ppm) 8.0 , 7.8 , 7.6 , 7.4 , 7.2 , 2.6 , 2.1 . 13C NMR  δ (ppm) 171.0, 167.5 , 142.3 (C3), 141.8 (C5), 135.6, 133.6, 133.0, 132.4, 130.5, 130.1, 128.4, 128.1, 127.7, 126.3, 114.1 (C4), 30.8(Ar-CH3), 22.8 (Ar-CH3). (+)ESI-HRMS m/z calculated for [C19H14Cl2N2O2 + H+] 374.2438, observed 374.2439.Compound 2  was added to the reaction flask that included excessive thionyl chloride and refluxed at 80 °C. After 6 h, the reaction mixture, which dissolved over time, was cooled down to room temperature. Excessive thionyl chloride was evaporated. The remaining oily product was purified in dry ether and then was crystallized from toluene, to yield 75%. Color milk white. FT-IR  was dissolved in anhydrous tetrahydrofuran (10 ml). The p-phenylene-diamine (0.028 g 0.25 mmol) was added to the reaction pot and the mixture was refluxed for 24 h under an atmosphere of nitrogen. Then, it was cooled to room temperature. The solvent was evaporated. The precipitated product was washed with diethyl ether. Then, it was filtered and dried. Yield 80%. Color brown. FT-IR  3385 , 2967 (aromatic -CH), 1731 , 1652 , 1595–1579 (C=N), 1536–1449 , 1313 (C-N). 1H NMR  δ (ppm) 8.7, 8.4 , 7.9–6.3 , 4.4 , 2.5, 2.2, 2.1, 2.0, 1.7 . 13C NMR  δ (ppm) 172.5, 165.8, 162.3, 159.9 (-C=O), 149.8 (C3), 142.1 (C5), 140.6, 139.6, 139.3, 139.2, 134.5, 130.4, 130.2, 130.1, 129.4, 127.2, 123.1, 121.7, 117.5, 114.1, 111.3, 110.3, 108.3 (C4’), 30.9, 30.3, 22.4, 22.7, 21.5 (Ar-CH3). Mn = 1333 g/mol, Mw = 2347 g/mol, .Pyrazole-3,4-dicarbonyl dichloride 4 (0.05 g) was added to 1 mL of DMF and the resulting solution was stirred at room temperature for 1 h. Any insoluble oligo-pyrazole was removed by filtration. The surface of a glass substrate was cleaned using piranha solution (sulfuric acid and hydrogen peroxide) and then rinsed with water. The solution of oligo-pyrazole was added dropwise on the glass substrate and left to dry to form a thin film of oligo-pyrazole 4. To obtain films with different thickness levels, the process was repeated several times for each film. Using this method, three films with thickness values of 20, 21, and 24 μm were obtained. The thickness values of the films were measured using a micrometer (sensitivity = 0.001 mm), as shown in Fig. Oligo-pyrazole 1 was synthesized according to a literature procedure [2 was obtained from the basic hydrolysis of 1 , 3064 cm−1 (Ar-C-H), 1721 and 1710 cm−1 .Poly-pyrazoles are the most extensively studied subject in polymer chemistry and organic chemistry because of their reliability, accessibility, and chemo-selectivity. Pyrazole-3-carboxylic acid rocedure  and pyra3, was prepared upon heating 2 with excess thionyl chloride. All the new compounds (1–3) were confirmed using spectroscopic methods, which were consistent with the previous studies [4 was synthesized upon the reaction of 3 and p-phenylene-diamine in refluxing THF for 1 day under an atmosphere of argon gas. The average molecular weight (Mn), average molecular weight (Mw), and polydispersity index (PDI) of the as-synthesized oligo-pyrazole 4 were determined by GPC using poly(methyl methacrylate). The Mn, Mw, and PDI of the as-synthesized oligo-pyrazole were observed to be 1333, 2347, and 1.872 g mol−1, respectively. As shown Scheme 4 was determined be a trimeric structure using gel permeation chromatography. The as-synthesized oligo-pyrazole 4 has three pyrazole structural units and ten phenyl groups. In addition, the as-obtained oligo-pyrazole was asymmetric due to the structure of the monomer. The better conjugation was used as a warranty since there were many aromatic structures. This also explains the low optical band gap value.The monomeric starting material, pyrazole-3,4-dicarbonyl dichloride  studies . The preompounds –3 were compounds –3 were c1H NMR, and 13C NMR spectra. The FT-IR bands at 3385 cm−1 correspond to the -NH amide groups. The bands corresponding to the amide (C=O) groups appear in the region of 1652–1731 cm−1. The band observed at 3066 cm−1 was attributed to the aromatic C-H stretching vibrations. The strong bands at 1595 and 1579 cm−1 correspond to the C-C stretching of the phenyl rings. In the case of the as-synthesized oligo-pyrazole 4, the correct structure was established by 1H NMR and 13C NMR spectroscopy in which the characteristic peak for the C=NH proton in was observed at δ = 9.94 ppm and the NH protons were observed as singlet peaks at δ = 6.0 and 5.1 ppm, and as multiplets in the region of δ = 6.9–8.0 due to the aromatic protons in the as-synthesized oligo-pyrazole 4.The structure of the as-obtained oligo-pyrazole was also confirmed by the FT-IR, The absorbance curves recorded for the oligo-pyrazole coated glass samples and uncoated blank glass were recorded and shown in Fig. The increase in absorbance could be greater if the oligo-pyrazole film was thick. The highest absorbance value was observed for the 24 μm film, while the lowest absorbance value was observed for the 20 μm film. The transmittance graph obtained for the 20, 21, and 24 μm films is shown in Fig. Eg value. The photon energy vs (α.hν)2 plot was used to obtain the direct band gap of oligo-pyrazole using the Tauc equation 2 plot was used to determine the indirect Eg values for the thin films of oligo-pyrazole  vs (αhν)1/3 was plotted for the oligo-pyrazole films with thickness values of 20, 21, and 24 μm  of the surface morphologies of the thin films deposited on the glass substrate were recorded at a scanning speed of 0.7 Hz using the AFM device in non-contact mode . The surThe height distribution graph was obtained using the AFM tool Fig. . The heiThis study synthesized a novel conjugated oligomer that was comprised of pyrazole skeleton. Thin films made from this oligomer with different thickness  were prepared and their optical properties were investigated. The band gap values of the oligo-pyrazole films were very low  and were suitable for optical applications. Furthermore, the band gap value was observed to decrease after increasing the thickness of the thin film, which showed that their optical applications could be controlled by adjusting the oligo-pyrazole film thickness in order to adjust the desired band gap. AFM was used to examine the surface morphology and properties of the organic films, and the two-dimensional and three-dimensional AFM images were used to see the gaps on the surface of the samples. The surfaces of the oligo-pyrazole films have extremely low roughness values. The as-prepared films exhibit good surface morphologies that are suitable for optoelectronic applications.ESM 1(PDF 711 kb)


Salidroside or p-tyrosol production by the endophytic fungus Rac56  was confirmed by UPLC/Q-TOF-MS and 1H-NMR. The fermentation conditions were optimized by orthogonal design using data processing system software. The broth fermentation results showed that salidroside and p-tyrosol extraction yields from Rac56 were stable and reached 1.729 ± 0.06 mg and 1.990 ± 0.05 mg per mL of culture medium, respectively. The optimal conditions for salidroside and p-tyrosol production in fermentation culture of Rac56 were determined to be 25 °C, pH values of 7 and 5, Czapek-Dox culture medium volumes of 150 mL and 50 mL in 250 mL flasks, rotation speeds of 100× g and 200× g, and fermentation durations of 7 and 15 days, respectively. Under these optimal conditions, stable yields of 2.339 ± 0.1093 mg and 2.002 ± 0.0009 mg per mL of culture medium of salidroside and p-tyrosol, respectively, were obtained, indicating that the P. fortinii Rac56 strain is a promising source of these compounds.2-(4-Hydroxyphenyl)ehyl-β- It was renulata . With inxploited . Howevermponents . In recemponents . As a reTaxomycesandreanae was isolated from its host plant Taxusbrevifolia in 1993 − and m/z 345.1194 [M − H + HCOOH]－of the chemical component produced by Rac56 (m/z 299.1128 [M − H]− and m/z 345.1185 [M − H + HCOOH]− of standard SAL (SAL and TYR produced by the Rac56 strain were further identified by UPLC/Q-TOF-MS. Results showed that the retention times (Rt) of the two metabolites produced by Rac56 were 2.15 min and 2.51 min a, corresby Rac56 c was condard SAL d.1H-NMR spectroscopic analysis. The aromatic region  and the sugar region  were found to contain aromatic compound and glycosidic constituent peaks identical to those found in commercially available SAL and TYR, respectively , potato dextrose medium (PD), Sabouraud medium (SD), and lactose-beef extract-peptone medium (LBP) media were 1.715 ± 0.007 mg/mL, 1.368 ± 0.021 mg/mL, 1.046 ± 0.018 mg/mL and 1.140 ± 0.023 mg/mL, respectively, and the yields of TYR in CD (1.863 ± 0.041 mg/mL) was higher than that in PD (1.551 ± 0.030 mg/mL), SD (1.419 ± 0.008 mg/mL), or LBP (1.067 ± 0.016 mg/mL). Thus, the CD medium was chosen as the best medium for subsequent optimization of the fermentation conditions.5)) orthogonal experimental design. The results of F test showed that temperature, culture volume, and fermentation time had significant effects on SAL and TYR production. The significance of each factor was determined by the corresponding F values and P values  a typical DSE fungus which is a widespread fungal endophyte of plants [P. fortinii exhibits versatile capacities of promoting elemental nitrogen (N) and phosphorus (P) absorption by host plants [P. fortinii have also been shown to aid the enhancement of disease resistance in hosts, controlling Verticillium yellows in Chinese cabbage [P. fortinii plays a key role in symbiosis with host plants.Among the four fungi we studied, the Rac56 isolate exhibited the most stable and highest yield of SAL and TYR. By ITS sequence based genotyping we found our isolate Rac56 to be similar to f plants ,18. Sevet plants  and syntt plants  by co-ex cabbage , the cap cabbage  and othe cabbage . All of Rhodiola plants, and were synthesized from phenylalanine or tyrosine in the cinnamic acid pathway  [O-β-d-glucoside of TYR and its glycosylation is dependent on UDP-glucosyltransferase catalysis [Rhodiola plants [SAL and TYR were originally derived from pathway) . TYR is atalysis ,31. As ca plants . HoweverR. crenulata samples were collected from Mila Pass , and R. sachalinensis and R. angusta were collected from Changbai Mountain  of China, and they were identified by Jinlong Cui and voucher specimens were deposited in the Institute of Applied Chemistry, Shanxi University. Isolation and purification of fungus from plant samples was done as described earlier in Sun et al. [3, 3 g; K2HPO3, 1 g; MgSO4·7H2O, 0.5 g; KCl, 0.5 g; FeSO4, 0.01 g; sucrose, 30 g; and deionized water to a total volume of 1000 mL). All flasks were incubated under constant shaking (150× g) at 25 ± 2 °C for 10 days. The fungal broth was filtered to separate the culture broth and mycelia using Whatman No. 1 filter paper. The mycelia and filtrate were extracted following the SAL and TYR extraction method as described in Chinese Pharmacopoeia [w/v) methanol by ultrasonication for 30 min, after which the three methanol extracts were combined. The culture filtrate was then evaporated under reduced pressure (8 × 103 Pa) to yield an extract, which was weighed and extracted three times with 10-fold (w/v) methanol for 10 min and extracts pooled. The extraction solution was then filtered using Whatman No. 1 filter paper and centrifuged at 5000× g for 10 min. Lastly, the supernatants were harvested to obtain the mycelia and filtrate extracts.Six pieces (5 mm diameter) of mycelial agar plugs from the edge of growing cultures of endophytic fungi were each inoculated into 500 mL Erlenmeyer flask with 200 mL of liquid CD medium , a 10 µL manual injector, a thermostatted column compartment, and a UV detector. The mobile phase consisted of a methanol/water mixture (30:70) at a flow rate of 1.0 mL/min at 30 °C and monitored using a UV detector at 276 nm. HPLC-grade methanol was purchased from Fisher Scientific . Ultrapure water was prepared using an ultrapure water system from Arium Comfort I . The SAL and TYR standards were purchased from the National Institute for the Control of Pharmaceutical and Biological Products . Detections of SAL and TYR in fungal extracts were based on their retention times. Quantifications of SAL and TYR were done using a linear regression equation fitted in a calibration graph as y = 0.88325x + 0.44806 (r = 0.99951) and y = 2.59700x + 2.18000 (r = 0.99960), respectively. The concentration ranges of SAL and TYR were 2 µg/mL to 200 µg/mL and 1.6 µg/mL to 160 µg/mL, respectively.The mycelia and filtrate extracts prepared were filtered through a 0.45 µm Millex-HV filter membrane  before determination of SAL and TYR contents by RP-HPLC analyses based on the method described in Chinese Pharmacopoeia . The ana18 column , a quaternary pump, and an autosampler. The mobile phase contained two solutions, namely (A) 0.1% (v/v) formic acid in high-purity water and (B) chromatographic grade acetonitrile. A timed gradient program of T (min)/B (%)  was used for elution, with a flow rate of 0.4 mL/min. The column temperature was maintained at 40 °C, the sample injection volume was 3 µL, and the detection wavelength was set to 277 nm. MS analyses were performed on a Waters Xevo G2 Q-TOF , equipped with an electrospray ionization source. Lock Spray™ was used to ensure mass accuracy and reproducibility. The deprotonated leucine-enkephalin (C28H37N5O7) at m/z 554.2615 was used as the lock mass in negative-ion detection mode. The source block and desolvation lamp were kept at 100 °C and 400 °C, respectively. The nebulization gas was set to 800 L/h at 400 °C. The cone gas was 20 L/h, and the source temperature was 100 °C. The capillary voltage and cone voltage were set at 2700 V and 40 V, respectively. The collision energy was 6 V, and the MS data were acquired from a mass-to-charge ratio (m/z) range of 100–1500. MS/MS experiments were performed by ramping collision energies from 35 V to 45 V. A dwelling time of 0.2 s was employed with an inter-acquisition delay of 0.01 s. Data acquisition and processing were performed using Masslynx v 4.1 software .The identity of SAL and TYR produced by the fungi was confirmed by determination of their molecular masses using ultra-performance liquid chromatography/quadrupole time–of–flight mass spectrometry (UPLC/Q-TOF-MS) analyses. Analytical UPLC separation was performed on a Waters Acquity I-Class system , equipped with a UV detector, a BEH C1H nuclear magnetic resonance (1H-NMR) spectra were determined on dried fungal extracts dissolved in methanol-d4 (CD3OD) using an AVANCE 300 MHz spectrometer . Chemical shifts were reported as δ values relative to tetramethylsilane used as an internal reference. Spectra related compounds were assigned by comparing fungal extracts and authentic SAL and TYR in CD3OD following the method in reference [To confirm the authenticity of fungal SAL and TYR, eference .http://www.ncbi.nlm.gov/BLAST). Sequences with high homology with the submitted isolate were retrieved and analyzed using Clustal W, and a neighbor-joining (NJ) phylogenetic tree was constructed from the evolutionary distance data using MEGA 4.0. The ITS rDNA sequences of the Rac12, Rac30, Rac56, and Rac63 strains that were identified by the molecular method are deposited in GenBank under accession numbers KJ542268, KJ542219, KJ542292, and KJ542358, respectively. Morphological identification of SAL and TYR-producing fungi was based on colony appearance and sporulation. Mycelial characteristics, such as color, septum, transparency, shape, and sporogenous structure and spores’ characteristics, such as shape, size, and conidial fructification, were observed through specimens mounting in 2% KOH under a light microscope . To authenticate the results of morphological identification, molecular method was adopted to verify the taxonomic status of fungi. The ITS sequence  was subjected to sequence similarity search performed on the NCBI database (5)) was adopted to optimize the culture conditions for SAL and TYR production using Data Processing System software , as shown in Single-factor design was employed to evaluate the effects of four culture media: CD, PD , SD , and LBP on SAL and TYR production by Rac56. An orthogonal design of five factors and four levels (L16(4All experiments were performed in triplicate and data are presented as mean ± standard deviation (SD). Statistical difference was analyzed with multi-way ANOVA using DPS 9.50 software .


Epimedium pseudowushanense B.L.Guo is used in traditional medicine as an aphrodisiac and to strengthen muscles and bones. Several recent reports have shown that flavonoids from Epimedium also significantly affect the treatment of breast cancer, liver cancer, and leukemia. However, few studies have examined the medicinal-ingredient yield of Epimedium, a light-demanding shade herb, under different light intensities. To investigate the effects of light intensity on medicinal-ingredient yields, Epimedium was exposed to five levels of light intensity until harvest time. Leaf dry biomass under L4 was the highest among different light treatments. L4 was also associated with the highest net photosynthetic rate. Quantification of epimedin A, epimedin B, epimedin C, and icariin showed that L3 produced the highest amount of epimedin C, and that flavonoid content responded to light levels differently. Results indicated that L3 and L4 were the optimal light levels for medicinal-ingredient yield. Epimedii Folium, commonly called Yinyanghuo, is an important perennial shade herb in traditional Chinese medicine. Epimedium belongs to the Berberidaceae family and is widely used as an aphrodisiac and to strengthen muscles and bones. Epimedium leaves contain high amounts of flavonoid glycosides, such as epimedin A, epimedin B, epimedin C, and icariin [Epimedium significantly affect the treatment of breast cancer, liver cancer, and leukemia [Epimedium has increased its market demand. However, its resource recycling rate is low and environmentally dependent. Furthermore, its natural sources are endangered, further increasing prices. Commercial culture can address resource constraints of Epimedium. E. pseudowushanense B.L.Guo is an important resource of Epimedii Folium and is the first Epimedium species introduced and cultivated in Guizhou Province, China. icariin ,2. Recenleukemia ,4,5. TheCamptotheca acuminata grown under 75% irradiance had significantly greater height, net photosynthetic rate (Pn), stomatal conductance (gs), total aboveground biomass, and chlorophyll fluorescence than plants grown under 100% (1500 ± 30 μmol·m−2·s−1), 50%, and 25% irradiance.Light is an important environmental factor influencing plant growth, development, and secondary metabolism . Under hO- and quercetin 3-O-glycosides) but does not influence the biosynthesis of monohydroxy B-ring-substituted flavonoids (pigenin 7-O- and kaempferol 3-O-glycosides) [Piper aduncum grown under 50% natural light irradiance had higher total flavonoid concentration than those grown under 100% natural irradiance. Based on these reports and the harvest time of E. pseudowushanense, the present study exposed Epimedium to five different light intensities for 60 days.The function of different flavonoids varies according to the structure and specific flavonoids are involved for instance in limiting exogenous microbial growth, helping generate new fruits or leaves, providing light protection, and enhancing antioxidant defense ,10. Qualcosides) ,12,13,14cosides)  reportedE. pseudowushanense grows in woodlands, scrub, and forest edges. In commercial culture, E. pseudowushanense seedlings are usually shade-grown by a simulated wild cultivation method. However, our understanding of the photosynthesis and flavonoid content of E. pseudowushanense under shade management is limited. This study aims to determine the optimal light conditions for commercial Epimedium production. This study also aims to investigate flavonoid accumulation in Epimedium under different light intensities to improve its propagation and cultivation. The results of the present study may contribute to the scientific culture and management of Epimedium, as well as provide a reference for other Epimedium species. We anticipate that our results will improve our understanding of the changes in photosynthetic parameters and concentrations of secondary metabolites in Epimedium under different light treatments. Our results will provide a sound theoretical foundation for the standardized cultivation of this important medicinal plant.In the natural environment, E. pseudowushanense growth: L3, L4, and L5 resulted in higher number of branches. On the contrary, L1 and L2 resulted in higher SPAD and water content. The number of branches under L3, L4, and L5 were 156.3% (p < 0.05), 187.5% (p < 0.05), and 203.1% (p < 0.05) higher, respectively, than of plants grown under L1. SPAD values under L1 and L2 were 53.1% (p < 0.05) and 54.3% (p < 0.05) higher, respectively, than in plants grown under L5. Water content responded similarly. The highest values were observed in plants under L1 and the lowest were in plants under L5 , leaf areas were higher under low light intensities (L1 and L2). L2 resulted in the highest leaf area. Light intensity had different effects on under L5 .p < 0.05), 107.6% (p < 0.05), and 173.7% (p < 0.05), respectively. The highest gs values were observed under L4. No significant differences were observed between other treatments (p > 0.05). Compared with L1, intercellular CO2 concentration (Ci) decreased by 33.1% (p < 0.05), 34.3% (p < 0.05), and 36.2% (p < 0.05) under L3, L4, and L5, respectively.Epimedium  and 95.8% (p < 0.05) higher, respectively, than under L1. Furthermore, epimedin C content increased as light intensity increased from L1 to L3, whereas epimedin C content decreased as light intensity increased from L3 to L5. Thus, L3 increased epimedin C by 483.6% (p < 0.05) compared with L1. Interestingly, icariin content was unaffected by light treatment. Icariin contents in plants under L4 and L1 were significantly higher than in plants under L5. Icariin contents under L2 and L3 were intermediate. Epimedin C was the major flavonoid in Epimedium.p < 0.05) compared with L1. Interestingly, leaf dry biomass decreased as light intensity increased from L4 to L5.Different light intensities were associated with differences in leaf dry biomass . The higEpimedium. Medicinal-ingredient yield was calculated as the product of flavonoid glycoside content and leaf dry biomass per plant. p < 0.05). Medicinal-ingredient yield gradually increased as light intensity increased from L1 to L3, whereas it decreased by 40.3% (p < 0.05) as light intensity increased from L3 to L5.Given that the total of epimedin A, epimedin B, epimedin C, and icariin content in flavonoid glycosides was more than 90% in any treatment (data not shown), these four components represented flavonoid glycosides in Epimedium is a shade-tolerant species, light intensity plays an important role in its survival, early growth, development of photosynthetic apparatus, and production of secondary metabolites [Epimedii Folium [Epimedium. Meanwhile, content of epimedin C, the major Epimedium flavonoid, was lower under low and high light intensity treatments, therefore reducing the medicinal-ingredient yield of individual plants under L1, L2, and L5 treatments  had larger leaves compared with those grown under high light intensities (L3–L5)  . These f content . In E. punder L4 . DecreasL3 to L5 , suggestsleyanum . Interessleyanum .E. pseudowushanense chloroplasts were damaged under high light intensities (L5), thus decreasing flavonoid glycoside content because chloroplasts synthesize flavonoids in the plant cell [Camptotheca acuminata seedlings [Chloroplasts are the only organelles in which photosynthesis occurs. Photoreactions are localized in the internal chloroplast membrane . Thylakoid structure and integrity are critical for effective photosynthesis . In thisant cell ,27. Simieedlings .E. pseudowushanense is most commonly utilized in the treatment of osteoporosis, liver cancer, and leukemia because of its high flavonoid content [Epimedium species [E. sagittatum ranged from 40–160 μmol·m−2·s−1. We set the L1 to L5 light regimes from 9.1–127.3 μmol·m−2·s−1. We conclude that L3 (54.6 ± 2.5 μmol·m−2·s−1) and L4 (90.9 ± 2.5 μmol·m−2·s−1) treatments achieved higher medicinal-ingredient yields after 60 days, and are therefore recommended for Epimedium cultivation. The present study provides a better understanding of the responses of growth, photosynthesis, and secondary metabolite accumulation in E. pseudowushanense seedlings exposed to various light intensities. Our results could serve as a theoretical basis for the standardized cultivation of E. pseudowushanense. content ,28,29. B species , the mosE. pseudowushanense buds germinate in February. Flowering occurs from March to April and fruiting occurs in May [E. pseudowushanense is cultivated under 10% to 30% irradiance in greenhouses. Its rhizome buds develop during winter. New leaves and stems with flowers develop in the following early spring. For this study, healthy, homogenous two-year-old E. pseudowushanense seedlings were collected from Lei Shan County  in Guizhou Province. Each stem had two compound leaves. Each compound leaf had three leaflets (−2·s−1), L2 (18.2 ± 2.5 μmol·m−2·s−1), L3 (54.6 ± 2.5 μmol·m−2·s−1), L4 (90.9 ± 2.5 μmol·m−2·s−1), and L5 (127.3 ± 2.5 μmol·m−2·s−1). Light intensities were measured by LI-6400 external quantum sensor  system. Sufficient water was provided for plants once every four days until the end of experimentation.s in May . E. pseuleaflets . SeedlinThe number of newly germinated branches was recorded after 60 days of light treatment. Leaf areas of three leaflets were measured by LI-3000C . Fresh leaf weights were measured by an electronic balance. Leaf dry weights were measured after oven drying leaves at 60 °C to constant weight. Water content was calculated as: water content = 1 − dry weight/wet weight. Chlorophyll content was measured by a chlorophyll meter SPAD-502 . Measurement light wavelengths were 650 nm and 940 nm. At the end of each treatment, all leaves of intact plants were collected to calculate leaf dry biomass.2, 500 μmol (CO2)·m−2·s−1 air concentration, 60% relative humidity, 200 μmol (CO2)·s−1 air flow, and 20 ± 0.5 °C temperature. Vapor pressure deficit (VPD) was calculated with leaf and air temperatures and relative humidity. VPD was similar among the five experimental treatments.Photosynthetic measurements were taken between 9:00 AM and 11:00 AM. Healthy, fully-developed, topmost leaflets of fronds were taken from three randomly selected plants per replicate treatment. Photosynthesis (Pn) was measured by a LI-6400 portable photosynthesis system  with a standard leaf chamber equipped with a 6400-02B LED light source. Data were recorded under 21% OE. pseudowushanense seedlings subjected to different light intensities for 60 days, a fully expanded leaflet from a randomly selected plant from each replicate per treatment was collected. Fresh leaves were cut into 4–6 slices (1 mm2) at their adaxial surfaces for transmission electron microscopy. Sections were placed immediately in 2.5% (v/v) glutaraldehyde , then de-gassed and fixed for at least 4 h. Samples were then post-fixed with 1% (v/v) osmium acid. After fixation, samples were washed three times with the same buffer for 15 min. Specimens were dehydrated by a graded ethanol series  then embedded in epoxy resin, from which ultrathin sections were prepared. The ultrathin sections were sequentially stained with uranyl acetate and lead citrate and then examined with a transmission electron microscope .To observe the chloroplast ultrastructure of mesophyll cells in −1 flow rate, 25 °C column temperature, 270 nm detection wavelength, and 20 μL injection volume. Six gradient concentrations of epimedin A (3.1–32.3 μg·mL−1), epimedin B (3.1–32.3 μg·mL−1), epimedin C (20.5–520.6 μg·mL−1), and icariin (3.1–32.3 μg·mL−1) were prepared. A Zorbax SB-C18 column  was utilized as the chromatographic column in chromatographic analysis . HPLC analysis data were processed by PerkinElmer ChemStation software .One ternate leaf was collected per plant after 30, 60, 90, and 120 days of exposure to different light intensity treatments. Fresh leaves of 10 seedlings per replicate were fixed for 3 min in a microwave, then dried to a constant weight at 60 °C. Sample pretreatment was performed according to the Chinese Pharmacopoeia Commission (2010). Samples were crushed and sifted through a No. 3 pharmacopoeia sieve. Approximately 200 mg of sample was added to 50 mL 70% ethanol then extracted by ultrasonication for 30 min. The extract was then filtered with a 0.45 μm microfiltration membrane for HPLC analysis. Eluent A contained double distilled water and eluent B contained acetonitrile. The gradient elution program was as follows: 0–17 min (25%–26% B), 17–26 min (26%–100% B). The column was washed with 100% eluent B between every two samples for 15 min and then re-equilibrated with 25% eluent B for 10 min. Elution conditions were as follows: 1.0 mL·minMedicinal ingredient yield was calculated as: medicinal-ingredient yield = (epimedin A content + epimedin B content + epimedin C content + icariin content) × leaf dry biomass.https://www.r-project.org/about.html). Differences between means were detected with Tukey HSD test. p value was set at 0.05 and 0.01 for ANOVA and Tukey HSD tests, respectively.Statistical analysis was conducted with one-way ANOVA software (R language version 3.1.1,


There was no significant difference of the metal ions’ effect on Mal-mPEG5000-SPA and SPA.The sweet potato β-amylase (SPA) was modified by 6 types of methoxy polyethylene glycol to enhance its specific activity and thermal stability. The aims of the study were to select the optimum modifier, optimize the modification parameters, and further investigate the characterization of the modified SPA. The results showed that methoxy polyethylene glycol maleimide  was the optimum modifier of SPA; Under the optimal modification conditions, the specific activity of Mal-mPEG5000-SPA was 24.06% higher than that of the untreated SPA. Mal-mPEG5000-SPA was monomeric with a molecular weight of about 67 kDa by SDS-PAGE. The characteristics of Mal-mPEG5000-SPA were significantly improved. The K SweetChemical modification of molecules is an effective means to increase enzymatic stability and biological activity. It can also effectively prolong the half-life of enzymes. Polyethylene glycol (PEG) is a good non-irritant amphipathic organic solvent without immunogenicity, antigenicity and toxicity ,9. The sWith similar properties to PEG, methoxy polyethylene glycol (mPEG) also works as a protein modifier with the active hydroxyl group at one end of PEG being blocked by the methoxy group. Modification through mPEG can change the relevant biological characteristics of enzymes or proteins, including hydrophobicity, surface charge, stability and water solubility ,14,15. IMethoxy polyethylene glycol N-hydroxylsuccinimide ester , Methoxy polyethylene glycol tosylate  and Methoxy polyethylene glycol maleimide  were adopted in this study for chemical modification on SPA to screen the optimum modifier. The response surface method was applied to optimize the molar ratio of the optimum modifier to SPA, as well as the modification temperature, pH value and other parameters. The enzymatic properties of the modification enzyme under optimal parameters were studied to improve its catalytic activity and thermal stability, so as to help lay the foundation for its industrial application.p < 0.05) in the same molar ratio. The specific activity of Mal-mPEG5000-SPA reached the maximum value at (2.081 ± 0.050) × 104 U/mg. There was a significant difference between Mal-mPEG5000 and other modifiers (p < 0.05). However, there was no significant difference between Ts-mPEG10000, Ts-mPEG20000 and NHS-mPEG20000 (p > 0.05). Therefore, Mal-mPEG5000 was selected as the optimal modifier for the next experiments.The change in SPA specific activity modified by 6 different modifiers was shown in 4 U/mg when the molar ratio was 1:4, an increase by 23.66% compared with that of the untreated SPA. As the concentration of Mal-mPEG5000 increased further, and the molar ratio increased to 1:6, the enzymatic specific activity decreased to (2.016 ± 0.051) × 104 U/mg, and there was a significant difference between the molar ratio of 1:4 and 1:6 (p < 0.05). Therefore, the optimal response molar ratio of SPA to Mal-mPEG5000 was concluded to be 1:4.The results from 4 U/mg at 55 °C. As the temperature increased to 65 °C, the specific activity dropped to (1.801 ± 0.055) × 104 U/mg, and decreased by 15.49%, and there was a significant difference between 55 °C and 65 °C (p < 0.05). Therefore, the optimal modification temperature was concluded to be 55 °C.The results from 4 U/mg at pH 6.0. As pH increased to 7.0, the specific activity was (1.925 ± 0.045) × 104 U/mg and decreased by 10.69%, and there was a significant difference between pH 6.0 and 7.0 (p < 0.05). Therefore, the optimal modification pH was concluded to be 6.0.The results from 4 U/mg at 10 min. As the modification time further increased, no significant change was noted in the enzymatic specific activity (p = 0.05). The ANVOA results showed no significant difference of enzymatic specific activity between 10 min and 30 min. Therefore, taking into account of practical application, the optimal modification time was concluded to be 10 min.The results from According to the effect of the molar ratio, modification temperature, pH and time, the time has little effect on modification, and modification pH (A), the molar ratio (B) and modification temperature (C) were selected as three factors to optimize the modification procedure by central composite design (CCD). With modification time set at 10 min, and Mal-mPEG5000-SPA specific activity (Y) as the response value, Box-Behnken design principles were followed to conduct a test of three factors and three levels. Results were shown in Multiple regression analysis was adopted to the experimental data by using the software Design-Expert V8.0.6. The response, Y , was selected as the test variables, and by the second order, a polynomial equation was developed (Equation (1)).p < 0.01), while the lack of fit was not significant (p = 0.317 > 0.05) and the value of the determination coefficient (R2) was 0.996, which indicated the goodness of fit of the regression model [The analysis of variance (ANOVA) for the response Y, the Mal-mPEG5000-SPA specific activity, was shown in on model . Based oIn order to further comprehend the interaction between the parameters, the response surfaces were obtained using Equation (2), which were plotted between two independent variables and the other independent variable was set at the zero-coded level. The analysis on the response was shown in 4 U/mg. under modification pH 6.08, the molar ratio 1:4.04, and modification temperature 58.85 °C. Considering the practicality of the validation test, the optimum parameters were corrected into modification pH 6.0, the molar ratio 1:4, and modification temperature 58 °C. The specific activity of Mal-mPEG5000-SPA was determined under the corrected parameters to be (2.150 ± 0.055) × 104 U/mg, an increase by 24.06% than that of the untreated one. The validation test showed that the optimization results from the response surface method were reliable, and adopting response surface method to optimize the modification process of Mal-mPEG5000 on SPA was feasible.Through the analysis by the software Design-Expert, the optimized combination for maximum Mal-mPEG5000-SPA specific activity was determined through canonical analysis of the response surfaces and contour plots as A = 6.08, B = 4.04 and C = 58.85 °C, i.e., the predicted value of Mal-mPEG5000-SPA specific activity of 2.247 × 10TM10 with SuperdexTM75 gel column  × 104 U/mg and (0.533 ± 0.036) × 104 U/mg respectively as shown in 1 was collected, centrifuged and concentrated for further purification.Mal-mPEG5000-SPA was separated and purified by AKTA purifierl column a–c. Sepa2 and S3 a, and thTM75 gel column, and there were elution peaks S11 and S12 (11 and S12 was determined to be (1.422 ± 0.057) × 104 U/mg and (0.061 ± 0.009) × 104 U/mg respectively  × 104 U/mg at 45 °C, an increase by 22.38% compared to the untreated one. Moreover, the activities of Mal-mPEG5000-SPA at 50 °C, 55 °C and 60 °C were determined as (2.074 ± 0.060) × 104 U/mg, (2.057 ± 0.062) × 104 U/mg, and (2.031 ± 0.064) × 104 U/mg respectively, all significantly higher than that of untreated SPA (p < 0.05). These results indicated that the enzymatic specific activity of modification enzyme was significantly improved after Mal-mPEG5000 modification.According to p < 0.05), which indicated high sensitiveness of SPA to temperature. No significant difference in the specific activity of Mal-mPEG5000-SPA from 20 °C to 45 °C for 1 h was found. But when the temperature rose to 50 °C, the specific activity of Mal-mPEG5000-SPA started to decline (p < 0.05), which suggested significant improvement of the thermal resistance of Mal-mPEG5000-SPA. These results showed that hydrolyzed starch by Mal-mPEG5000-SPA has a wider range of application in the food industry, which can significantly enhance enzymatic efficiency to reduce production costs.4 U/mg and (1.733 ± 0.050) × 104 U/mg, and the specific activity was improved by 18.92% after modification.p < 0.05), while the difference for Mal-mPEG5000-SPA was not significant (p = 0.05), which indicated higher adaptability of Mal-mPEG5000-SPA to a pH environment. In addition, the specific activity of Mal-mPEG5000-SPA was constantly higher than that of SPA under pH value from 4.0 to 7.5, which indicated lower susceptibility of Mal-mPEG5000-SPA to pH environment. This means Mal-mPEG5000-SPA could applied more broadly and is more suitable for industrial application.m value for sweet potato starch, potato starch, corn starch, soluble starch, amylase and amylopectin by Mal-mPEG5000-SPA hydrolysis was respectively (1.63 ± 0.033) mg/mL, (2.06 ± 0.028) mg/mL, (2.36 ± 0.063) mg/mL, (1.84 ± 0.025) mg/mL, (2.18 ± 0.029) mg/mL and (2.11 ± 0.052) mg/mL. The Vmax value was respectively (32.06 ± 0.61) mmol/min/mL, (16.23 ± 0.32) mmol/min/mL, (10.66 ± 0.37) mmol/min/mL, (20.88 ± 0.78) mmol/min/mL, (13.35 ± 0.38) mmol/min/mL, (13.89 ± 0.41) mmol/min/mL. The aE value was respectively (11.07 ± 0.43) kJ/mol, (18.24 ± 1.12) kJ/mol, (26.52 ± 1.21) kJ/mol, (14.71 ± 1.15) kJ/mol, (21.16 ± 1.12) kJ/mol and (21.66 ± 0.8) kJ/mol. These results indicated that Mal-mPEG5000-SPA had the lowest Michaelis constant for sweet potato starch. Therefore, Mal-mPEG5000-SPA was determined to have the strongest binding affinity for sweet potato starch. Next only to sweet potato starch, soluble starch was followed by potato starch, amylase and amylopectin. Compared with the SPA kinetic parameters reported in earlier studies [m declined by 12.95%, Vmax increased by 26.87%, and Ea dropped by 12.63%, which suggested Mal-mPEG5000-SPA showed stronger affinity for sweet potato starch than SPA.The results of kinetic parameters of Mal-mPEG5000-SPA were shown in 2+, K+, Zn2+ and Ca2+ was respectively (143.48 ± 6.25)%, (115.49 ± 4.51)%, (111.28 ± 4.82)% and (102.88 ± 4.32)%, which demonstrated that Mn2+, K+, Zn2+ and Ca2+ had good activation effect. Mg2+ showed a slight inhibitive effect on Mal-mPEG5000-SPA, with relative specific activity standing at (95.25 ± 4.14)%. However, Cu2+, NH4+, Fe3+, Al3+, Ba2+ and EDTA demonstrated a relatively strong inhibitory effect on Mal-mPEG5000-SPA, with relative specific activity reduced to 55–80%. Meanwhile, under the effect of Cd2+, Hg+ and Ag+, the relative specific activity were all below 30%, among which Hg+ and Ag+ indicated the strongest inhibitory effect on Mal-mPEG5000-SPA. Compared with the effect of metal ions on SPA reported in earlier studies [p = 0.05).As shown in  studies , the effSweet potato, molar weight 56.043 kDa, harvested in Xinxiang City, Henan Province, China, its cultivar was Shangshu 19. NHS-mPEG5000, NHS-mPEG20000, Ts-mPEG5000, Ts-mPEG10000, Ts-mPEG20000 and Mal-mPEG5000 were from Nanocs . All other reagents were of analytical reagent grade and used without further purification.TM10, General Electric Company, Boston, Massachusetts, MA, USA) was used to purify the enzyme, and Mono Q anion exchange chromatographic column and SuperdexTM75 gel column were adopted for separation and purification with detection wavelength set at 280 nm. The buffers for purification were as follows. Buffer A: 20 mM pH 5.8 disodium-hydrogen phosphate-citric acid, Buffer B: 1 mol/L NaCl. The buffers were filtered through a 0.22 μm membrane, ultrasound treated for 20 min, and stored in a refrigerator at 4 °C. Buffer A was used to equilibrate the chromatographic column. The sample was injected after equilibration of buffer to the baseline. The column was washed with 5 column volumes of buffer A, and then eluted with a gradient from 0–100% Buffer B at the flow rate of 1.0 mL/min, and maximum back-pressure of 4 MPa for 30 min. A collector was used to obtain 1 mL fractions. After the elution was complete, the enzymes in the collection tube corresponding to the peak position were collected, frozen and dried in a vacuum freeze dryer  and stored at 4 °C for later usage. The separation and purification of mPEG5000-β-SPA: 70% ammonium sulfate was added into the reaction liquid to precipitate. The rest steps were the same as those of the β-amylase separation and purification.Using the methods described by Liang et al.  with minw/v) polyacrylamide gel was adopted.The molecular mass and the purity of the enzyme was conducted by SDS-PAGE according to the Laemmli  method, Protein content was measured as described by Lowry et al. , and bovThe molar ratio of SPA to modifiers was determined at 1:4. SPA and 6 different types of modifiers  were placed in the buffer , respectively, and then kept in a water-bath thermostatic metal oscillator at 55 °C for 10 min. The reaction mixture was obtained for dialysis. After the dialysis was complete, the enzyme specific activity was measured to obtain the optimal modifier.SPA was modified by Mal-mPEG5000 at the molar ratio of SPA to Mal-mPEG5000 from 1:1 to 1:6, for modification temperature from 25 °C to 75 °C and for modification pH from 3.0 to 8.0. The enzyme specific activity of modified enzyme  was used as the index for evaluating the effects of the different modification parameters to be optimized via response surface methodology (RSM). A central composite design (CCD) were used with three variables and three levels for optimizing the modification conditions. These three factors were modification pH (A), the molar ratio of SPA to Mal-mPEG5000 (B) and modification temperature (C) . A secon0 is the intercept; α1, α2, α3, linear coefficients; α11, α22, α33, squared coefficients; and α12, α13, α23, interaction coefficients. Analysis of variance was used to evaluate the model’s adequacy and to determine the regression coefficients and their statistical significance. The response surface contour plots showed how the independent variables interacted and how those interactions influenced the overall response [Y is the predicted response, αresponse .Influence of temperature on Mal-mPEG5000-SPA and SPA: The optimum temperature of Mal-mPEG5000-SPA was investigated at pH 5.8 over a temperature range of 20–75 °C. SPA and Mal-mPEG5000-SPA was incubated in 20 mM disodium-hydrogen phosphate-citric acid buffer (pH 5.8) during 60 min, respectively. The thermostability of the enzyme residual specific activity were determined under different temperature conditions (20–75 °C).Influence of pH on Mal-mPEG5000-SPA and SPA: The optimum pH of Mal-mPEG5000-SPA was investigated at 50 °C over a pH range of 3.0–8.5. Mal-mPEG5000-SPA was preincubated for 60 min under 50 °C, the residual specific activity was determined at a pH range of 3.0–8.5 to evaluate the pH stability.2+, Mg2+, Cu2+, Zn2+, Mn2+, K+, NH4+, Hg+, Al3+, Fe3+, Ba2+ and Cd+), AgNO3 and EDTA at 40 °C for 30 min, and the residual activities were determined respectively. An enzyme that did not contain metal ions was chosen as control (100%).Incubation of the enzyme was carried out by 10 mM of metal ions salts (chlorides including Cam) and the maximum velocity (Vmax) of Mal-mPEG5000-SPA and SPA was defined by Lineweaver-Burk plot. Different concentrations of substrates (1–20 mg/mL) in 20 mM disodium-hydrogen phosphate-citric acid buffer pH 5.8 were used and specific activity was assessed by the DNS method. The values of Km and Vmax were calculated respectively based on the double reciprocal plot [The Michaelis constant , and the temperature was set from 25 °C to 65 °C. Representing 1/T in K on the axis of x and natural log of specific activity on the axis of y, Arrhenius plot was generated. aE was defined based on the value of the slope.p < 0.05) was performed by Duncan new complex method. All analyses were done in triplicate, and the statistical significance was determined using the mean values ± standard deviation.All experiments were carried out in triplicate. SPSS 22.0 statistical software  was used for the analysis of variance, and the significance test , the specific activity of Mal-mPEG5000-SPA was (2.150 ± 0.055) × 10


Fusarium spp. and Aspergillus spp. were found to be the fungi most likely to produce mycotoxins throughout the growing season. In particular, A. flavus and A. niger were found only rarely both in field and in post-harvest, while A. versicolor was always present although in low concentrations. Penicillium spp. strains were isolated sporadically and were found to be irrelevant in Italian rice fungal contamination. Sterigmatocystin (STC) was the main mycotoxin found in Italian rice, while aflatoxin (AFB1), deoxynivalenol (DON) and ochratoxin A (OTA) were rarely detected. Contamination generally increased from post-flowering to ripening; considering rice varieties, significant differences (p ≤ 0.01) were found in fungal contamination and STC production; no differences were observed between sowing densities. During storage, an increase in STC content was observed in higher temperature regimes, while all the other considered mycotoxins remained unchanged. These results indicated that contamination by STC, an emerging mycotoxin not legislatively regulated by the European Union, can be relevant in rice.Mycotoxigenic fungi and relative mycotoxins contamination were monitored in Italian paddy rice samples both in field during the growing season and the first five months of storage. Three experimental fields, nine rice varieties and three sowing densities were considered; then, different lots of paddy rice were stored in warehouses at different temperature regimes.  Italy is the main rice producer in Europe, accounting for about 50% of the entire European production. Rice cultivation is mostly located in Northern Italy , where water is relatively abundant and the rice crop can be cultivated in flooded fields; in 2018, the area dedicated to rice growing covered about 217,000 hectares ha and rice production amounted to nearly 1,451,319 tons -sterigmatocystin (96.4% 13C) was purchased from Biopure  as standard solution in acetonitrile . Stock and working STC standard solutions were prepared and calibrated spectrophotometrically as reported by Bertuzzi et al. [The STC analytical standard was obtained from Sigma-Aldrich , while the internal standard [i et al. .v/v using a rotary-shaking stirrer for 60 min. After purification through an immunoaffinity column, the extract was filtered (Millex HV 0.45 mm) before HPLC-FLD analysis ; the average recovery for AFB1 was 95.8 ± 3.4% . The limit of detection (LOD) and quantification (LOQ), defined at those levels resulting in signal-to-noise ratios of 3 and 10, were 0.05 and 0.15 µg kg−1, respectively. OTA was extracted from a 10 g of sample with 100 mL of a mixture of 0.13 M sodium bicarbonate-methanol (5 + 5 v/v) for 45 min using a rotary-shaking stirrer. After purification through an immunoaffinity column, the eluate was concentrated under a gentle stream of nitrogen, brought to 2 mL with acetonitrile-water (41 + 59 v/v), vortex-mixed for few seconds and filtered before HPLC-FLD analysis. The mean recovery (mean of 3 replicates at 2 spiking levels) was 95.2 ± 3.4%; LOD and LOQ were 0.02 and 0.06 µg kg−1, respectively. DON was extracted from samples (25 g) with 100 mL of acetonitrile-water (86 + 14 v/v); an aliquot (6 mL) of the filtrate was slowly pressed through a MycoSep 227 column. An aliquot (200 µL) of the internal standard diacetoxyscirpenol (DAS 10 mg L−1) was added to 2 mL of the eluate. The solution was evaporated to dryness and derivatized with 200 µL of trimethilsilylimidazole-trimethilclorosilane (1 + 0.2 v/v) for 15 min in subdued light. Then 0.8 mL hexane was added, and the solution was washed with 1 mL 0.2 M phosphate buffer pH 7.5, and the hexane phase was used for GC-MS. GC-MS analysis was carried out using a TraceGQ Ultra coupled with an ISQ single quadrupole mass spectrometry . The analysis was carried out using a capillary column Rtx-5MS, 30 m × 0.25 mm i.d., 0.25 µm film thickness. LOD and LOQ were 5 and 15 µg kg−1, respectively; the average recovery was 92.4 ± 2.6%. STC was extracted from an aliquot of 20 g taken from the milled sample with 100 mL acetonitrile-water 8 + 2 v/v using a rotary-shaking stirrer for 60 min. After purification through an immunoaffinity column, the extract was concentrated under a gentle flow of nitrogen and brought to 1 mL with acetonitrile-water 4 + 6 v/v. An aliquot of 900 µL of cleaned extract was transferred into an autosampler vial and mixed with 100 µL of isotopically labelled STC (12 µL L−1). A volume of 20 µL of the extract was injected into an LC-MS/MS system consisting of a LC 1.4 Surveyor pump, a Quantum Discovery Max triple-quadrupole mass spectrometer  and a PAL 1.3.1 sampling system . STC was chromatographed on a Betasil RP-18 column  with a gradient acetonitrile-water ; the ionization was performed using positive atmospheric pressure chemical ionization (APCI). The matrix effect was low, less than 10%; the LOD and the LOQ were 0.05 and 0.15 µg kg−1, respectively. The average recovery was 90.4% ± 4.2%.The analyses were carried out using methods developed in our department; in particular, AFs were determined by HPLC-FLD as reported by Bertuzzi et al. ; OTA by p ≥ 95%) to highlight significant differences.Data on fungal incidence were arcsine transformed, while mycotoxin content in flour was ln transformed before statistical analysis . The sta


Moreover, the new analogue, 9-methylfascaplysin, was designed and synthesized from 5-methyltryptamine. Interestingly, 9-methylfascaplysin is a more potent inhibitor of Aβ fibril formation than fascaplysin. Incubation of 9-methylfascaplysin with Aβ directly reduced Aβ oligomer formation. Molecular dynamics simulations revealed that 9-methylfascaplysin might interact with negatively charged residues of Aβ42 with polar binding energy. Hydrogen bonds and π–π interactions between the key amino acid residues of Aβ42 and 9-methylfascaplysin were also suggested. Most importantly, compared with the typical Aβ oligomer, Aβ modified by nanomolar 9-methylfascaplysin produced less neuronal toxicity in SH-SY5Y cells. 9-Methylfascaplysin appears to be one of the most potent marine-derived compounds that produces anti-Aβ neuroprotective effects. Given previous reports that fascaplysin inhibits acetylcholinesterase and induces P-glycoprotein, the current study results suggest that fascaplysin derivatives can be developed as novel anti-AD drugs that possibly act via inhibition of Aβ aggregation along with other target mechanisms.β-Amyloid (Aβ) is regarded as an important pathogenic target for Alzheimer’s disease (AD), the most prevalent neurodegenerative disease. Aβ can assemble into oligomers and fibrils, and produce neurotoxicity. Therefore, Aβ aggregation inhibitors may have anti-AD therapeutic efficacies. It was found, here, that the marine-derived alkaloid, fascaplysin, inhibits Aβ fibrillization  Alzheimer’s disease (AD) is a chronic and debilitating neurodegenerative disease that normally occurs in the elderly. AD is clinically characterized by cognitive impairments, mainly due to the loss of synapses and neurons in the brain, representing the greatest of health challenges . It is pTwo features of AD are the accumulation of extracellular senile plaques and intracellular neurofibrillary tangles. The amyloid hypothesis is now recognized as the core pathology of AD. This hypothesis explained that β-amyloid (Aβ), the main component of senile plaque, is a chief pathological element of dementia . Aβ itseFascaplysinopsis sp. [The ocean is a valuable resource for the discovery of new biologically active natural products . Marine psis sp. . Fascaplpsis sp. . Manda epsis sp. . With th3a) and 9-methylfascaplysin (3b) were synthesized by a two-step process from commercially available tryptamine and 5-methyltryptamine, respectively, as shown in 1H NMR and 13C NMR, and HRMS of fascaplysin and 9-methylfascaplysin, were shown in Fascaplysin  was used to further study the morphology of the 9-methylfascaplysin-modified Aβ sample. The unmodified Aβ oligomer was almost round, and the diameter was about 10–100 nm, which is consistent with previous reports . InteresAβ oligomer can bind to the postsynaptic membrane of neurons, causing neuronal death at low concentrations . To evalFDA/PI staining is another method that is typically used to assess the cell protective effects of small molecules such as 9-methylfascaplysin. Using this method, living cells and dead cells are respectively stained green and red. The portion of viable cells in the group treated with Aβ oligomer was less than that in the control group . FurtherN-terminus of Aβ42. It is noted that the absolute values of the binding free energy of 9-methylfascaplysin is larger than that of fascaplysin. Thus, 9-methylfascaplysin is suggested to be more effective than its lead compound to decrease Aβ aggregation, which is consistent with previous ThT and TEM results.MD simulations were performed to investigate the possible interactions between Aβ and 9-methylfascaplysin. The binding free energies of the Aβ-9-methylfascaplysin complex in the final 95–100 ns were calculated. The parent molecule, fascaplysin, was used as a control for comparison. As shown in 42 and 9-methylfascaplysin in the final 5 ns were determined, as demonstrated in 42. This interaction of 9-methylfascaplysin with negatively charged residues of Aβ42 mainly contribute to the calculated polar binding energy. Additionally, in the same binding mode, 9-methylfascaplysin molecules can have contact with hydrophobic residues F4, Y10, F20, V39, and I41 of Aβ42, which contributes to the determined nonpolar binding energy. However, no atomic contact was observed between the nearby positively charged residues  and 9-methylfascaplysin molecules, likely due to the fact that 9-methylfascaplysin contains a positively charged quaternary nitrogen atom  was sealed in a stainless-steel hydrothermal reactor under N2, and heated to 230 °C for 80 min. The reaction mixture was dissolved in MeOH and filtered through nylon membrane to remove carbonized particles. The filtrate was precipitated in ethyl acetate. The obtained precipitate was dried under vacuum to afford product 3b in 80% yield. The purity of 3a and 3b were determined to be 99.0% by HPLC  δ 9.62 , 9.14 , 8.57 , 8.49 , 8.07 , 8.01 , 7.89 , 7.82 , 7.74 , 7.53 . 13C NMR : δ 182.83, 147.63, 147.57, 141.01, 137.50, 134.77, 131.80, 131.66, 127.20, 126.04, 124.84, 124.53, 123.48, 123.31, 120.79, 119.96, 116.09, 114.27. HRMS (ESI) (positive mode): m/z calculated for C18H11N2O: 271.0866; M+, found: 271.0864.Fascaplysin and 9-methylfascaplysin were synthesized via a two-step process from tryptamine and 5-methyltryptamine in 50–55% overall yields, according to published methods, as shown in  reports . 2b : δ 9.32 , 8.85 , 8.33 , 8.19 , 8.06–7.96 , 7.75 , 7.69–7.57 , 2.51 . 1H NMR : δ 13.36 , 9.62 , 9.08 , 8.49 , 8.33 , 8.09–7.97 , 7.73 , 7.73–7.62 . 13C NMR : δ 182.74, 147.56, 145.79, 140.51, 137.49, 136.39, 132.86, 131.73, 131.46, 127.08, 126.01, 124.48, 123.95, 123.09, 120.62, 120.05, 116.08, 113.88, 21.37. HRMS (ESI) (positive mode): m/z calculated for C19H13N2O: 285.1022; M+, found: 285.1014.42  was dissolved in hexafluoroisopropanol . Furthermore, 100 μL Aβ was added to a clean tube and diluted with 900 μL Milli-Q water. HFIP in the solution was evaporated under N2 to until a final concentration of Aβ of about 50 μM. A portion of Aβ was mixed with different concentrations of 9-methylfascaplysin, or a vehicle blank, to obtain an Aβ solution at 10 μM. The Aβ solution was kept for two days at room temperature under constant stirring before it was centrifuged at 14,000× g for 15 min at 4 °C. The supernatant was collected, and this mainly contained Aβ oligomer.Aβ oligomer was prepared as previously reported . Synthet42 power was dissolved in HFIP to form Aβ monomers and diluted with Milli-Q water. After completely evaporating the HFIP with N2, Aβ monomer was diluted with NaOH to form 1 mM Aβ solution. Next, 2 μL Aβ solution and 10 μL 9-methylfascaplysin was added to PBS that contained 5 μM ThT . The mixture (200 μL) was incubated in the dark for 6 days at 37 °C. Fluorescence intensity at the excitation and emission wavelengths of 440 nm and 485 nm, respectively, was used to evaluate the quantity of Aβ fibrils.Aβ fibrils were also prepared as previously described . Aβ42 poDot blotting analysis was also performed according to literature methods. Briefly, 2 μL co-incubated Aβ and 9-methylfascaplysin was spotted onto the nitrocellulose membrane before it was dried by air. After blocking the membrane in 1% TBST solution containing 5% BSA overnight, the membrane was incubated with anti-oligomer antibody A11  or anti-Aβ1-17 antibody 6E10  for 1 h with shaking. Subsequently, the membrane was washed by 1% TBST before it was re-incubated with antibody for 1 h. The membrane was finally developed with an Enhanced chemiluminescence plus kit after 3 washes in 1% TBST.TEM samples were performed by placing 2 μL samples on a carbon-coated grid. The samples were stained by 1% uranyl acetate, and the excess staining solution was removed by placing the grid onto a clean paper. The grid was examined using a TEM .2 at 37 °C. The medium was refreshed every two days. Before experiments, the medium of SH-SY5Y was changed to DMEM supplemented with 1% FBS, for 24 h.SH-SY5Y cells  were maintained in high glucose modified Eagle’s medium (DMEM) which was supplemented with 1% penicillin (100 U/mL)/streptomycin (100 µg/mL) and 10% fetal bovine serum (FBS) with 5% CO3--2,5diphenyltetrazolium bromide (MTT) assay was performed to measure the cell viability. An aliquot of 5 μL Aβ oligomer or 9-methylfascaplysin-modified oligomer was added to the wells of 96-well plates. After incubating the cells for 24 h at 37 °C, 10 μL MTT was added. Next, 100 µL solvate (0.01 N HCl in 10% SDS) was added after 4 h. The absorbance of the samples was measured at a wavelength of 570 nm with 655 nm after 16 h.In this protocol, viable cells were stained by fluorescein diacetate (FDA), which form fluorescein in viable cells to be visualized. By contrast, propidium iodide (PI) stained nonviable cells by entering their cell membranes. Briefly, after staining with 5 μg/mL of PI and 10 μg/mL of FDA for 15 min, the cells were examined. The images were captured by a UV light microscopy and compared with photos taken under phase contrast microscopy. The photos of each well were captured from five random fields in order to measure cell viability quantitatively. The number of cells stained by PI and FDA were counted to calculate the cell viability as follows: % of cell viability = the number of cells stained by FDA/(the number of cells stained by PI + number of cells stained by FDA) .42 monomer used in the simulation were taken from the solution NMR structures  in Protein Data Bank (PDB) (https://www.rcsb.org/), and the conformation of Aβ42 monomer is shown in https://atb.uq.edu.au/) [42 monomer was put in an 8 × 8 × 8 cubic box with periodic boundary conditions. Then, eight inhibitors were randomly placed around the Aβ42 monomer. The minimum distance among any molecule was set to 15 Å. The concentration of 9-methylfascaplysin and fascaplysin used in this system was 26.94 μM. The remaining space was filled with water molecules. Finally, in order to neutralize the simulation system, 5 Cl− ions were added, in random locations, to replace the corresponding water molecules.The initial coordinates for Aβedu.au/) . The SPC42 monomer. Short-range van der Waals interactions and short-range electrostatic interactions were smoothly truncated at 10 Å. Newton’s equation of motion was integrated using the leapfrog algorithm with a time step of 2 fs for equilibrium dynamics [It was necessary to minimize the simulation system, and the collision of any atoms in the simulation system and incorrect geometry was removed. The steepest descent minimization method with 50,000 steps was used to allow reasonable spacing among water molecules, 9-methylfascaplysin, and the Aβdynamics ,45. The dynamics . Long-radynamics . The findynamics . For all42 monomer and 9-methylfascaplysin molecules. The command of gmx mindist was used to analyze the number of contacts between each residue of Aβ42 monomer with 9-methylfascaplysin molecules. The binding energy between inhibitors with the Aβ42 monomer was calculated using g_mmpbsa software [Several auxiliary programs in GROMACS 5.1.4 package were used to analyze the simulation trajectories. The gmx hbond command was used to calculate the number of hydrogen bonds between Aβsoftware ,50. Tempsoftware .p < 0.05 were considered to be statistically significant.Data were expressed as means ± SEM. Statistical significance was determined by one-way ANOVA and Tukey’s test for post hoc multiple comparisons. Results with In conclusion, it was demonstrated that 9-methylfascaplysin inhibits the aggregation of toxic Aβ in vitro, possibly directly via hydrogen bonding and π–π interactions. The interaction of 9-methylfascaplysin with Aβ could explain the inhibition of the neuronal toxicity of Aβ oligomers in SH-SY5Y cells. Based on these results, it is suggested that fascaplysin derivatives might be developed as potential new drugs for AD treatment. Further studies, including medicinal chemistry structural optimization and in vivo toxicity, should be conducted using the 9-methylfascaplysin scaffold as a lead molecule.


Chloroflexus aggregans is a thermophilic filamentous anoxygenic phototrophic bacterium frequently found in microbial mats in natural hot springs. C. aggregans often thrives with cyanobacteria that engage in photosynthesis to provide it with an organic substrate; however, it sometimes appears as the dominant phototroph in microbial mats without cyanobacteria. This suggests that C. aggregans has the ability to grow photoautotrophically. However, photoautotrophic growth has not been observed in any cultured strains of C. aggregans. We herein attempted to isolate a photoautotrophic strain from C. aggregansdominated microbial mats in Nakabusa hot spring in Japan. Using an inorganic medium, we succeeded in isolating a new strain that we designated “ACA-12”. A phylogenetic analysis based on 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer (ITS) region sequences revealed that strain ACA-12 was closely related to known C. aggregans strains. Strain ACA-12 showed sulfide consumption along with autotrophic growth under anaerobic light conditions. The deposited elemental sulfur particles observed by microscopy indicated that sulfide oxidation occurred, similar to that in photoautotrophic strains in the related species, C. aurantiacus. Moreover, we found that other strains of C. aggregans, including the type strain, also exhibited a slight photoautotrophic growing ability, whereas strain ACA-12 showed the fastest growth rate. This is the first demonstration of photoautotrophic growth with sulfide in C. aggregans. The present results strongly indicate that C. aggregans is associated with inorganic carbon incorporation using sulfide as an electron donor in hot spring microbial mats. Chloroflexus species are thermophilic filamentous anoxygenic phototrophic bacteria generally found in neutral to alkaline hot springs. In these natural hot springs, Chloroflexus organisms form microbial mats with cyanobacteria , ely 65°C , 17. Culggregans , 17, 23.ggregans , which sChloroflexus, two strains, i.e., C. aurantiacus strain OK-70-fl  was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen . C. aggregans strain NBF was previously isolated from Nakabusa hot spring (−1) 0.1 g sodium glutamate, 0.1 g disodium succinate, 0.1 g sodium acetate, 0.1 g yeast extract , 0.1 g Bacto casamino acids , 0.1 g Na2S2O3·5H2O, 0.1 g (NH4)2SO4, 75 mg KH2PO4, 78 mg K2HPO4, 0.2 mL of a vitamin mixture (−2).t spring . These s mixture , and 1 m mixture . CultureChloroflexus-dominated microbial mats are always found in Nakabusa hot spring in Nagano Prefecture, Japan . The thickness of these mats that developed at 65°C is approximately 3 mm. Sampling was performed on November 4, 2016. Each single piece of the collected mat (approximately 10 mg wet weight) was incubated in a 100-mL vial containing a 40-mL enrichment medium anaerobically at 55°C under illumination from an incandescent lamp.−1) 0.5 g Na2S2O3·5H2O, 0.5 g (NH4)2SO4, 0.75 g KH2PO4, 0.78 g K2HPO4, 1 mL of a vitamin mixture (2 and CO2 (80%:20% [v/v]) and then autoclaved. The gas phase of the vials was replaced with the same gas composition mixture. Before the inoculation, a filter-sterilized NaHCO3 solution  and a Na2S solution  were added to the vial.The enrichment medium (pH 7.0) was prepared as follows. The vial containing the solution consisted of  c and a  by absorption spectral observations of the pigment extract ; and gliding motility on agar plates.Strain ACA-12 was isolated from the enrichment culture using an agar plate. An aliquot of the enrichment culture was streaked on an agar plate of modified PE medium (see above) supplemented with 1.5% Bacto agar (Difco Laboratories), and the inoculated plate was incubated under aerobic dark conditions at 55°C. Under these conditions, C. aggregans strains ACA-12, MD66T, and NBF were initially pre-cultured photoheterotrophically. Pre-cultures were serially transferred to the inorganic medium twice for the photoautotrophic growth experiment. The inorganic medium (pH 7.0) for the growth experiment was prepared as follows. The vial containing the solution consisting of (L−1) 0.1 g (NH4)2SO4, 0.15 g KH2PO4, 0.16 g K2HPO4, 0.2 mL of a vitamin mixture (2 and CO2 (80%:20% [v/v]) and then autoclaved. Prior to the inoculation, the filter-sterilized NaHCO3 solution  and Na2S solution  were added to the vial.The cells of  mixture , and 2 m mixture  was gassg, 4°C, 5 min) and then washed once with the wash solution containing (L−1) 0.75 g KH2PO4, 0.78 g K2HPO4, 1 mL of a vitamin mixture . The mixture was centrifuged  and the supernatant was collected. The absorption spectrum was recorded between 400 and 1,000 nm using a UV-1800 spectrophotometer . To calculate BChl c (absorbance at 666 nm) concentrations, we used the extinction coefficient of 74 mM−1 cm−1  and resuspended them in 1 mL of phosphate-buffered saline (PBS) (pH ~7.4) with 0.1% Tween 80. Cells were dispersed by sonication  at 4°C for 10 min in a sonication bath . BChl in vivo absorption of BChl c to estimate the growth curves of strain MD-66T and NBF. The in vivo spectra of the bacterial cultures were recorded by a UV-1800 spectrophotometer (Shimadzu). To cancel the effects of turbidity from extracellular elemental sulfur, we used the value of the quadratic differential of the absorption spectra at 740 nm in the calculation. The smoothing of the original spectrum and the quadratic differential of the absorption spectrum were calculated by the Convolution (Savitzky-Golay) method using UVProve 2.42 software (Shimadzu).We used the g for 5 min. The supernatant (150 μL) was collected and fixed with 150 μL of 1.8 mM zinc acetate, and stored at 4°C until analyzed. Sulfide concentrations were assessed by the methylene blue formation method according to Cline  to prevent hydrogen sulfide from evaporating, and cells were removed by centrifugation at 13,982×Taq polymerase . Polymerase chain reaction (PCR) amplification was performed by a T100 Thermal Cycler  under the following conditions: initial denaturation at 94°C for 3 min; 30 cycles of denaturation at 94°C for 30 s, primer annealing at 55°C for 45 s, and extension at 72°C for 1 min; and terminal elongation at 72°C for 4 min.We used a Wizard Genomic DNA Purification kit  to extract the genomic DNA of strain ACA-12. The 16S rRNA gene of strain ACA-12 was amplified using the 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGY TACCTTGTTACGACTT-3′) primers  with Ex-T in the present study. PCR amplification was performed under initial denaturation at 95°C for 3 min; 35 cycles of denaturation at 94°C for 1 min, primer annealing at 55°C for 1 min, and extension at 72°C for 1 min; and terminal elongation at 72°C for 3 min.The 16S-23S rRNA gene internal transcribed spacer (ITS) region sequence of strain ACA-12 was amplified using a forward primer (5′-GACACACACGCTACAATGGC-3′) and reverse primer (5′-TGCCCCATTCGGAAATCTCC-3′). The PCR primer set was designed based on the 16S–23S rRNA gene information of strain MD-66The SQR sequence of strain ACA-12 was amplified using a forward primer (5′-AAGCCGATTACATCCCACCG-3′) and reverse primer (5′-AACGGGAAGCTCTCTTTGGG-3′). The PCR primer set targeting the majority of the open reading frame (ORF) was designed according to the gene information of strain NBF (LC439416) in the present study. PCR amplification was performed under initial denaturation at 94°C for 2 min; 30 cycles of denaturation at 94°C for 30 s, primer annealing at 60°C for 30 s, and extension at 72°C for 1 min; and a terminal elongation at 72° C for 5 min.These PCR products were sequenced by the Big Dye Terminator v3.1 Sequencing kit  with an ABI3130xl Genetic Analyzer (Applied Biosystems).C. aggregans ACA-12, LC438939, LC439412, and LC439413; C. aggregans NBF, LC439414, LC439415, and LC439416.The partial 16S rRNA gene, ITS region, and SQR gene sequences obtained in the present study have been deposited in the DDBJ/EMBL/GenBank nucleotide sequence databases with the following accession numbers: Taq Buffer , dNTP mixture (0.2 mM each), 0.4 μM of each primer, and 1.25 U of Takara Ex Taq Hot Start Version (Takara). PCR amplification was performed under initial denaturation at 94°C for 3 min; 4 cycles of denaturation at 95°C for 5 min, primer annealing at 40°C for 5 min, and extension at 72°C for 5 min; 30 cycles of denaturation at 94°C for 1 min, primer annealing at 55°C for 1 min, and extension at 72°C for 2 min; and terminal elongation at 72°C for 10 min. Five microliters of the PCR reaction was analyzed on a 1.5% agarose gel. DNA bands were stained with GelGreen  and visualized on an LED transilluminator .The rep-PCR oligonucleotide primers evaluated in the present study were ERIC 1R (5′-ATGTAAGCTCCTGGGGATTCAC-3′) and ERIC 2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′), BOXA1R (5′-CTACGGCAAGGCGACGCTGACG-3′), and (GTG)5 (5′-GTG GTGGTGGTGGTG-3′) , 29. TheC. aggregans strain MD-66T, such as a filamentous morphology (c and a (data not shown), similar to other Chloroflexus strains  clearly distinguished the isolate from strain NBF . SQR is a membrane-associated protein that catalyzes the oxidation of sulfide to elemental sulfur and transfers electrons to the membrane quinone pool (T (1.1% difference), the SQR amino acid sequences of these three strains showed 100% identity  based on the BChl c concentration was calculated from the exponential growth data plotted against time . The cells of C. aggregans strain MD-66T grew slightly under these conditions, and the growth rate was approximately 0.035 doublings h−1 . The growth rate of strain NBF was approximately 0.043 doublings h−1.t spring  and Nakat spring . In the ings h−1 . Cell grings h−1 . In the C. aggregans and has the following properties that are similar to those of C. aggregans: a filamentous cell morphology and cell size, the presence of BChl a and c, and the formation of microbial mat-like cell aggregates in a liquid medium. Although our analysis based on 16S rRNA gene and ITS sequences did not distinguish the new strain from C. aggregans strain NBF  to support the photoautotrophic growth of C. aggregans (C. aggregans is widely distributed below the surface layer of a microbial mat (C. aggregans strains in natural environments has not yet been conducted, each strain appeared to have grown at sites under segregated conditions, e.g., with or without cyanobacteria and different temperatures.In Nakabusa hot spring, a microbial mat containing ggregans , 22. C. bial mat . Since oC. aurantiacus strain OK-70-fl. Similarly, we observed the deposition of bright particles in cultures of C. aggregans (Chloroflexus, the presence of sulfur dioxygenase, which oxidizes elemental sulfur and converts it to sulfite, has not been reported, and Chloroflexus does not appear to have the ability to use elemental sulfur as an electron donor in a photosynthetic electron transport system.Madigan and Brock  reportedggregans . In the Chloroflexus species, including strain ACA-12, contain a gene for Type II SQR (Chloroflexus species. The present study is the first to demonstrate the photoautotrophic growth on sulfide of strains of C. aggregans that contain the Type II SQR gene in their genomes. This may be indirect evidence for Type II SQR having the function of dissimilatory sulfide oxidation, similar to other types of SQR.The enzyme SQR is classified into six groups. The genomes of e II SQR . Type IIe II SQR . Althouge II SQR , it may C. aggregans strains are considered to basically be photoheterotrophs that require organic compounds produced by neighboring cyanobacteria in natural environments. However, this may not be accurate because photoautotrophic growth was detected in C. aggregans strains in the present study. Therefore, C. aggregans is not just a consumer that depends on cyanobacteria, it may thrive independently with carbon fixing, and, hence, cannot be ignored as one of the primary producers in hot springs, as expected based on the biological activities of the microbial mats (ial mats . However


Acupuncture is effective for irritable bowel syndrome (IBS); however, the mechanisms of action are not fully understood. We aim to explore the mechanism of electroacupuncture (EA) in the dual regulation of disorders of gut-brain interaction. Tianshu), ST36 (Zusanli), SP6 (Sanyinjiao), and LR3 (Taichong) for 15 min every day. Rats in the CUMS+PB group were treated by the administration of gavage with 2.7 mg/mL pinaverium every day. Visceral pain threshold, the percentage of time spent in open arms (OT%) in the elevated plus maze test (EPMT), and the sucrose preference (SP%) in the sucrose preference test (SPT) were measured at baseline, day 15, and day 30. The expression of zonula occludens-1 (ZO-1), the morphology of the connective structure of intestinal epithelium, the CRF and CRF-R1 mRNA expression in the hypothalamus, and the double staining of intestinal mucosal mast cells (IMMC) and CRF-R1 were measured at the end of the experiment.  A rat model of IBS was generated by chronic unpredictable mild stress (CUMS). Eight of 32 rats were assigned to the blank control group. The remaining 24 rats received CUMS for 14 days. Then, the rats surviving and successfully modelled were randomly divided into the CUMS group, the CUMS+EA group, and the CUMS+PB (pinaverium bromide) group. In the next 14 days of treatment, rats in the CUMS+EA group were acupunctured at ST25  to repair the intestinal mucosal barrier. The above roles suggest that EA may play a dual role in alleviating the gastrointestinal and psychological symptoms of IBS, suggesting a potentially dual therapeutic role for EA in regulating disorders of gut-brain interaction in IBS rats. Rome IV suggests that the pathogenesis of IBS is mainly due to disorders of gut-brain interaction [IBS is a common, chronic gastrointestinal disorder characterized by recurrent lower abdominal discomfort and altered bowel habits . It has eraction .A considerable number of studies found that acupuncture can improve gastrointestinal motility, decrease intestinal permeability, and reduce visceral hypersensitivity , 8. Our Accumulating evidence has shown that CRF, which is mainly produced in the hypothalamic paraventricular nucleus, can induce altered bowel habits, visceral hypersensitivity, and emotional disorders, such as anxiety-like or depression-like behaviours, by combining with central CRF-R1 .Mast cells (MCs) are important immune cells that are widely distributed in intestinal mucosa. The number and activity of IMMC in IBS patients were both positively correlated with intestinal permeability  and abdoWe hypothesized that EA may decrease the expression of CRF and CRF-R1 in the hypothalamus and gastrointestinal mucosa while upregulating ZO-1 expression, adjusting TJs to repair the intestinal mucosal barrier. Thus, EA may play a dual role in alleviating the gastrointestinal and psychological symptoms of IBS and regulating disorders of gut-brain interaction in IBS rats.SD rats  were obtained from the Sichuan Academy of Medical Sciences, Chengdu, China. They were housed in individual cages , maintained on a 12 h light/dark cycle (lights on at 8:00) with food and water available ad libitum. The experiment was approved by the Experimental Animal Welfare Ethics Committee at the Chengdu University of Traditional Chinese Medicine (reference no. 2016-02). The experiments were performed according to the National Guideline for the Care and Use of Laboratory Animals, Amendment 2 . All efforts were made to minimize suffering.After 1 week of adaptation, 8 of 32 rats were assigned to the blank control group. The remaining 24 rats received CUMS for 14 days. Colorectal distension (CRD) pressure, OT%, and SP% were taken as markers for model establishment. Then, the rats surviving and successfully modelled were randomly divided into a CUMS group, a CUMS+EA group, and a CUMS+PB group. After 14 days of treatment, the above behavioural tests were performed. All animals were then euthanized under isoflurane anaesthesia. Throughout the entire experiment, animals were housed in groups of 2-4 except when they were subjected to isolation, high-density housing, or behavioural testing.The CUMS procedure included the following seven stress methods: high-density housing for 24 h, separation of housing for 24 h, restraint for 2 h, 45°C warm swim for 5-10 min, tail pinch for 20 min, inescapable shock for 15 min , and 240 Hz shaking-crowding for 1 h . All theFour acupuncture points commonly used in the treatment of IBS were selected. They are ST25, ST36, SP6, and LR3. ST25 is 5 mm lateral to the anterior midline at the navel level. ST36 is located at the posterior and lateral side of the knee joint, 5 mm below the capitulum fibulae. SP6 is located 10 mm above the prominence of the lateral malleolus of the hind limb. LR3 is located between the first and second metatarsal bones on the dorsum of the foot. EA was performed by a well-trained acupuncturist once a day for 14 consecutive days. During treatment, rats were restrained but conscious. Stainless steel disposable acupuncture needles  were inserted at ST25 and ST37 to a depth of 4-5 mm and at SP6 and LR3 to a depth of 2-3 mm and connected to an EA apparatus . Electrical stimulation was applied in the form of disperse-dense waves at alternating 2 Hz and 15 Hz frequencies, respectively, and 1 mA intensity for 15 min , 19, 20.Pinaverium  was dissolved in sterile distilled water at a concentration of 2.7 mg/mL and administered by gavage into rats of the pinaverium group at 10 mL/kg once daily. Other animals received only distilled water intragastric administration , 22.Rats were deprived of food but had free access to water 24 h before behavioural assessments. The prepared colorectal distension balloon covered with saxoline was completely inserted into the descending colon and rectum under ether anaesthesia. Rats were placed into a transparent acrylic box (20 cm × 8 cm × 6 cm) that could not escape or turn around for 30 min until fully recovered from the anaesthesia. The tube of the balloon was connected via a three-limb tube to a desk model sphygmomanometer. The balloon that inserted into the rat intestinal tract was inflated with air in increments of 5 mmHg at intervals of 5 s until a visible contraction of the abdominal wall was observed. The distension duration and intervals between two distensions were selected for 20 s and 5 min. Five abdominal withdrawal reflex (AWR) scores (AWR 0 to AWR 4) were used to assess the intensity of visceral stimuli : AWR 0: After animals habituated to two water bottles for 3 days in home cages, water bottles were removed 6 h before treatment. A free choice between plain water and 2% sucrose solution was provided to each animal for 12 h. The positions of bottles were counterbalanced across the left or right side of the testing cages. Water intake and sucrose intake were measured during the 12 h dark cycle. The preference for sucrose over water was used as a hedonic measure .The EPMT was conducted on a plus-shaped apparatus consisting of two 50 × 10 × 1.5 cm open arms and two 50 × 10 × 40 cm enclosed arms elevated 50 cm from the floor. The EPM was conducted in a room illuminated by a single red light bulb over the centre of the maze. Each rat was placed on the maze for 5 min. The apparatus was novel to the rats at the time of testing, and each rat was tested only once. After each rat test, the maze was wiped with ANNE Q (pet deodorant) to prevent odours causing disturbance to the following rats. Sessions were video recorded and scored for (1) time spent in the open arms, (2) time spent in the closed arms, (3) time spent in the central area, and (4) the total time of each subject by an observer blind to the animal's group. A rat was considered to have entered or spent time in an arm only when all four paws were in the respective arm. The percentage of time in the open arms to the total time deducted from the central area was analysed , 25.The hypothalamus and distal colon  were rapidly collected after the rats were executed. The intestinal contents were washed with 0.9% normal saline. Then, the cleaned colon was quickly placed in 4% polyoxymethylene and fixed at room temperature for 48 h for subsequent immunohistochemical analysis. The hypothalamus, located between the posterior margin of the optic chiasma and the anterior pituitary, was wrapped in tinfoil and quickly placed in liquid nitrogen. Then, the hypothalamus was removed from the liquid nitrogen and stored at −80°C for subsequent fluorescence quantitative- (fq-) PCR analysis.β-actin mRNA: forward: 5′-ACAACCTTCTTGCAGCTCCTC-3′; reverse: 5′-CTGACCCATACCCACCATCAC-3′. The fq-RT-PCR of CRF mRNA and CRF-R1 mRNA was performed in 20 μL volumes with SYBR Green PCR Master Mix  at 95°C for 2 min and 40 cycles at 95°C for 15 s, 60°C and 60°C for 15 min, using a LightCycler 480 .Using TriPure Isolation Reagent  according to the instructions to extract total CRF RNA and CRF-R1 RNA from hypothalamus tissue, RNA quantity was assessed using a NanoDrop 2000 ultramicrospectrophotometer . cDNA was synthesized using a reverse transcriptase kit . Sequences of gene-specific PCR primers  used were as follows: CRF mRNA: forward: 5′-TCTCTGGATCTCACCTTCCACCTT-3′; reverse: 5′-AGTTTCCTGTTGCTGTGAGCTTGC-3′; CRF-R1 mRNA: forward: 5′-TCGGGAGAAGGCTACCAGAC-3′; reverse: 5′-GGCTTCGCACCCTTCCG-3′; and μm thick sections. After being washed in TBS, sections were incubated for 30 min in 0.5% Triton X-100 and blocked with 10% goat serum in TBS for 2 h at 37°C.The colon tissues were postfixed in 4% paraformaldehyde and immersion fixed for 72 h, embedded in paraffin, and then sliced into 5 The sections were incubated for 1 h at 37°C with Goat anti-CRHR1/CRF-R  and Rabbit Anti-Mast Cell Tryptase antibody  diluted in TBST with 10% goat serum before incubating overnight at 4°C. The following day, the sections were washed and incubated with donkey anti-goat IgG H&L  and donkey anti-rabbit IgG H&L , diluted in TBS with 10% goat serum, for 1 h at 37°C. After washing, the sections were coverslipped with Antifade Mounting Medium and examined at 200x magnification under a fluorescence microscope .The sections were incubated for 1 h at 37°C with anti-ZO-1  diluted in TBST with 10% goat serum before incubating overnight at 4°C. The following day, the sections were washed and incubated with biotin-labelled secondary antibody, diluted in TBS with 10% goat serum, for 1 h at 37°C. After washing, the sections were coverslipped with Antifade Mounting Medium and examined at 400x magnifications by an optical microscope .After colon tissue was cut into 1 × 1 × 1 mm, the sample was prefixed in 2.5% glutaraldehyde at 4°C for 2 h and then rinsed with 0.1 M phosphate buffer 3 times for 45 min each time. The sample was then postfixed with 1% osmium tetroxide for 1 h and was rinsed as described before and was dyed with 1% uranyl acetate for 2 h. After dehydration with acetone, tissues were soaked in a solution of acetone and epoxy resin and then embedded polymerization at 45°C for 3 h and 65°C for 48 h, respectively. The sample was cut into ultrathin sections (70 nm) and stained with uranyl acetate for 40 min and then lead citrate for 15 min. The samples were examined under TEM  for ultrastructural analysis of colon tissue.U tests were used for pairwise comparisons following parametric and nonparametric tests, respectively. p < 0.05 was considered statistically significant.Statistical analysis was conducted using SPSS V.15.0 Software . The Shapiro-Wilk test was used to test for the normality of data. Normally and nonnormally distributed data are presented as the mean ± SD and median (IQR), respectively. One-way analysis of variance (ANOVA) followed by a post hoc least significant difference (LSD) test was used for normality assumption and homogeneity of variance data. A nonparametric Kruskal-Wallis test was applied when the normality assumption and homogeneity of variance were violated. The Duncan and Mann-Whitney n = 8), CUMS group (n = 7), CUMS+EA group (n = 8), and CUMS+PB group (n = 8). There were no other fatalities or exclusions from the study.One rat died after 17 days, which resulted in the following final sample sizes: blank control group (p > 0.05). After CUMS, CRD pressure was markedly decreased in the other three groups compared with the blank control group (21.67 (10.84) vs. 40.00 (16.67), p < 0.01; 15.00 (13.34) vs. 40.00 (16.67), p < 0.01; and 20.00 (9.16) vs. 40.00 (16.67), p < 0.01). Additionally, CRD pressure was significantly elevated in the CUMS+EA and CUMS+PB groups compared with the CUMS group after treatment (23.33 (6.67) vs. 18.34 (11.67), p < 0.05; 26.67 (6.67) vs. 18.34 (11.67), p < 0.01). No significant difference was found between the two receiving treatment groups (23.33 (6.67) vs. 26.67 (6.67), p > 0.05).As shown in p < 0.01). The CUMS+EA and CUMS+PB groups both significantly improved the mean optical density of ZO-1 compared with the CUMS group (2.12 (0.10) vs. 1.54 (0.18), p < 0.01; 2.00 (0.18) vs. 1.54 (0.18), p<0.01). Furthermore, the mean optical density of ZO-1 in the CUMS+EA group increased more obviously than the CUMS+PB group (2.12 (0.10) vs. 2.00 (0.18), p<0.01).As shown in As shown in As demonstrated in p > 0.05). All groups receiving CUMS displayed a statistically significant decrease in OT% compared to the blank control group (10.37 (12.48) vs. 29.10 (21.70), p < 0.01; 13.94 (13.86) vs. 29.10 (21.70), p < 0.01; 11.99 (14.54) vs. 29.10 (21.70), p < 0.01). After treatment, the OT% was markedly elevated in the CUMS+EA and CUMS+PB groups compared with the CUMS group (32.60 (43.12) vs. 3.67 (8.05), p < 0.01; 15.43 (14.62) vs. 3.67 (8.05), p < 0.01). There was no significant difference between the two treatment groups (32.60 (43.12) vs. 15.43 (14.62), p > 0.05).As shown in p > 0.05). After modelling, the SP% in the other three groups had a significant reduction compared with the blank control group (38.91 (34.77) vs. 79.51 (22.57), p < 0.01; 44.93 (10.72) vs. 79.51 (22.57), p < 0.01; and 44.06 (18.58) vs. 79.51 (22.57), p < 0.01). After the treatment, the SP% of the CUMS+EA and CUMS+PB groups were obviously higher than the CUMS group (71.69 (29.39) vs. 53.35 (29.77), p < 0.01; 53.92 (37.35) vs. 53.35 (29.77), p > 0.05). No significant difference was found between the two receiving treatment groups (71.69 (29.39) vs. 53.92 (37.35), p > 0.05).As shown in p < 0.01). CUMS+EA and CUMS+PB both obviously inhibited CRF expression compared with the CUMS group (1.25 (0.45) vs. 1.75 (0.77), p < 0.01; 1.02 (0.58) vs. 1.75 (0.77), p < 0.01). No significant difference was found between the CUMS+EA and CUMS+PB groups (1.25 (0.45) vs. 1.02 (0.58), p > 0.05).As shown in p < 0.01). CUMS+EA and CUMS+PB both significantly suppressed CRF-R1 expression compared with the CUMS group (0.92 (0.87) vs. 5.18 (8.02), p < 0.01; 1.15 (0.55) vs. 5.18 (8.02), p < 0.01). There was no significant difference between the CUMS+EA and CUMS+PB groups (0.92 (0.87) vs. 1.15 (0.55), p > 0.05).As shown in p < 0.01). CUMS+EA and CUMS+PB both significantly reduced the number of cells double stained for CRF-R1 and MC compared with the CUMS group (57.00 (12.00) vs. 90.50 (9.00), p < 0.01; 56.50 (16.75) vs. 90.50 (9.00), p < 0.01). No significant difference was found between the CUMS+EA and CUMS+PB groups (57.00 (12.00) vs. 56.50 (16.75), p>0.05).As shown in To evaluate the association between MC and CRF-R1 in gastrointestinal mucosa, double labelling of MC and CRF-R1 was performed on sections of the rat gastrointestinal mucosa. A high proportion of CRF-R1 colocalized with MC was found in colon mucosa. CRF-R1 immunoreactivity within cell bodies showed green cytoplasmic staining, and positive IMMC staining was observed in red, while yellow was the double staining of CRF-R1 and IMMC, as demonstrated in Rome IV Standards of Functional Gastrointestinal Disorders that IBS is the disorder of gut-brain interaction, further elaborating the importance of the interinfluence between intestinal and emotional symptoms in IBS [It is pointed out in s in IBS . Visceras in IBS .AWR is a major indicator of intestinal sensitivity. A previous study found that the pain threshold of IBS patients was obviously lower than that of healthy patients when the duodenum received intense distention stimulation . In thisDamage to TJs could destroy the intestinal epithelial barrier and increase intestinal permeability , 30. ZO-As for mental disorders, rats' anxiety-like behaviours were evaluated by OT% of EMPT and depression-like behaviours by SP%. In this experiment, OT% and SP% of rats decreased significantly after modelling, and both EA and PB treatments can effectively increase OT%. EA can distinctly improve SP% to a relatively normal level, while PB had no effective influence on it. As a result, EA may be superior to PB in improving depression-like behaviours, although there was no significant difference between the two treatments. This result is consistent with the following finding: EA can enhance the SP% of depressing rats and relieve rats' anxiety and depression-like behaviours, with a similar effect to PB, an antidepressant medicine .According to previous studies, central CRF-R1 activation is related to growing mental disorders, visceral hypersensitivity, and intestinal movements ; intestiIn conclusion, our study indicates that EA can decrease the expression of hypothalamic CRF and CRF-R1, relieve mental disorders, meanwhile reduce the expression of CRF-R1 in the gastrointestinal mucosa, decrease IMMC, increase ZO-1 expression, and adjust TJs to repair the intestinal mucosal barrier, suggesting a potentially dual therapeutic role for EA in alleviating the gastrointestinal and psychological symptoms of IBS, meaning that EA may regulate disorders of gut-brain interaction in IBS rats.