Average FD recoveries from spinach, tap water, and soil samples were 89.3–112.3%, 93.0–102.1%, and 86.9–97.6%, respectively. Meanwhile, FD detection results of icELISA were compared with those of ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The comparable results verified that icELISA was suitable for rapid detection of FD residue in environmental and agricultural samples.Flubendiamide (FD), the first commercial phthalic acid diamide that targets insect ryanodine receptor (RyRs), has played an important role in pest management. With its extensive worldwide application, a rapid and convenient method to detect its existence in the environment is necessary. In this study, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed to analyse FD residue on environmental and food samples. The established icELISA showed a half maximal inhibition concentration (IC Developed by Bayer Crop Science and Nihon Nohyaku in 2007, it became the first commercialised phthalic diamide insecticide that targets insect RyRs. It is characterised by its broad insecticidal spectra, extremely high intrinsic potency, remarkable selectivity, low ecotoxicity, and low residue levels3. At present, FD is used to control lepidopterous insects, including Helicoverpa spp., Heliothis spp., Spodoptera spp., Plutella spp., Trichoplusia spp., and Hyrotis spp., as part of pesticide resistance management and integrated pest management programs4. Therefore, it is applied extensively on crops, vegetables, and fruits5.Flubendiamide , the continued use of FD will result in unreasonable adverse effects on the environment due to one degradation product of FD in water is highly toxic to fish. The FD registrations of over 200 crops in the US and rice in China were cancelled in 2016, its products still ranked in the top ten of the global pesticide market10, HPLC with photodiode array detection (DAD)12, and tandem mass spectrometry (MS/MS)13. Conventional instrumental methods exerted remarkable sensitivity, selectivity, accuracy, and reliability, but usually require expensive instruments, large consumption of solvents, highly skilled professionals, and time-consuming sample preparation.Many analytical methods have been developed for FD residue detection in environmental and food samples, including high-performance liquid chromatography (HPLC) with UV-visible detection (UVD)16. The indirect competitive enzyme-linked immunosorbent assay (icELISA) is based on the competition between the immobilised antigen and an unknown amount of analyte to bind with a small fixed amount of antibody17. The antibody bound to the immobilised antigen would also bind to a specific enzyme-labelled second antibody. The colour intensity obtained is inversely proportional to the amount of analyte being investigated18. Such immunoassay is suitable for monitoring trace amounts of pesticides in a large number of environmental and food samples.The immunology-based enzyme-linked immunosorbent assay (ELISA) has been extensively applied in the detection of environmental contaminants. Compared to conventional instrumental methods, ELISA is a rapid and sensitive method which requires only small quantities of test materials and simple pre-treatment of sampleTo our knowledge, there is no report of monoclonal antibody (mAb)-based ELISA to monitor residual FD in the environment and food samples. In this study, a hapten was designed and synthesised based on FD structural characteristics, and anti-FD monoclonal antibody (mAb) and icELISA for the analysis of FD were developed. Through verification by analytical method based on UPLC-MS/MS, the developed icELISA is a confirmed sensitive and effective method to quantitatively determine trace FD in the environment and agricultural samples.21. Based on these findings, carboxylic acid group was introduced to the terminal of aliphatic amine for binding to carrier protein, whereas phthalic acid and aromatic amine moieties were kept. With this method, we designed and synthesized the hapten (compound 10), as shown in Fig. 3 containing an amine group was prepared by the reaction of ethyl 3-mercaptopropanoate (compound 1) with 2, 2-dimethylaziridine (compound 2) in the 1, 2-dimethoxyethane, and then reacted with 3-iodophthalic anhydride (compound 4) in the dichloromethane with triethylamine as the acid acceptor to obtain compound 5. In the presence of trifluoroacetic anhydride as dehydrating agent, compound 5 was converted to corresponding N-substituted isoimide compound 6 at 0–5 °C, and subsequently reacted with 2-methyl-4-(perfluoropropan-2-yl)aniline (compound 7) at 25 °C with a yield of 54% to give the compound 8, which was oxidized by m-chloroperbenzoic acid (m-CPBA) to obtain compound 9. Finally, compound 9 was hydrolyzed by aqueous lithium hydroxide to form compound 10 (FD-PA) containing a carboxylic acid group at yield of 85%.In the design and synthesis of hapten, keeping the maximal extent of the parent structural characteristics is crucial to the ultimate acquisition of antibody with high sensitive and selectivity. Since FD, a novel molecule that targets ryanodine receptors with skeleton phthalamides, was developed as a commercial insecticide, many studies were conducted to evaluate the structure modification on the three moieties of FD’s, namely (a) phthalic acid, (b) aliphatic amine, and (c) aromatic amine. The previous study showed that the modification in phthalic acid and aromatic amine had negative influence on FD insecticidal activity, whereas modification in the aliphatic amine terminal could retain or improve FD insecticidal activityWe conjugated hapten (FD-PA) with BSA and OVA via the active ester method as immunogen and coating antigen, respectively. The UV-vis spectra were used to monitor the conjugation of haptens and carrier proteins. As shown in Fig. Antisera collected from the mice after the fourth immunisation were screened against the coating antigen. The titre of the antibody was defined as the fold dilution with an absorbance of 1.0 in icELISA. The mouse with the highest titre and the best percentage inhibition in icELISA was used for further study. Three positive hybridoma cells were cloned by limiting dilution. A positive clone, designated as 4B3, was found to secret mAbs against artemisinin. Subsequently, 4B3 was cultivated and used to produce ascites.23. Regarding to the optimal combination of coating antigen and mAb, a checkerboard titration was performed in icELISA (see Supplementary Table −1) and mAb 4B3 (1.0 mg mL−1) were at 1:500 and 1:2000, respectively. Thus, the concentration of FD-PA-OVA as the coating antigen was fixed as 2 µg ml−1 and the concentration of the mAb 4B3 was fixed at 0.5 µg ml−1 in this study.To make icELISA work at optimal conditions, some influence parameters were optimized as belowmax), IC50, and Amax/IC50 ratio were used to assess the optimum condition. As shown in Fig. max/IC50 value at pH 8.4 reached the best with the lowest IC50 (28.4 µg L−1) compared to that at pH 6.5, 7.5 and 9.6, so pH 8.4 was selected as the optimal one. Regarding to the effect of ionic strength on established icELISA, as shown in Fig. −1) had a little effect on Amax values. However, the IC50 decreased with increasing concentrations of NaCl from 0 to 0.8 g L−1, and when the NaCl concentration was increased to 1.6 g L−1 from 0.8 g L−1, the change of IC50 was very small. Therefore, 0.8% NaCl was chosen as the optimal ionic strength for the assay buffer.The effect of pH values and NaCl concentrations on icELISA were studied, too. The maximum absorbance , and the working range between 4.06 and 103.59 µg L−1 .Under the optimal conditions obtained above, a typical inhibition curve see Fig.  was obta25. Meanwhile, the other FD analogues also showed no cross-reactivity with FD, indicating the validity of the novel haptens, and that the antibody was sufficiently specific for FD analysis.To evaluate the specificity of icELISA for mAb 4B3, the cross-reactivity of icELISA with FD, chlorantraniliprole, cyantraniliprole, and other FD analogues were measured  in the spectrogram confirmed the presence of FD  between icELISA and UPLC-MS/MS was 0.9984 , which was 5-fold below the EPA minimum MRL. Thus, the sensitivity of the developed assay is sufficient for screening residual FD in practical applications and it could be used for quick and reliable detection of FD residues in environmental and agricultural samples.According to the European Food Safety Authority (EFSA), the maximum residue limit (MRL) of FD in spinach and the minimum MRL in foods or fruits are 15 µg g50 value of 17.25 µg L−1 for FD and a working range of 4.06–103.59 µg L−1 based on 20–80% of inhibition. In addition, the fortified FD detected by icELISA in spinach, tap water, and soil samples had average recoveries of 86.9–112.3%. Moreover, the amount of residual FD in actual spinach and soil samples detected by icELISA was in agreement with that detected by UPLC-MS/MS (R2 > 0.99). In conclusion, the results displayed that this new immunoassay was suitable for the quick and convenient determination of residual FD in environmental and agricultural samples.In this study, FD haptens were designed and synthesised. Corresponding mAbs were also produced and used in icELISA development for FD detection. The established assay exhibited no cross-reactivity with insect ryanodine receptors-targeting chlorantraniliprole and cyantraniliprole, and showed no cross-reactivity with several FD analogues. Developed based on mAb 4B3, the icELISA method had an IC1H NMR, 13C NMR and HRMS. FD suspension concentrate  was purchased from the China National Agricultural Means of Production Group Corporation. Chlorantraniliprole and cyantraniliprole were purchased from Shanghai DuPont (China). Reagents purchased from Sigma-Aldrich  included the serum-free cell-freezing medium dimethylsulphoxide (DMSO), bovine serum albumin (BSA), ovalbumin (OVA), hypoxanthine, aminopterin, thymidine (HAT), penicillin, streptomycin, L-glutamine, complete and incomplete Freund’s adjuvant, polyoxyethylene sorbitan monolaurate (Tween-20), and N-hydroxysuccinimide (NHS). Polyethyleneglycol (PEG)-2000, o-phenylenediamine (OPD), N, N-dicyclohexylcarbodiimide (DCC), 3-iodophthalic anhydride, ethyl 3-mercaptopropanoate, 2,2-dimethylaziridine, 2-methyl-4-(perfluoropropan-2-yl)aniline and m-chloroperbenzoic acid (m-CPBA) were purchased from J & K Chemical . Goat anti-mouse IgG conjugated with horseradish peroxidase (IgG–HRP) was purchased from Jackson Immuno Research Laboratories . Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco BRL . Chromatography-grade acetonitrile was purchased from Thermo Fisher Scientific . All other reagents were obtained from Beijing Chemical Reagents Co. .FD was purchased from Nichino Shanghai Co., Ltd. FD analogues were synthesised in our previous work and characterised by using 2 incubator , automated plate washer (Wellwash 4 Mk2), and microplate reader (Multiskan MK3) were purchased from Thermo Fisher Scientific . A KH-500E ultrasonic cleaner was purchased from Kunshan Hechuang Ultrasonic Apparatus Co., Ltd. . The electric heating constant temperature incubator was purchased from Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd. .Cell culture plates and 96-well polystyrene microplates were purchased from Costar . The direct heat CO2HPO4, pH 5.5), substrate solution (4 mL 30% H2O2 added to 10 mL citrate-phosphate buffer containing 2 mg mL−1 OPD), and stop solution (2.0 M H2SO4). FD standard solutions of various concentrations were prepared by diluting the 1 mg mL−1 stock solution (FD powder dissolved in DMSO) with methanol. All reagents and solvents were of analytical grade. Deionised water used for diluting buffers and solutions was produced by using Millipore Water Purification System .The buffers and solutions included coating buffer , phosphate-buffered saline (PBS) , PBS with 0.1% (v/v) Tween-20 (PBST), PBST containing 0.5% (w/v) gelatin (PBSTG), citrate-phosphate buffer  were obtained from the China Institute of Veterinary Drug Control . For cultivation of myeloma and hybridoma cells, DMEM containing 10–20% (v/v) FBS was supplemented with 0.2 M glutamine, 50 000 units L28 according to the synthetic route illustrated in Fig. Hapten was synthesised1  and compound 2  were added into 100 mL 1, 2-dimethoxyethane in a 250 mL flask. The mixture was heated at 85 °C and stirred for 3 h. The solvent was vacuum-evaporated, whereas the residue was then purified by silica gel column chromatography  to obtain compound 3 in light yellow liquid form . 1H NMR : δ 4.07 , 2.75 , 2.56–2.48 , 1.44 , 1.19 , 1.09 .Compound 3  and triethylamine  in dichloromethane (80 mL) was slowly added to a compound 4 solution  in dichloromethane (80 mL) at 25 °C. The reaction mixture was stirred for 5 h, poured into water (100 mL), and acidified with diluted hydrochloric acid  to pH 2–3. The aqueous layer was extracted three times with dichloromethane (30 mL) and dried over anhydrous sodium sulphate. The solvent was vacuum-evaporated and the residue was used in the subsequent reaction without further purification.A mixture of compound 5 residue in 60 mL anhydrous toluene was cooled with an ice bath. Trifluoroacetic anhydride  was added dropwise to the mixture with stirring. Following completion of the addition, the mixture was further stirred for 2 h. The progress of the reaction was monitored by using TLC (petroleum ether/ethyl acetate = 1:1). After the reaction was complete, the solvent was vacuum-evaporated, whereas the residue was used in the subsequent reaction without further purification.Slurry of compound 7  was added to compound 6 residue solution in dichloromethane (30 mL). Two drops of trifluoroacetic acid was added to the mixture, which was subsequently stirred for 1 h at 25 °C. The progress of the reaction was monitored by using TLC . Following completion of the reaction, the solution was washed with diluted hydrochloric acid , aqueous sodium carbonate solution , and water (30 mL), respectively, and dried over anhydrous sodium sulphate. The solvent was vacuum-evaporated, whereas the residue was purified by silica gel column chromatography  to obtain compound 8 in white solid form . 1H NMR : δ 9.57 , 8.15 , 8.03 , 8.01–7.98 , 7.68 , 7.53 , 7.26 , 4.06 , 2.93 , 2.62 , 2.47 , 2.38 , 1.32 , 1.18 . 13C NMR : δ 171.30, 167.67, 165.75, 141.59, 140.79, 139.63, 136.14, 131.98, 130.12, 127.49, 127.32, 127.18, 124.01, 123.50, 123.36, 121.30, 121.02, 95.21, 89.93, 89.50, 60.06, 54.72, 42.05, 34.56, 28.63, 25.44, 18.03, 14.08. HRMS (m/z): [M-H]− calcd. for C27H28F7IN2O4S, 735.0630; found, 735.0633.Compound 8 solution  in dichloromethane (60 mL). The reaction was carried out at 25 °C and the progress of the reaction was monitored by using TLC (petroleum ether/ethyl acetate = 1: 2). Following completion of the reaction, the mixture was poured into water and extracted with chloroform. The organic layer was washed with an aqueous sodium hydrosulphite solution  and an aqueous sodium carbonate solution , respectively, and dried over anhydrous sodium sulphate. The solvent was vacuum-evaporated, whereas the residue was purified by silica gel column chromatography  to obtain compound 9 in white solid form . 1H NMR : δ 9.78 , 8.44 , 8.03 , 7.89 , 7.74 , 7.53 , 7.29 , 4.10 , 3.70 , 3.39–3.34 , 2.68 , 2.39 , 1.54 , 1.19 . 13C NMR : δ 170.16, 167.81, 165.71, 141.50, 140.90, 139.52, 136.09, 133.00, 130.26, 127.42, 127.25, 125.00, 123.49, 123.36, 121.71, 121.44, 120.43 , 95.45, 91.26 , 79.28, 60.68, 59.03, 52.66, 50.46, 26.72, 26.31, 18.09, 14.01. HRMS (m/z): [M-H]− calcd. for C27H28F7IN2O6S, 767.0528; found, 767.0533.M-Chloroperoxybenzoic acid  was added to compound 9 solution  in THF (40 mL) at 0 °C. The reaction mixture was stirred for 12 h. The solvent was vacuum-evaporated and the residue was acidified with diluted hydrochloric acid . The aqueous layer was extracted three times with dichloromethane (20 mL) and dried over anhydrous sodium sulphate. The solvent was vacuum-evaporated and the residue was purified by silica gel column chromatography (ethyl acetate: methanol = 20:1) to obtain compound 10 in white solid form . 1H NMR : δ 9.79 , 8.43 , 8.00 , 7.85 , 7.71 , 7.51 , 7.26 , 3.67 , 3.30 , 2.59 , 2.36 , 1.51 . 13C NMR : δ 171.65, 167.78, 165.71, 141.49, 140.87, 139.51, 136.07, 132.99, 130.26, 127.41, 127.27, 124.99, 123.53, 123.40, 121.70, 95.47, 79.30, 58.97, 52.67, 50.73, 26.82, 26.29, 18.10. HRMS (m/z): [M-H]− calcd. for C25H24F7IN2O6S, 739.0215; found, 739.0212.LiOH  was added dropwise to compound 29. The mixture was dialysed against 3 L PBS for 3 days with three solvent changes per day, and stored at -20 °C. The conjugates of FD−OVA and FD−BSA were used as a coating antigen and immunogen, respectively. By using UV-vis spectroscopy, the final structures of the protein-conjugated haptens were confirmed30.Through the carbodiimide method, hapten was conjugated with BSA and OVA to produce immunogen (FD–BSA) and coating antigen (FD–OVA)31. Five female Balb/c mice aged seven weeks old were immunised with subcutaneous injection of 100 µg FD-OVA dissolved in 0.1 mL PBS and 0.1 mL complete Freund’s adjuvant. Incomplete Freund’s adjuvant was used in the two subsequent FD-OVA injections at 2-week intervals. Two weeks after the third injection, the mouse with the best immune performance was injected with 100 µg FD-BSA in 100 µL PBS. Three days later, spleen cells were collected from the mouse, mixed with the SP2/0-Ag14 myeloma cells at a ratio of 10:1, and fused with PEG-2000. Next, the cells were cultured in complete medium . The hybridoma cells were selectively cultured in complete medium supplemented with HAT for 7 days. Next, the supernatant was subjected to icELISA, in which positive hybridoma cells were cloned by limiting dilution. The clones were further selected by icELISA. The clone with high antibody titre and good sensitivity in the culture supernatant was designated as 4B3 and cultured in mice for production of mAb in ascites. Anti-FD mAbs were purified from ascite fluid by ammonium sulphate precipitation.A mAb against artemisinin was prepared according to the procedures described previously by Zhao in 2011A checkerboard titration procedure was used to optimize coating antigen and anti-FD mAb interaction. All plates were washed four times with washing buffer (PBST) and all reactions were incubated in a constant-temperature incubator at 37 °C. First, the microplate was coated with FD–PA-OVA (100 µL) in coating buffer at 37 °C for 3 h. After the microplate was washed, standard sample or analytes in PBSTG (50 µL) was added to each microplate well, followed by addition of mAb in PBSTG (50 µL). Following incubation for 0.5 h, the microplate was washed with PBST. Each well was incubated with goat anti-mouse IgG–HRP (1 mg/L) diluted in PBSTG (100 µL) at a ratio of 1: 1 000 for 0.5 h. Next, the plate was washed again and substrate solution (100 µL) was added to each well. Finally, 15 min later, the reaction was terminated by adding stop solution  to each well, and the absorbance was measured by using a microplate reader at 492 nm.Experimental parameters, including buffer pH and ionic strength, were studied to improve the sensitivity of the immunoassay. The influences of pH values were evaluated using different PBSTG solutions with pH values . The effects of ionic strength were determined using PBSTG buffer at different NaCl concentration . Standard curves were obtained using Origin 2018 software.The mAb specificity of icELISA was evaluated by cross-reactivity (CR) with a set of FD structural analogues. The relative CR was calculated by the following formula:−1 on the spinach cultivated in a greenhouse and an outdoor experimental field of China Agricultural University. On the third and fifth days after spraying, spinach samples and soil samples were collected. Meanwhile, three spinach samples were purchased from different supermarkets and three soil samples were separately collected from Xiaotangshan (Beijing), Wuqiao (Hebei), and Jiaozuo (Henan). All samples were stored at −40 °C until use.FD  was sprayed according to label directions at dosage of 250 g a.i.hag for 5 min. The organic layer was dehydrated by filtration through anhydrous sodium sulphate. The same extraction steps were repeated three times. All extracts were combined and dried by a gentle stream of nitrogen gas. The residues were dissolved in PBSTG (1.0 mL) and were diluted 90- or 45-folds for use in icELISA. Each analysis was conducted in triplicate.Chopped and homogenised spinach leaves (1 g) and grinded soil (1 g) were placed into 10 mL polytetrafluoroethylene centrifuge tubes. Following addition of 3 mL acetonitrile, the mixture was allowed to stand at 4 °C for 3 h. Next, the mixture was stirred on a vortex shaker for 3 min, monitored by ultrasonic extraction for 10 min, and centrifuged at 4 000 × −1. Tap water was spiked at concentrations of 20, 30, 40, 60, 80 ng mL−1. The grinded soil (1 g) was spiked at concentrations of 500, 1 000, 1 500, 2 000 and 3 000 ng g−1. FD in spiked samples were analysed with the established icELISA.Spinach leaves (1 g) was cut into pieces and then spiked with FD at concentrations of 1 000, 1 500, 2 000, 3 000 and 4 000 ng g32. The analysis was carried out by a Thermo Fisher TSQ Quantum Access MAX system . Chromatographic separation was performed by a DionexTM UltiMateTM 3000 Open Sampler XRS UHPLC system with a Hypersil GOLD C18 column . The mobile phase was acetonitrile/water  at a flow rate of 0.3 mL min−1. The injection volume was 5 µL. A triple quadrupole mass spectrometer with an electrospray ionisation source was applied for analysis under the following conditions: spray voltage of 3000 V, capillary temperature of 350 °C, vaporiser temperature of 200 °C, sheath gas pressure of 35 psi, and aux valve flow of 15 arb. The collision gas was Argon (99.999%) with pressure at 1.5 mTorr. All analytes were ionized in negative mode. The UPLC-MS/MS results were compared with those of icELISA to evaluate the reliability of the established immunoassay.FD standards, as well as spinach and soil samples were analysed with UPLC-MS/MS according to the procedure described by Caboni with slight modificationAll procedures performed in studies involving animals were in strict accordance to the standards described in the “Guide for the Care and Use of Laboratory Animals” . Animal studies were approved by the independent Animal Ethical Committee at China Agricultural University.Supplementary information


Pseudomonas putida MCCC 1A00316 was originally isolated from an Antarctic soil and has demonstrated potential nematicidal activity. Thus, it has promising applications for the biological control of Meloidogyne incognita. The larval mortality and egg-hatching inhibition rates of M. incognita will increase with the rising concentration of culture filtrates of P. putida MCCC 1A00316 and the duration of exposure. Thus, this study aimed to separate, purify, and identify nematicidal compounds from P. putida MCCC 1A00316 and to validate their anti-M. incognita activities. Compounds were purified through silica gel column chromatography and thin-layer chromatography combined with high-performance liquid chromatography (HPLC). Structural identification was conducted through liquid chromatography time-of-flight mass spectrometry, 1H nuclear magnetic resonance (NMR) spectroscopy, 13C-NMR, and Marfey’s method. The isolated compounds were identified as cyclo(l-Pro–l-Leu) on the basis of the results of the above analyses and previously reported data. The effects of various concentrations of cyclo(l-Pro–l-Leu) on the mortality rates of second-stage juveniles (J2) of M. incognita were investigated. Results showed that HPLC-purified cyclo(l-Pro–l-Leu) displayed nematicidal activities. The mortality rate of M. incognita J2 reached 84.3% after 72 h of exposure to 67.5 mg/L cyclo(l-Pro–l-Leu). The lowest egg-hatching rate (9.74%) was observed after 8 days of incubation with 2000 mg/L cyclo(l-Pro–l-Leu). An egg-hatching rate of 53.11% was obtained under the control treatment (sterile distilled water). However, cyclo(l-Pro–l-Leu) did not elicit chemotaxis activity to M. incognita. This is the first work to investigate the anti-M. incognita characteristics of cyclo(l-Pro–l-Leu). Meloidogyne incognita, is one of the most important phytoparasitic nematode species; infections by this species induce root galling, prevent plant growth, and cause nutrient deficiency [M. incognita infections result in yield losses of 25.6% [Phytoparasitic nematodes are ubiquitous microscopic soil pests that cause severe crop losses by parasitizing plant roots . The rooficiency ,3. M. inof 25.6% . The gloof 25.6% ,8.The most common methods for the control of root-knot nematodes include the use of resistant cultivars, crop rotation, and chemical nematicidal agents. Nevertheless, resistance to root-knot nematodes is unstable and often reduces yields , and croPasteuria penetrans is mainly based on the parasitization of target nematodes [Bacillus cereus S2 has demonstrated intense nematicidal activity against M. incognita [Chaetomium globosum, can adversely affect the survival rates of juvenile M. incognita with an LC50 of 77 μg/mL [Biocontrol agents have attracted considerable attention in recent years because of their safety to the environment, humans, especially food and vegetable security and animals . Variousematodes . Sphingoncognita . Chaetog77 μg/mL . Pseudomonas putida MCCC 1A00316 produces various nematicidal factors, such as protein alkaline metalloproteinase AprA; the secondary metabolites hydrogen cyanide and cyclo(l-Ile–l-Pro); and several volatile organic compounds. Guo et al. reported that 96 h of exposure to 500 mg/L cyclo(l-Ile–l-Pro) from P. putida resulted in a mortality rate of 50% [Z)-hexen-1-ol acetate, 2-undecanone, and 1-(ethenyloxy)octadecane, presented nematicidal activity against second-stage juveniles (J2) of M. incognita and inhibited the egg hatching of M. incognita when applied as a direct-contact nematocide and as a fumigant [fumigant . NumerouPseudomonas species are ubiquitous in nature and produce a large number of secondary metabolites with activities against important plant diseases [P. putida MCCC 1A00316 from an Antarctic soil showed nematicidal potential and thus has promising applications for the biological control of M. incognita. Therefore, we separated and purified potential nematicidal compounds from P. putida MCCC 1A00316. We isolated and identified one nematicidal compound and characterized its anti-M. incognita activity. diseases . In the M. incognita was affected by exposure to the culture filtrates of MCCC 1A00316 in a dose-dependent manner. The emergence of J2 was inversely related to filtrate concentration and exposure duration  provided a retention factor value (Rf) of 0.5 (p < 0.001) (The fermentation supernatant was extracted by using ) of 0.5  and exhi< 0.001) A. M. incognita J2. Exposure to F1-1 at the concentration of 5 and 25 mg/mL resulted in >70% and 100% J2 mortality rates, respectively , which were then screened through TLC (dichloromethane:methanol 15:1 and iodine spraying)  and agaiectively B. F1-1 wectively . M. incognita J2. Results revealed that P2, P3, and P4 had higher nematicidal activity than P1 and P5  δ:6.46 , 4.06 , 3.96 , 3.47 , 3.39 , 2.21 , 2.13 , 1.82-1.92 , 1.39 , 1.23 , 1.02 , and 0.89 . 13C-NMR  δ: 170.8 (C-1), 165.6 (C-1′), 60.4 (C-2), 59.0 (C-2′), 45.2 (C-5), 35.9 (C-3′), 28.7 (C-3), 24.5 (C- 4′), 22.6 (C-4), 15.2 (C-6′), and 12.1 (C-5′). Thus, P3 was identified as cyclo(L-Pro–L-Ile), which was originally reported by Guo et al. [The peak data of P3 was consistent with those of cyclo(Pro–Ile). The data were as follows: 1H-NMR  δ:6.33 , 4.12 , 4.01 , 3.42 , 2.19 , 1.82-1.91 , 1.42 , and 0.94 , 0.92 . 13C-NMR  δ: 171.0 (C-1), 167.0 (C-1′), 59.3 (C-2), 53.4 (C-2′), 45.5 (C-5), 38.7 (C-3′), 28.2 (C-3), 24.8 (C- 4′), 23.0 (C-5′), 22.9 (C-4), and 21.3 (C-6′).The peak data of P4 was consistent with that of cyclo(Pro–Leu)  and werel-Pro, d-Pro, l-Leu, and d-Leu were 11.974, 13.099, 22.403, and 26.963 min, respectively, whereas those of the FDAA derivatives of hydrolysates of P4 were 11.932 and 22.410 min  displayed strong nematicidal activities. J2 mortality reached 42.85% and 63.64% after 48 h of exposure to 67.5 and 135 mg/L cyclo(l-Pro–l-Leu), respectively, and reached 84.30% and 100% after 72 h of exposure to 67.5 and 135 mg/L cyclo(l-Pro–l-Leu), respectively (l-Pro–l-Leu) concentration increased (l-Pro–l-Leu). An egg-hatching rate of 53.11% was observed under treatment with SDW. Therefore, an appropriate concentration of cyclo(l-Pro–l-Leu) could be used for the effective control of nematodes by drastically reducing J2 mortality and egg-hatching rates.The nematicidal effects of the two cyclodipetides from MCCC 1A00316 isolates were evaluated through a direct-contact bioassay. The HPLC-purified cyclo(ectively . In addincreased . Egg-hatl-Pro–l-Leu) are presented in l-Pro–l-Leu) had C.I. values of 0.035 and 0.021 (0 < C.I. <1), respectively, and that 1000 and 500 mg/L cyclo(l-Pro–l-Leu) had C.I. values of −0.014 and −0.048 (−1 < C.I. <0), respectively. Analyzing these data through a Wilcoxon rank–sum test, however, revealed that the number of attracted or repulsed nematodes per side did not show statistically significant differences. These data suggest that different concentrations of cyclo(l-Pro–l-Leu) did not induce chemotactic activity.The chemotaxis responses of nematodes to cyclo by using various chromatographic techniques, NMR analysis, and Marfey’s method. Cyclo(l-Pro–l-Leu) is a member of the diketopiperazine class of molecules, which includes the smallest cyclic peptides. This compound is synthesized through the head-to-tail folding of linear dipeptides and exhibits an extensive range of biological properties, including antibacterial, antifungal, and antiviral effects [l-Pro–l-Leu) from Streptomyces sp. has been identified as an antifungal agent with activity against the rice blast fungus Pyricularia oryzae and a range of other fungi [l-Pro–l-Leu) produced by the bacterium Achromobacter xylosoxidans remarkably inhibits the production of highly toxic, carcinogenic, and teratogenic aflatoxins by the fungus Aspergillus parasiticus [l-Pro–l-Leu) was active against 12 vancomycin-resistant enterococci strains and possesses antileukemic activity [l-Pro–l-Leu) produced by P. putida WCS358 could be used as biosensors for quorum sensing [d-Pro-l-Leu) purified and identified from Bacillus amyloliquefaciens Y1 also has nematicidal activity against M. incognita J2 and egg hatching inhibition activity, but the nematicidal activity of cyclo(d-Pro-l-Leu) was significantly lower than that of cyclo(l-Pro–l-Leu) in this study [l-Pro–l-Leu) isolated from P. putida MCCC 1A00316 has nematicidal and egg-hatching inhibition activities. It was previously noted that nematode mortality rate reached 46.19% after 72 h of treatment with 500 mg/L cyclo(l-Ile–l-Pro) in our former study [l-Pro–l-Leu). However, different concentrations of cyclo(l-Pro–l-Leu) failed to elicit chemotactic activity from nematodes. Therefore, it was confirmed that P. putida MCCC 1A00316 and its metabolites cyclic dipeptides displayed potential as biocontrol agents against nematodes.We isolated and purified a nematicidal compound from the crude extract of  effects . 2,5-diker fungi . Cyclo(lasiticus . It was activity . Moreove sensing . Interesis study . To our M. incognita eggs were collected from the roots of infested tomato plants (Solanum lycopersicum L.) that had been previously infected with nematodes. The plants were maintained under greenhouse conditions at 23–26 °C and relative humidity of 40–60%. The plants were watered manually once a day. After 45 days, the plants were uprooted, and their roots were rinsed free of soil with tap water. Egg masses were transferred to a bottle by using a dissecting needle and shaken with 1% NaOCl solution for 3 min. The egg mass suspension was then passed through a series of filters with pore sizes of 74, 45, and 25 μm. Sterilized eggs that were retained on the 25 μm filter were sprayed with sterile distilled water (SDW) for collection. J2 were obtained under sterile conditions by using a modified Baermann funnel method [l method ,29.P. putida on the basis of 16S rDNA sequence homology and physiological and biochemical characteristics [2, 0.75 g of MgSO4, 1 g of CaCl2, 0.55 g of KCl, 0.16 g of NaHCO3, 0.08 g of KBr, 34 mg of SrCl2, 22 mg of H3BO3, 4 mg of Na2SiO3, 2.4 mg of NaF, 8 mg of Na2HPO4, 0.5 mg of MnCl2, 0.5 mg of CuSO4, and 10 mg of ZnSO4. The pH and temperature of the seed medium were maintained at 7.6–7.8 and 28 °C, respectively. The medium was shaken at 180 rpm. After 18 h, 1% seed liquid (2.5 mL) was transferred into a 500 mL flask containing 250 mL of 2216E medium. The flask was then shaken for 48 h at 28 °C at 180 rpm. The fermentation culture was centrifuged at 8000 r/min for 10 min at 4 °C to obtain the supernatant. Filtrates were passed through a 0.22 μm filter to remove bacterial cells and were used at the original concentration or to prepare 1×, 3×, 5×, 6×, 7×, 9× dilutions with SDW.Strain MCCC 1A00316 was isolated from Antarctic soil and identified as eristics . The streristics . The 221M. incognita egg suspensions (10 μL) were prepared (180 eggs/well) as described, transferred to separate wells of a 24-well tissue culture plate, and mixed with 500 μL of culture filtrates with various concentrations. Next, 4 μL (10 mg/mL) of chloramphenicol was added to each well to inhibit bacterial growth. Then, 1× and 3×, 5× and 6×, 7× and 9× dilutions of culture filtrates were transferred to different 24-well tissue culture plates. 2216E medium and SDW were used as controls. Experiments were performed three times, and every treatment was replicated three times. Plate lids were sealed with parafilm, and the plates were maintained at 28 °C. An inverted microscope  was used to observe egg-hatching rates after 2, 4, 6, 8, and 10 days of incubation. M. incognita J2 were suspended in SDW. The J2 suspensions (3 μL) were transferred to the wells of a 96-well tissue culture plate (50 J2/well) containing 200 μL of different dilutions of culture filtrates. A total of 3 μL (10 mg/mL) of chlorampheniol was added to the wells to inhibit bacterial growth. Next, 1× and 3×, 5× and 6×, 7× and 9× dilutions of culture filtrates were placed in different 96-well tissue culture plates. 2216E medium and SDW were used as controls. The experiments were performed three times, and every treatment was replicated three times. Plate lids were sealed with parafilm, and the plates were maintained at 28 °C. Juveniles were observed with the aid of an inverted microscope (XDS-1B COIC) under 10× and 40× after 24, 48, and 72 h and classified as motile or immotile/paralyzed.P. putida MCCC 1A00316 was mixed with equal volumes (250 mL) of n-butyl alcohol through 3 min of shaking in a separatory funnel. The extracted organic phases were evaporated to dryness at 58 °C by rotary evaporation, dissolved with 2 mL of methanol, and filtered using the funnel filter paper method. The procedure used to isolate pure compounds from strain MCCC 1A00316 is illustrated in v/v). Bands were visualized through spraying with iodine. Similar fractions were combined and tested against J2. The fraction that showed high nematicidal activity was loaded on a small silica gel column (32 mm × 26 mm × 305 mm) containing 45 g of silica gel (60–80 mesh), eluted with various ratios  of dichloromethane:methanol, screened through TLC (dichloromethane:methanol 15:1 and spraying with iodine), and tested against J2. Filtrate dissolved in methanol was mixed with a small quantity of silica gel (200–300 mesh) and then evaporated to dryness at 58 °C. The dry powder was eluted through a big silica gel column (60 mm × 53 mm × 457 mm) containing 170 g of silica gel (200–300 mesh). Elution was performed by using 400 mL of ethyl acetate:methanol at varying ratios . Fractions were monitored through thin-layer chromatography (TLC) on silica gel plates  with ethyl acetate:methanol  was used as the mobile phase. A variable-wavelength recorder set at 210 nm was applied to detect the compounds eluted from the column at a flow rate of 3 mL/min [The fraction exhibiting the most intense nematicidal activity was freeze-dried and then dissolved in ultrapure water. The dissolved fraction was filtered through a 0.22 μm filter. Next, 100 μL samples were loaded for high-performance liquid chromatography (HPLC) analysis by injection onto an semi-preparative chromatographic column . Acetonitrile:water  at a flow rate of 0.3 mL/min. The HPLC eluate was introduced into a mass spectrometer via an ESI interface at a spray voltage of 4.0 kV. The mass spectrometer was operated in positive-ion mode with a capillary temperature of 325 °C and a capillary voltage of 130 V. Mass spectra were acquired by scanning the mass range from m/z 100 to m/z 1700.The three compounds with intense nematicidal activity were analyzed through liquid chromatography time-of-flight mass spectrometry (Agilent Technologies 6540UHD Accurate-Mass Q-TOF LC-MS). Next, 1 μL samples were analyzed via reverse-phase HPLC  with a gradient of acetonitrile:water  by using C2D3N as the solvent unless otherwise indicated.The structures of the compounds were determined by using a DRX 600 NMR instrument  equipped with a 5 mm microprobe. Approximately 5 mg of the purified compound was dissolved in acetonitrile-d3 (C2D3N) and then subjected to spectral analysis. NMR spectra were recorded on the instrument, which was operated at room temperature and at 600 and 125 MHz for 2O (100 μL), transferred to a 1 mL reaction vial, and treated with a 2% solution of FDAA (200 μL) in acetone and then with 1.0 M NaHCO3 (40 μL). The reaction mixture was heated at 45 °C for 1 h, cooled to room temperature, and acidified with 2.0 M HCl (20 μL). In a similar fashion, standard d- and l-amino acids were derivatized separately. The derivatives of the hydrolysates and standard amino acids were subjected to HPLC analysis  at 30 °C with the following gradient program: solvent A, water + 0.2% TFA; solvent B, MeCN; linear gradient 0 min 25% B, 40 min 60% B, 45 min 100% B; and UV detection at 340 nm [A solution of three different compounds (1.5 mg) in 6 M HCl (1 mL) was heated to 120 °C for 24 h. The solution was then evaporated to dryness. The residue was redissolved in Ht 340 nm .M. incognita were suspended in SDW. J2 suspensions (3 μL) were added to the wells of a 96-well tissue culture plate (50 J2/well) containing 100 μL of cyclodipetide solutions with different concentrations. SDW was used as the control. The experiments were performed three times, and every treatment was replicated three times. Plate lids were sealed with Parafilm. The plates were maintained at 28 °C. Juveniles were observed with the aid of an inverted microscope (XDS-1B COIC) under 10× and 40× magnification after 24, 48, and 72 h of exposure and categorized as motile or immotile/paralyzed.The hatched J2 of M. incognita was examined in a 96-well microtiter plate containing 60 eggs. Various concentrations of the purified compounds in SDW were added to each well to the final concentrations of 125, 500, 1000, and 2000 mg/L. SDW was used as the control. The number of juveniles that hatched from eggs at 2, 5, and 8 days of incubation was counted under an inverted microscope.The influence of the purified compound on the egg-hatching rates of l-Pro–l-Leu) was placed on one side (area A) of a 35 mm Petri dishes containing 2% water agar. A filter paper immersed in SDW was placed on the the opposite side of the plate (area B) as the control. Subsequently, 100 J2 M. incognita were added to the center (area D) of the Petri dish. The dish was then incubated in a dark cabinet at 20 °C for 8 h. The numbers of M. incognita J2 in areas A and B were then counted under a dissecting microscope for the calculation of the chemotaxis index (C.I.) [The chemotaxis assay was performed in accordance with the method of Zhai et al. . A 5 mm x (C.I.) , which iM. incognita J2 was considered attracted to the tested sample if 0 < C.I. < 1, repelled if −1< C.I. < 0, and unaffected if C.I. = 0. Experiments were performed in triplicate, and treatments were replicated three times.M. incognita in in vitro bioassays were corrected by Abbott’s formula. Data from all assays were analyzed through by one-way analysis of variance with SPSS 20 . Data from the chemotaxis assay were analyzed through a homogeneity test of variance. Paired Student’s t-test was performed if variance was homogeneous (p ≤ 0.05). Otherwise, the Wilcoxon rank–sum test was used. Duncan’s multiple range test was employed to test for significant differences among the anti-M. incognita activities of the culture filtrates of strain MCCC 1A00316. Statistical comparisons between two experimental values were performed via t-test, and significant differences were determined in accordance with a threshold of * p < 0.05; ** p < 0.01; *** p < 0.001.The mortality rates of


Tenofovir alafenamide (TAF) has similar efficacy compared to tenofovir disoproxil fumarate (TDF), but a less favorable effect on lipids. Aim of this retrospective multicentre study was to evaluate the impact on lipids of switching from rilpivirine (RPV)/ emtricitabine (FTC)/TDF to RPV/FTC/TAF in a cohort of HIV-1 infected patients. Total cholesterol (TC), high density lipoproteins (HDL) and low density lipoproteins (LDL) were compared at the moment of the switch and at the first following evaluation, by using paired t-test. Overall, 573 patients were considered, 99% with HIV-RNA <50 copies/ml, with mean age of 49.7 (±0.4) years and median 13.4 (6.9–22.5) years of HIV infection. In the study population with available data , mean TC changed from 173 ±1.7 to 188 ±1.8 mg/dl; mean HDL from 46 ±0.7 to 51± 0.7 mg/dl; mean LDL from 111 ±1.5 to 120 ±1.8 mg/dl . Neither LDL/HDL nor TC/HDL ratio changed significantly, with LDL/HDL from 2.6 ±0.5 to 2.5 ±0.5 (p = 0.12) and TC/HDL from 4.0 ±0.6 to 3.9 ±0.6 (p = 0.11). In patients with baseline diagnosis of hypercholesterolemia , there was no significant change in TC  or LDL , while HDL increased from 51 ±1.6 to 55 ±1.7 mg/dl (p<0.0001) and both LDL/HDL and TC/HDL ratio decreased significantly, from 3.2±0.1 to 3.0 ±0.1 (p = 0.025) and from 4.7±0.1 to 4.4 ±0.1 (p = 0.004). In this real life study, a slight increase in lipids was found after switching from RPV/FTC/TDF to RPV/FTC/TAF, but these results were not confirmed in people with hypercholesterolemia, in which lipids did not change and LDL/HDL and TC/HDL ratio decreased. Tenofovir disoproxil fumarate (TDF) has demonstrated high efficacy in all stages of HIV infection and in different drug combination regimens . However3.This is a retrospective, observational, multicentre study, conducted in two Infectious Disease Centres in Northern Italy, ASST Papa Giovanni XXIII Hospital in Bergamo and Policlinico S. Martino Hospital in Genoa. Data for Policlinico S. Martino were retrieved from MedInfo, an online database for anonymous and automatic data collection . Data frBaseline was considered the day of the switch or the nearest blood exam preceding that date. The second timepoint was the first available evaluation immediately successive to the switch. Repeated evaluation performed after less than 6 weeks were excluded. Missing LDL were calculated with Friedewald equation . HIV-1 R3).Continue variables were expressed as mean  or median , while categorical variables were expressed as frequencies. At baseline, a comparison between patients with or without HC was performed using unpaired two-samples t-test for variables with normal distribution  and by using Mann-Whitney U test for skewed variables . Categorical variable were compared at baseline between non-HC and HC patients by Chi squared test  or by Fisher’s exacts test  were subsequently compared in the same subjects at baseline and follow up through paired t-test, while data with skewed distribution  were compared using paired samples Wilcoxon test. Changes in TC, LDL, HDL, LDL/HDL and TC/HDL were compared in patients with and without HC by using unpaired two-samples t-test, while changes of TG were compared in patients with or without HT by using Mann-Whitney U test.A linear regression model was used to evaluate correlations between baseline values of TC, LDL, HDL and TG and their changes at follow up, that were expressed with regression coefficients, R, and 95% Confidence Intervals, 95%CI. Chi squared test was used to compare the frequencies of HC and HT before and after the switch.All data were collected from December 2017 to April 2018. All patients followed in both Policlinico S. Martino Hospital and ASST Papa Giovanni XXIII Hospital signed an informed consent form in which they agreed use of their clinical data, in anonymous form, for scientific purposes. The use of the Ligurian HIV Network database for scientific purposes was approved by the Ligurian Ethics Committee . The study has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments and in accordance with Italian national laws.3, and HIV-RNA was < 50 copies/ml in 99.5%. The general characteristics of the study population are given in Overall, 573 patients had switched from RPV/FTC/TDF to RPV/FTC/TAF, of whom 145 (25%) were females. Patients had mean age of 49.7 (±0.4) years and a history of median 13.4 (6.9–22.5) years of HIV infection. Risk factors for HIV acquisition were sexual intercourse in 374 (65%) patients , intravenous drug use in 181 (32%) and other/unknown factors in 18 (3%). In the whole population who switched from RPV/FTC/TDF to RPV/FTC/TAF, baseline mean CD4 were 777 ±13.7 cells/mm3, p = 0.4 and CD8 from 920 ±19.0 to 916 ±20.0 cells/mm3, p = 0.6, respectively), mean CD4/CD8 ratio slightly increased . When limiting the analysis to patients with baseline CD4<500 cells/mm3 , both CD4 and CD8 increased significantly from 368 ±9.2 to 412 ±12.8 cells/mm3 (p<0.0001) and, from 747 ±35.5 to 787 ± 39.5 cells/mm3, p = 0.037, respectively. CD4/CD8 ratio increased slightly, but significantly, in this group, from mean 0.6 ±0.03 to 0.7 ±0.04, p = 0.038.After a median follow up of 12 (8–24) weeks, although mean CD4 and CD8 remained stable, (CD4 from 777 ±13.7 to 787 ±14.5 cells/mmThree patients (0.5%) with baseline HIV-RNA >50 copies/mL  achieved HIV-RNA < 50 copies/mL at follow up. Conversely, nine patients (1.5%) with baseline undetectable viremia had HIV-RNA >50 copies/mL at follow up .In the same time frame the serum creatinine levels slightly but significantly decreased, from 0.96 (± 0.01) to 0.92 (± 0.01) mg/dL, p<0.0001.The evaluation of TC at both baseline and follow up was available for 431 out of the 573 patients (75%), HDL for 426 (74%), LDL for 423 (74%), TG for 430 (75%). At baseline, 45 patients were on statin therapy, and no patient added or stopped a lipid-lowering agent during the observation period. All lipids increased significantly : mean TCDuring the study period, the proportions of subjects with HC and HT increased, the first one from 20.1 to 36.4% (p<0.0001) and the second from 11.4 to 13.0% (p<0.0001).Patients who developed HC had baseline TC levels slightly higher compared to patients who maintained normal TC levels .When analyzing separately patients with baseline diagnosis of hypercholesterolemia , the change in TC and LDL resulted significantly different from that of other patients , p<0.0001 for both, Both LDL/HDL ratio  and TC/HDL ratio  reduced significantly in patients with HC, and the reduction of both was more marked than that seen in other patients , while LDL/HDL ratio  and TC/HDL ratio  did not change significantly.The variation in TC resulted inversely correlated to baseline TC value  did not experience significant change in TG levels during the study period, from median 238.5 (215–310) mg/dl to 212 (163–292) mg/dl, p = 0.088. TG trend was opposite to that seen in non-HT patents, with median -25  mg/dl in HT vs. + 0  mg/dl in non-HT patients Click here for additional data file.


Blastobotrys (Arxula) adeninivorans (Ba) encodes 88 glycoside hydrolases (GHs) including two α-glucosidases of GH13 family. One of those, the rna_ARAD1D20130g-encoded protein  was overexpressed in Escherichia coli, purified and characterized. We showed that maltose, other maltose-like substrates  and sucrose were hydrolyzed by BaAG2, whereas isomaltose and isomaltose-like substrates  were not, confirming that BaAG2 is a maltase. BaAG2 was competitively inhibited by a diabetes drug acarbose (Ki = 0.8 µM) and Tris (Ki = 70.5 µM). BaAG2 was competitively inhibited also by isomaltose-like sugars and a hydrolysis product—glucose. At high maltose concentrations, BaAG2 exhibited transglycosylating ability producing potentially prebiotic di- and trisaccharides. Atypically for yeast maltases, a low but clearly recordable exo-hydrolytic activity on amylose, amylopectin and glycogen was detected. Saccharomyces cerevisiae maltase MAL62, studied for comparison, had only minimal ability to hydrolyze these polymers, and its transglycosylating activity was about three times lower compared to BaAG2. Sequence identity of BaAG2 with other maltases was only moderate being the highest (51%) with the maltase MalT of Aspergillus oryzae.Genome of an early-diverged yeast  Blastobotrys adeninivorans (syn. Arxula adeninivorans) belongs to a basal clade of Saccharomycotina subphylum and diverged in the evolution of fungi long before Saccharomyces  and ScMAL62  were computed at ExPASy Proteomics Server (http://expasy.org). Purified proteins were maintained in 100 mM K-phosphate buffer (pH 6.5) at 4 °C.The pURI3-BaAG2Cter and pURI-ScMAL62Cter containing the um as in  was usedribed in . ProteinpNPG was assayed as in [p-nitrophenol. 100 mM K-phosphate buffer (pH 6.5) was used and reactions were conducted at 30 °C if not stated otherwise. The purified enzyme was unstable if diluted therefore 5 g/L bovine serum albumin (BSA) was added to the dilution buffer as a protein stabilizer to retain its full catalytic activity. For preliminary assay of substrate specificity of BaAG2, the enzyme was reacted with 100 mM concentration of various potential substrates: maltose, sucrose, maltotriose, isomaltose, melezitose, maltulose, turanose, palatinose or α-MG. At fixed time points, aliquots were withdrawn, combined with three volumes of 200 mM Tris buffer (pH 8.3) and subsequently heated at 96°C for 5 min to stop the reaction. The content of the released glucose was determined spectrophotometrically as in [p-nitrophenol or glucose release from substrates were measured at four to seven concentrations ranging from 0.1–3.0 mM for pNPG and 2.5–250 mM for di- and trisaccharides. At least three independent measurements for each substrate and concentration were made. BaAG2 content in reaction mixtures ranged from 0.02 to 3.5 µg/mL. The initial velocity data analysis with enzyme kinetics module of the SigmaPlot  yielded kinetic parameters  for the enzyme. kcat and kcat/Km were calculated from these data. The theoretical Mw value of 67,901 Da for the kcat calculation was computed in ExPASy Proteomics Server (http://expasy.org).Hydrolysis of ed as in ,40 accorly as in ,47. The BaAG2 was studied by incubating a suitable amount of enzyme (0.035–0.13 µg/mL) with 0.2–2.0 mM pNPG in the presence of following potential inhibitors: 5 μM acarbose, 0.1 mM Tris, 5 mM palatinose, 10 mM isomaltose, 10 mM glucose, 100 mM α-methylglucoside, 100 mM trehalose or 400 mM fructose. The Ki values were calculated using enzyme kinetics module of the SigmaPlot .Inhibition of BaAG2 in the presence and absence of ligands. The concentration of BaAG2 was used with 2 µM and for ligands: 100 mM fructose, 50 mM palatinose, 50 mM glucose, 5 mM Tris and 5 mM acarbose. The reaction was conducted in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer (pH 7.0) with 150 mM NaCl. The experiment was based on [Differential scanning fluorimetry (DSF) was used to evaluate the thermostability of based on ,47,65 wiw 20 kDa and 110 kDa, was assayed in K-phosphate buffer  containing 0.2 g/L Na-azide. Polysaccharide concentration in the reaction mixture was 5 g/L, and 20 µg/mL of the enzyme was used. At desired time points  aliquots were withdrawn and heated to stop the reaction. A negative control containing 20 µg/mL BSA instead of the enzyme was incubated alongside. Concentration of released glucose was measured as described above and 3 µl of the samples were analysed on TLC. Maltase from S. cerevisiae (ScMAL62), amyloglycosidase from Aspergillus niger  and α-amylase from Aspergillus oryzae  were reacted with tested glucose polymers for comparison.Degradation of glucose polymers, i.e., amylopectin-free amylose from potato, amylopectin from potato, glycogen from oysters and dextrans of MBaAG2 and ScMAL62, 50 mM of the sugar was reacted with 2.6 µg/mL of the enzyme in K-phosphate buffer  containing 0.1 g/L of Na-azide and samples were withdrawn at fixed time points. 20 mM isomelezitose (transglycosylation product from sucrose of Ogatae polymorpha MAL1) was also tested as a potential substrate. The reaction samples were analyzed using TLC.To assay the hydrolysis of malto-oligosaccharides (DP 4‒7), panose and melezitose by pNPG hydrolysis by BaAG2 was measured at 30 °C in BSA-supplemented buffers of varied pH (from 3.8 to 8.5) using McIlvaine’s buffer (Na-phosphate/citrate buffer) [pNPG was measured at varied temperatures from 20 to 65 °C. Initial velocity data were plotted against the temperature to reveal the temperature optimum. For thermostability determination, BaAG2 was incubated in 100 mM K-phosphate buffer (pH 6.5) buffer containing 5 g/L BSA for 30 min at temperatures 10, 20, 30, 37, 45 and 50 °C. After cooling the samples on ice, residual activity of the enzyme was determined according to the hydrolysis of 1 mM pNPG at 30 °C. Every temperature point was assayed in triplicate. The activity measured after incubation at 10 °C was taken as 100%.Initial velocity of 1 mM  buffer)  and 100 BaAG2 or ScMAL62) was incubated in 100 mM K-phosphate buffer (pH 6.5) with 0.2 g/L Na-azide and 250 mM or 500 mM maltose at 30 °C up to 72 h. The samples with BaAG2 also contained 5 g/L BSA. Samples were withdrawn at fixed intervals, heated at 95 °C to stop the reaction and analysed on TLC and by HPLC.20 µg/mL of the enzyme . 0.5 µl of the stopped reaction mixture were spotted onto the plate and sugars were separated with two runs in chloroform:acetic acid:water  [To visualize hydrolysis and polymerization products, the TLC analysis was conducted as in  on Silic, v:v:v) . For the, v:v:v) .HPLC analysis was performed similarly as in . GlucoseGene sequences from domains 1 and 2 (D1/D2) of large subunit ribosomal RNA were aligned to build a neighbor-joining phylogenetic tree  of yeastS. cerevisiae maltase MAL62 (UniProtKB: P07265), S. cerevisiae isomaltase IMA1 (UniProtKB: YGR287C), O. polymorpha maltase-isomaltase MAL1 (UniProtKB: Q9P8G8) and B. adeninivorans AG2 were aligned using Clustal Omega [S. cerevisiae IMA1 structure in complex with isomaltose (PDB: 3AXH) [Protein sequences of al Omega ,15 were B: 3AXH)  was visuB: 3AXH)  and aminBlastobotrys (Arxula) adeninivorans belonging to the basal group of Saccharomycotina diverged in the evolution of yeasts hundreds of millions of years before Saccharomyces and can be considered as a yeast species with deep phylogeny. The genome of B. adeninivorans encodes two putative α-glucosidases. In current work, one of them, BaAG2, was produced in E. coli and characterized in detail. BaAG2 was proven to be a maltase—hydrolysing α-1,4 and α-1,3 but not α-1,6 linkages in glucose-containing substrates. Interestingly, BaAG2 was strongly and competitively inhibited not only by acarbose, a diabetes drug and a well-known inhibitor of α-glucosidases, but also by Tris. Importantly, at high maltose concentrations, BaAG2 exhibited transglycosylating ability producing potentially prebiotic di- and trisaccharides: isomaltose, panose and maltotriose. Thus, BaAG2 may have a biotechnological value. In contrast to yeast maltases, BaAG2 showed exo-hydrolytic activity on starch, amylose, amylopectin and glycogen. S. cerevisiae maltase MAL62 assayed for comparison had only minimal ability towards these polymers and its transglycosylating activity was much lower.According to the literature data, a non-conventional yeast


Moreover, GpIV cells had improved mRNA and protein expression of Rbfox3/NeuN and choline acetyltransferase (ChAT).The present data describe the relative neuro-2a cellular differentiation induced by reducing serum concentration (0.1% FBS) in DMEM in the presence/absence of 20 μM retinoic acid (RA). Neurite outgrowth was observed within 24 h in DMEM supplemented with reduced serum and retinoic acid (GpIV). The CFSE based proliferation assay data signified cessation of neuro-2a cellular proliferation in GpIV. An increase in the number of cells arrested at G Furthermore, mRNA and protein expressions of Rbfox3  and ChAT  were assessed using real-time PCR  were subjected to various nutrient culture conditions, namely DMEM + 10% FBS (GpI), DMEM + 10% FBS + 20 μM Retinoic acid (GpII), DMEM + 0.1% FBS (GpIII), DMEM + 0.1% FBS + 20 μM Retinoic acid (GpIV), to observe the effect on cellular differentiation. Phase contrast microscopy -A–D was s length E and bras length F was sees length -E, F. Thtime PCR -D, E andtime PCR -A–C. The22.12 conditions. For differentiation, the cells were plated at a density of 2 × 104 cells/cm2 and maintained in the standard growth media for 24 h. Next day, the standard growth media were replaced with the differentiation media of various compositions  supplemented with 10% FBS  at 37 °C, 5% COositions  in their2.22) was calculated using Analyze Particles function׳s parameters  in ImageJ.Neuro-2a cells were grown in 6-well multi-dishes  and differentiated as per above method. Images were captured with the Nikon Eclipse TS100 at 20× magnification using VEZU US300. Neurite length was measured using ImageJ-Simple Neurite Tracer plugin 2.36cells were harvested by trypsinization (0.25%), resuspended in 1 ml of ice-cold PBS . 100% ice-cold ethanol  was added dropwise, and the mixture was incubated at 4 °C for 20 min. Cells were then washed with ice-cold PBS and incubated in the staining solution . Scanning of samples was done using FACS Calibur™  and data were analyzed using BD CellQuest™ Pro.Cell cycle analysis was performed as per Lisa et al. 2.4TM .Before starting the process of neuro-2a cellular differentiation, cells were processed for CFSE as per manufacturer protocol . Briefly, cells were incubated at 37 °C in a staining mixture of CFSE for 20 min, followed by further incubation in complete media for 5 min. Subsequently, complete media were replaced with the differentiation media. After completion of differentiation protocol, cells were trypsinized and scanned with FACS Calibur2.5R method . The integrity of RNA was checked using NanoDrop 260/280 ratio (1.8–2.2) and RNA gel electrophoresis -ddCt method.RNA was extracted using Trizol2.6®CA-630, 0.5% Sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0; Sigma). ~ 50 μg proteins were electrophoresed on 12% PAGE and electroblotted  onto the nitrocellulose membrane. Blots were blocked with 5% BSA, 1 h at room temperature and then incubated with primary antibodies, i.e., ChAT , Rbfox3  respectively for overnight at 4 °C. Samples were further incubated with anti-mouse IgG-HRP  and anti-rabbit IgG-HRP  respectively for 1 hr. Blots were developed with ClarityTM Western ECL Substrate  and imaged with Fuji-LAS4000 luminescent image system .Proteins were extracted in RIPA Buffer (150 mM NaCl, 1.0% IGEPAL2.7P ≤ 0.05, P ≤ 0.01, P ≤ 0.001 were considered to be statistically significant.Values are presented as the mean±S.E. Statistical analysis was done with SigmaPlot 11.0 with one way ANOVA and post hoc analysis using Tukey׳s multiple comparison test.


EB was developed to detect SO32−/HSO3−. The probe displayed both colorimetric and ratiometric responses toward SO32−/HSO3−. It displayed a quick response (within 60 s), good selectivity and high sensitivity (a detection limit of 28 nM) towards SO32−/HSO3−. The SO32−/HSO3− sensing mechanism was confirmed as the Michael addition reaction by ESI-MS. Moreover, the probe could be applied to measure the level of sulfite in real samples, like sugar and chrysanthemum, and it could also be used to detect SO32−/HSO3− in HepG2 cells through confocal fluorescence microscopy, which proved its practical application in clinical diagnosis.Based upon the intramolecular charge transfer (ICT) mechanism, a novel ratiometric fluorescent probe  For example, based on the skeleton (E)-1,1,3-trimethyl-2-styryl-1H-benzo[e]indol-3-ium with acetyl substitution, a probe reported by Zhou et al. can usefully test carboxylesterase indol-3-ium iodide for sensing SO32−/HSO3−  and 4-hydroxybenzaldehyde were used to synthesize the probe as the intramolecular charge transfer (ICT) donor and the acceptor respectively indol-3-ium iodide  and anhydrous ethanol (20 mL) were added. Subsequently, compound 2, 4-hydroxybenzaldehyde  was added to the mixture. This mixture reacted at 80 °C for 12 h. Then, the solution was disposed in a rotary vacuum desiccator and the solid was extracted with CH2Cl2/H2O. The organic layer was collected and dried via anhydrous Na2SO4. The crude product was purified with the CH2Cl2/MeOH eluent using the silica-gel column, see m/z: 342.34 [M]+; 1H-NMR  δ 10.86 (s), 8.52 , 8.42 , 8.30 , 8.25 – 8.16 (m), 8.12 , 7.81 , 7.72 , 7.53 , 7.00 , 4.90 – 4.72 (m), 2.03 (s), 1.50 . 13C-NMR  δ 196.44, 138.68, 137.51, 133.51, 131.20, 130.21, 128.89, 127.76, 127.73, 123.90, 113.66, 55.94, 43.80, 21.95, 14.10.In a two-neck round-bottom flask, compound 32−, HCO3−, Ac-, PO43−, HPO42−, H2PO4−, Cl−, Br−, I−, NO2−, S2−, SO42−, Hcy, Cys, GSH and SO32− were all prepared by corresponding saline solution in purified water (10 mM). A small amount of stock probe EB (10 μM) and SO32− (500 μM) were transferred to a 5 mL volumetric flask, diluted with PBS (pH = 7.4) to volume as the test solution at room temperature, which was prepared to measure fluorescent spectra and UV-Vis absorption. The test solution should be shaken well and detected after 3 min. The same volume of SO32− solution could be replaced by various anion solutions mentioned above to detect interference using the same condition of spectra.The probe was dissolved in ethyl alcohol to prepare the 1 mM stock solution. The anion and amino acid solution such as CO32−  in water samples. Then, the emission intensities at 473 nm and 570 nm were detailed and the analysis results were also compared with that achieved by the titration method.Four kinds of samples: tap water, river water, sugar and chrysanthemum were prepared to detect sulfite respectively. Sugar and River water were diluted with PBS to a concentration of around 25 % (pH = 7.4) as the diluent solution. And the chrysanthemum sample was extracted with PBS solution (pH = 7.4) as the test solution (0.5 g/100 mL). Then transferred stock probe solution (10 μM) to a 5 mL volumetric flask, diluted with the diluent solution to volume. Incubated for 5 min at room temperature before detection. By the way, the recovery test also needed to add accurate SOEB and probe EB after reaction with SO32− were detected using Rhodamine B (Φs = 0.89 in ethanol) as a standard. The Fluorescent quantum yields were determined based on the Equation:The fluorescent quantum yields of probe 32−. The fluorescence intensity ratio of the probe was measured and the standard deviation of the blank measurement was achieved. The limit of detection was calculated according to the following formula: LOD = 3σ/kThe detection limit (LOD) was calculated based on the fluorescence titration of the probe (10 μM) in the presence of SO473/I570) of the probe scanning for 10 times; k: the slope of the line graph of fluorescence intensity ratio (I473/I570) and reactant concentration.LOD: detection limit; σ: the standard deviation of the fluorescence intensity ratio  added with 1% Penicillin-Streptomycin Solution (100×), 10% fetal bovine serum (FBS) at 37 °C under the atmosphere containing 5% CO32− (0–200 μM) at 37 °C for 2 h and then incubated with the probe (10 μM) for 2 h. Before the detection achieved by the apparatus of Fluorescence Microscope and a 63× oil-immersion objective lens (Leica), cells needed to be washed by PBS for three times. The cells were stimulated by the intensity of wavelengths at 405 nm, 488 nm, and collected emission on 450–490 nm, 550–590 nm.To inspect the sensitivity of the probe, cells were incubated in 10 μM probe for 2 h at 37 °C with culture medium . To detect exogenetic sulfite, cells were pretreated with different concentrations of SOEB for SO32−/HSO3− detection under physiological pH, which exhibited a colorimetric and ratiometric response to SO32−/HSO3−. It could demonstrate a quick response, high sensitivity and selectivity to SO32−/HSO3– in aqueous solution and real samples. The sensing mechanism of the interaction between the probe and SO32−/HSO3− was verified using ESI-MS. Moreover, it exhibited the excellent application of detecting SO2 derivatives in living cells with low cytotoxicity. All the results suggested this safe and harmless probe could be used conveniently.On the basis of the Michael addition reaction, we developed a new ratiometric fluorescent probe


Aspergillus sp. SCSIOW3, resulting in significant changes of the secondary metabolites. One new diphenylether-O-glycoside (diorcinol 3-O-α-D-ribofuranoside), along with seven known compounds, were isolated from the culture treated with a combination of histone deacetylase inhibitor (suberohydroxamic acid) and DNA methyltransferase inhibitor (5-azacytidine). Compounds 2 and 4 exhibited significant biomembrane protective effect of erythrocytes. 2 also showed algicidal activity against Chattonella marina, a bloom forming alga responsible for large scale fish deaths.Chemical epigenetic manipulation was applied to a deep marine-derived fungus,  The UV spectrum (MeOH) displayed absorption maxima at λmax (log ε) 281 (4.37) and 279 (3.01) nm. The 1H- and 13C-NMR, DEPT, and HMQC spectroscopic data revealed six olefinic methines, five olefinic quaternary carbons, four oxymethines, an oxymethylene, two methyl groups, and four hydroxyl groups  +, ; c 0.67, MeOH); UV (MeOH) λmax (log ε) 205 (4.21) nm; IR (film) νmax: 3391, 2918, 1584, 1462, 1323, 1155, 1038 cm−1.Compound 1 was hydrolyzed with 1 mL 2N HCl then heated at 80 °C for 3 h under refluxing. The solution was extracted with CHCl3 (1 mL × 3) to remove the aglycone. The water layer was neutralized with NaOH (2N), then concentrated to dryness. The dry powders hydrolysis mixture were dissolved in 1 mL of dry pyridine and then added with 1 mL of trimethylsilyl imidazole and heated at 60 °C under stirring for 1 h. After the reaction, an aliquot (0.4 µL) of the supernatant was removed and directly subjected to GC-MS analysis with the other sugar derivatives under the following conditions: Agilent GCMS, 7890A-5975C, Column HP-5MS (30 m × 0.25 mm × 0.25 µm). A temperature gradient system was used for the oven, starting at 100 °C and increasing up to 150 °C at rate 10 °C/min, then increasing up to 260 °C at rate 15 °C/min for 40 min carrier gas N2 (1.2 mL/min), injector and detector temp 260 °C, split ratio 1:20. The configurations of the sugar fraction were determined by comparison of the retention times of the corresponding derivative with standard D-ribose at 8.40 min.0.5 mg of m/v) DPPH ethanol solution, followed with a 30 min incubation in dark and detection at 517 nm. The biomembrane protective assay was tested in erythrocytes which were obtained by centrifuging whole rabbit heart vein blood with 3.2% citrate at 700 g for 10 min at 4 °C. 6 × 108 /mL of the washed erythrocytes were treated with the compounds for 30 min at 37 °C, followed with the addition of 100 mM, 2′-Azobis (2-amidinopropane) dihydrochloride (AAPH) to induce hemolysis for 60 min at 37 °C. The supernatant was measured at 545 nm after a centrifugation at 700 g, 2 min. The biomembrane protective activity was expressed as percentage of a negative control, in which intact erythrocytes in 0.9% saline were measured. Curcumin was used as a positive control.The direct antioxidative activity of the isolated compounds were evaluated by the modified 2,2-diphenyl-1- hydrazyl (DPPH) radical scavenging assay , in whicChattonella marina was cultured in YMNL GXZ Constant temperature illuminate incubator .The proliferation of algal was detected through OLYMPUS IX51 inverted microscope . C. marina was donated by the Algal Culture Collection of the Institute of Hydrobiology at Jinan University in Guangzhou, China. It was cultivated under a 12:12 h light-dark cycle with 50 μmol photons m−2·s−1 at 22 °C in a sterilized f/2 medium prepared with natural seawater. Exponential growth phase algal cultures were divided into aliquots for further study. C. marina in the exponential growth phase were divided into aliquots and plated in a 12-well plate at a density of 2.9 × 104 cells/well. The tested samples were dissolved in dimethyl sulfoxide (DMSO), and then two-fold serial dilutions were performed. 2 μL of each sample solution was then added in the culture medium to make the final concentrations of 100, 50, 25, 12.5 and 6.5 µM, respectively. 2 μL of DMSO was used as a solvent control. Four parallel repetition was set in each group. The alga was cultured under the conditions described above. Living alga cells in 100 μL culture were counted by a DengXun DSJ-01 counting chamber for zooplankton  to detect the proliferation of the algal cells over 15 min and 120 min.p values) of the results was calculated by Student’s t-test. The results were considered to be significant when p < 0.05 (*), and to be highly significant when p < 0.01 (**).All results were expressed as mean ± SD. Statistical significance , along with seven known compounds, diorcinal (2), 3,3′-dihydroxy-5,5′-dimethyldibenzofuran (3), cordyol (4), gibellulin B (5), cyclo-(L-Trp-L-Phe) (6), sydonic acid (7), and sydowic acid (8), were isolated from the culture treated with a combination of SBHA and 5-AZA. 1 was the first diphenyl ether glycosides incorporating D-ribose as a sugar component. The biomembrane protective effect of erythrocytes and DPPH radical scavenging activities were tested for compounds 1, 2, 4, 7 and 8. None of these compounds exhibited any radical scavenging activity at high concentrations  by DPPH assay. Compounds 2 and 4 exhibited the significant biomembrane protective effect of erythrocytes, which suggested their potential therapeutic use or preventive agents for ROS-associated diseases. In addition, 2 demonstrated algicidal activity against Chattonella marina. 7 did not show any algicidal activity.The present study aimed at activating silent biosynthetic pathways of secondary metabolites and identifying active novel compounds from fungi. In total, 72 marine derived fungi were tested by a chemical epigenetic approach, in which 28 strains (38.8%) showed significant changes on the chemical profiles. Strain


We found that the frequencies of circulating MAIT cells gradually increased in blood from newborns as they progressed into adulthood (20–40 years old) but then decreased during further progression toward old age (>60 years old). The lowered numbers of circulating MAIT cells in the elderly was correlated with a gradual increase of apoptosis. A majority of circulating MAIT cells expressed the chemokine receptors CCR5 and CCR6, and most also expressed CD8 and CD45RO. Few expressed CD69 in cord blood, but the frequency increased with age. Upon in vitro activation with PMA plus ionomycin or IL12 plus IL18, fewer MAIT cells isolated from the young adult group expressed IFN-γ, IL17A and Granzyme B then cells from other age groups while the proportion of cells that expressed TNF-α was similar. Taken together, our data provide information for guiding the assessment of normal levels and phenotypes of MAIT cells at different ages in healthy individuals and patients.Mucosal-associated invariant T (MAIT) cells, which are enriched in human blood and express a semi-invariant TCR chain, play important roles in conditions such as infectious diseases and cancer. The influence of age on levels and functional characteristics of circulating MAIT cells have not been fully addressed. Here we have collected blood samples from a large cohort of healthy Chinese individuals from newborn (cord blood) to the elderly and assessed the levels of circulating MAIT cells as well as their phenotype, activation and apoptosis status, and cytokine expression profiles after  Most MAIT cells express an invariant Vα7.2-Jα33 TCRα chain (TRAV1-2-TRAJ33) paired with a limited number of TCRβ chains  cells are the most abundant TCRαβhain TRAV-2-TRAJ33hain TRAV-2-TRAJ33β chains , which iβ chains . In addiβ chains , 7 and t−CD161−, CD27+CD161−, and CD27−CD161+. MAIT cells are enriched in healthy human blood, representing 1–10% of αβ T cells , following the manufacturer's protocol. To assess intracellular cytokine production, PBMCs were stimulated for 18 h with either 50 ng/ml PMA plus 500 ng/ml Ionomycin  or with 10 ng/mL IL-12  plus 100 ng/mL IL-18 , with the addition of brefeldin A in the last 4 h in complete medium. Independent experiments of samples from four age groups were performed in parallel for 3–5 times, and all the data were collected. As a control, samples without stimulation were used for setting gating regions in flow cytometric analysis.TM III flow cytometer and analyzed by FlowJo software .Staining of surface marker antigens was performed at 4°C for 15–20 min in the dark, using the following monoclonal fluorochrome conjugated antibodies: FITC-CCR6 (G034E3), FITC-CCR5 (HEK1/850), Percep5.5-TCRγδ(B1), PE-CD161 (HP-3G10), PE-Cy7, or FITC-TCRVα7.2 (3C10), Pacific Blue-CD62L (DREG-56), FITC-CD45RO (1CHL), Pacific Blue-CD69 (FN50), APC-CD127 (A019D5), APC-Cy7-CD3 (HIT3a), Pacific Blue or APC-CD8 (RPA-T8), and APC or FITC-CD4 (OKT4). All antibodies were purchased from BioLegend . Dead cells were excluded during FACS analysis. Intracellular cytokine staining was performed using fixation/permeabilization buffer , following the manufacturer's protocol. Pacific Blue-Annexin V was used for the detection of cell apoptosis immediately after isolation of PBMCs. Data was acquired using BD FACSAriaU-test or unpaired Student's t-test while the correlation analysis was performed using Spearman's test. A linear regression analysis was performed to compare the age-related changes in the level of MAIT cells and the expression of CD69 or PLZF in circulating MAIT cells. All analyses were performed using GraphPad Prism 6 . Data is expressed as mean ± SEM, mean values are shown with horizontal bars, and p < 0.05 are considered as statistically significant .The statistical significance was assessed using Mann–Whitney +TCRVα7.2+TCRγδ−CD161hi cells by flow cytometry   . Therefo+ T cells, we analyzed the CD3+ cells in PBMCs. As shown in + cells were the lowest in cord blood , the highest in the children group (60.32 ± 1.08%), and gradually decreased from youth to elderly .To determine whether age-associated changes in MAIT cells were due to similar changes in CD3+TCRγδ− T cells relative to the entire cohort did not reflect any relationship between age and the percentage of MAIT cells   was observed from child to youth while a negative correlation between age and the frequency of MAIT cell   . However 0.0001)  was show 0.0001) ; althoug 0.0001) .+, CD8+ and double negative (DN) subsets (−CD8+ MAIT cells accounted for about 50% (55.32% ± 4.72) in the CB group, 58–80% in the children group (78.51 ± 1.423%), 75% in the youth group (77.97 ± 1.54%), and 75 % in the middle-age group (78.66 ± 1.82%), but decreased to about 70% in the elderly group (71.86 ± 3.56%). CD4+CD8− MAIT cells were rare, accounting for about 10% in the CB but <10% in the other age groups. On the other hand, CD4−CD8− DN MAIT cells accounted for about 30% (30.32 ± 4.04%) of the MAIT cells in the CB group. In the children, young, middle-age and elderly groups, the percentages of DN MAIT cells were less than in the CB group, reflecting a lower percentage than CD8+ cells but a higher percentage than CD4+ MAIT cells, accounting for 15.78 ± 1.03, 15.61 ± 1.31, 12.96 ± 1.4, and 20.04 ± 3.96%, respectively , central memory , and effect memory  populations. MAIT cells in CB were predominantly naive (55.82% ± 5.42), with very low percentages of CM (3.15% ± 1.23) and EM (1.69% ± 0.76) populations. Naive MAIT cells rapidly differentiated into EM and CM cells in the children group and were thus very rare in the youth, middle-age and elderly groups ; i.e., about 15% regular CD8+ T cells were CM cells  (+ in the entire cohort. The percentage of CD127 in circulating MAIT cells was the highest in the CB group (97.04% ± 1.15) and the lowest in the elderly group (67.02% ± 3)  and elderly (87.16%) groups than that in the youth group, the differences among the three groups were not statistically significant (+ MAIT cells (14.56% ± 6.01). However, more than 85% MAIT cells expressed CCR5 in the other groups   .IL7 rece 0.0001) , 35. Con02% ± 3) . MAIT cenificant . Newbornr groups . PLZF, ar groups . An anal −0.618) .+ cells in MAIT cells   . Meanwhi= 0.562) . These din-vitro stimulation with PMA and ionomycin. We didn't detect any cytokine secretion of MAIT cells in the CB group with the naive phenotype (data not shown), so we compared the cytokine production in the children, youth, middle-age, and elderly groups. As the percentage of EM cells in MAIT cells gradually increased with age, we speculated that the cytokine secretion of MAIT cells might also gradually increase with age. Surprisingly, MAIT cells in the youth group had the lowest IFN-γ+ cells (26.24% ± 2.44%), with other groups being not obviously different    . The sam± 3.54%) . Differe ± 2.94) . These oWe further measured the expression of IL17A, granzyme B, and TNF-α in the MAIT cells from different age groups and found that the percentages of IL17A-expressing cells in the circulating MAIT cells of healthy subjects were very low (around 1%−3%). Similar to IFN-γ, MATI cells had the lowest percentage of IL17A-expressing cells in the youth group, while the other three groups had similar percentages of IL17A-expressing cells . The MAI+ MAIT cells and age in healthy children (+ and DN) from children to elderly  upon activation with different stimuli, circulating MAIT cells expressed multiple cytokines that were also deferentially regulated along with ages, showing the lowest expression in youth.MAIT cells belong to innate-like T lymphocytes. Until now, our understanding of peripheral blood MAIT cells in healthy individuals at different ages is still incomplete. Khaitan's group found that there is no significant correlation between the frequency of CD8children . Booth's elderly . These s elderly , were su elderly , no data+TCRVα7.2+CD161hi cells, which is largely coincident with the MR1 tetramer+ population in human according to the pioneer study on MR1 tetramer, which may equip these cells for rapid activation by IL18. Indeed, our data showed that IL12 plus IL18 could potently activate MAIT cells to produce IFN-γ.Upon activation, MAIT cells can produce several cytokines , 37, 38.E.coli , H.pylorH.pylori , Mycobacrculosis , and pulrculosis ], it wouPut together, our data demonstrated that circulating MAIT cells in healthy subjects from CB to elderly are regulated by intrinsic properties of MAIT cells, such as apoptosis, and by environmental factors, such as microbiota. Functionally, MAIT cells in youths produce lower levels of IFN-γ, IL17A, and granzyme B. Therefore, our findings provide important information about circulating MAIT cells, which is helpful to the understanding of MAIT cell changes in healthy individuals and patients in a variety of clinical settings.PC, WD, DZ, JL, and X-PZ designed the study. JinleW, HX, and HC recruited the study participants. PC, DL, TZ, LH, QW, JinliW, WZ, XY, DD, and WH performed the experiments and acquired data. PC, WD, and DL were involved in data analysis and interpretation. PC and WD wrote the manuscript. X-PZ and JG revised the manuscript. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pyrus ussuriensis Maxim, a pear commonly known as “Sandolbae” in Korea, is used as a traditional herbal medicine for asthma, cough, and fever in Korea, China, and Japan. P. ussuriensis Maxim leaves (PUL) have therapeutic effects on atopic dermatitis (AD). However, there are no reports on the efficacy of specific components of PUL. In the present study, activity-guided isolation of PUL was used to determine the compounds with potent activity. Astragalin was identified as the major component of the chloroform-soluble fraction of PUL (PULC) using High-performance liquid chromatography (HPLC) analysis. Astragalin and PULC were tested in vitro and in vivo for their effects against AD. PULC and astragalin dose-dependently inhibited the production of nitric oxide (NO) in mouse macrophage (RAW 264.7) cells, and interleukin (IL)-6 and IL-1β in tumor necrosis factor (TNF-α)/interferon γ (IFNγ) induced HaCaT cells. In the AD mice model, PULC and astragalin application significantly reduced dermatitis severity, scratching behavior, and trans-epidermal water loss (TEWL) when compared to that of 2, 4-dinitrochlorobenzene-treated NC/Nga mice. Additionally, they normalized skin barrier function by decreasing immunoglobulin E (IgE) levels in the serum. Filaggrin and involucrin protein levels were normalized by PULC treatment in HaCaT cells and skin lesions. These results indicate that PULC and astragalin ameliorate AD-like symptoms by alleviating both pro-inflammatory cytokines and immune stimuli in vitro and in vivo in animal models. Therefore, PULC and astragalin might be effective therapeutic agents for the treatment of AD. Atopic dermatitis (AD), a chronic inflammatory skin disease, is due to the complex linkage between environmental and genetic mechanisms . It is aThe associated skin changes that occur in human AD are induced in NC/Nga mice through exposure to several environmental aeroallergens ,14. In lPyrus ussuriensis is the major cultivated species of pear and is widely distributed in China, Korea, and Japan. It is known for its medicinal properties including anti-inflammatory, antioxidant, antitussive, and diuretic effects [P. ussuriensis reported that its leaf extract has antioxidant [P. ussuriensis leaf extract inhibited symptoms of AD in the DNCB-induced NC/Nga mouse model [P. ussuriensis Maxim leaves (PUL) was used to determine the compounds with potent activity and investigate the therapeutic effects of P. ussuriensis and its components in vitro in macrophages and keratinocytes and in vivo in the DNCB-induced AD-like skin lesion model using NC/Nga mice.Currently, various herbal medicines are used for the prevention of AD and asthma diseases ,19. Pyru effects . In Koreioxidant  and antise model . In the P. ussuriensis Maxim. leaf extract was prepared as described by Cho et al. [o et al. . The liqAs previously described by Cho et.al.,  HPLC ana5 U/L), streptomycin (100 mg/mL), and 10% of fetal bovine serum at 37 °C in a humidified CO2 (5%) incubator. RAW 264.7 and human HaCaT cells were purchased from the Korean Cell Line Bank  and were maintained in Dulbecco’s modified Eagle’s medium containing 1% of penicillin -2,5-diphenyltetrazolium bromide (MTT) assay according to Hwang et al. . RAW 2644 cells/well in a 96-well plate and were pretreated with PUL sub-fractions, followed by LPS activation for 24 h. Using Griess reagent, NO levels were measured in the culture media. Briefly, 50 µL Griess reagent was added to 50 µL conditioned medium and the absorbance was measured at 570 nm. Effect PUL sub-fractions on LPS-induced NO production in RAW 264.7 cells were performed according to Han et al. with some adjustments . RAW 2645 cells/well in a 24-well plate. When the cells reached a confluency of 70–80%, they were stimulated with 10 ng/mL of TNFα or IFN-γ, and with PULC and its sub-sub fractions. After 24 h, the supernatants were collected and IL-6 and IL-1β secretion was evaluated using the respective ELISA kit . IL-6 and IL-1β secretion was measured using enzyme-linked immunosorbent assay (ELISA). HaCaT cells were seeded at a density of 2 × 10NC/Nga female mice (4 weeks old) were purchased from Orient Bio  and were maintained in a pathogen-free environment at a temperature of 22 ± 2 °C and a 12-h light/dark cycle. The mice were randomly divided into four groups (9 per group): NOR, normal control vehicle , DEX (DNCB + positive control treated with dexamethasone 0.1%), PULC, and DNCB + PULC 0.5%. To induce AD-like skin lesions. The skin of the animals was shaved on day 6; 200 µL of DNCB (1%) was dissolved in acetone + olive oil mixture  and then applied to the dorsal skin on day 7. For five weeks, 200 μL of each sample was applied on the back of both the ears twice weekly. All the five groups were then treated according to the treatment group. Dorsal skin tissues obtained from the mice were subjected to histological and western blot analysis. All procedures were performed in accordance with the guidelines for the Care and Use of Laboratory Animals (LCDI-2017-0038) approved by the Institutional Animal Care and Use Committee of Gachon University .2/h. The measurements were performed from week 7 of the experiment. Dermalab Combo system  was used to measure TEWL, hydration and erythema of the epidermal layer of the skin of Nc/Nga mice. At approximately 21–22 °C and humidity of 50–55%, the measurements were made by placing a probe on the mice’s skin that was stabilized for approximately 30 s; data were recorded in g/mOnce a week, dermatitis severity score was checked as previously described . The sevOn the final day of the experiment, scratching behavior of each mice was recorded using a digital camera  as described  by countMeasurement of serum IgE level was analyzed as previously described . On the For histological examination, the dorsal skin of each mouse collected on the last day of the experiment was fixed with 10% neutral-buffered formalin and embedded in paraffin. Skin sections of 4 µm thickness were cut and transferred to slides. Before examination at 100× magnification, hematoxylin and eosin (H&E), IL-1β, and toluidine blue stains were used to stain the deparaffinized skin sections. The stained skin was observed under a Nikon Eclipse 80i Microscope  at 100× magnification. Cell lysates and/or dorsal skin tissues were lysed overnight in PRO-PREP™ protein extraction solution  at 4 °C. The lysates were centrifuged at 11,000× g for 30 min at 4 °C. The supernatants were collected, and protein concentrations were determined using Bio-Rad protein assay reagent  according to the manufacturer’s protocol. Proteins (20 μg) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 12% and blotted onto a polyvinylidene fluoride membrane (PVDF). The membranes were blocked for 1 h in 5% of skim milk at room temperature, followed by overnight incubation with primary antibodies against filaggrin, involucrin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at 4 °C. After incubation with the respective horseradish peroxidase-conjugated secondary antibodies, the blots were developed via enhanced chemiluminescence . Densitometric analysis was performed using Bio-Rad Quantity One software version 4.3.0 .p- value .The data are expressed as the mean ± standard error of the mean (SEM) using GraphPad Prism 5.0 . One-way analysis of variance (ANOVA) followed by Tukey’s honest significance test was used to evaluate the statistical comparison between control and various groups. The differences were considered statistically significant at a HPLC-UV analysis at 254 nm was used to determine the components of PULC. Astragalin was found to be the major constituent of PULC. Its retention time (Rt: 20.53 ± 0.28 min) matched with its standard. Content analysis indicated that PULC contained 0.03 ± 0.00 μg/100μg of astragalin .As shown in γ treated HaCaT cells. PULC and sub-sub fraction B both at a dose of 10 µg/mL lowered IL-6 levels to 67.60% and 59.41% and IL-1β levels to 50.43% and 45.68%, respectively compared to negative control. Also, 100 µg/mL astragalin lowered IL-6 levels to 60.44% and IL-1β levels to 43.60% compared to TNF-α/ IFN-γ treated group (CON). The inhibitory effect of inflammatory cytokines was dose-dependent and no cytotoxicity was detected (An inhibitory effect of PULC and C2 sub-sub fractions on IL-6 and IL-1β production was observed in TNF-α/ IFN-detected A–I.2/h) as compared with that in the normal control vehicle treated group (19.82 ± 2.63 g/m2/h). A decrease in skin TEWL was observed in the dexamethasone (0.1%) (26.72 ± 4.68 g/m2/h) and PULC (0.5%) (39.85 ± 5.63 g/m2/h) treated groups. By week 9, erythema level was increased in DNCB-treated NC/Nga mice (12.47 ± 1.58 RI) compared with the normal control group (7.58 ± 0.80 RI) while the erythema level was reduced in dexamethasone (0.1%) (11.85 ± 0.41 RI), and PULC (0.5%) (9.32 ± 0.68 RI) application in DNCB-treated mice  and PULC decreased AD-like symptoms in DNCB-treated NC/Nga mice. The increased dermatitis score in DNCB-treated NC/Nga mice (14.87 ± 2.22) was significantly decreased in the dexamethasone (0.1%) (10.5 ± 1.64) and PULC (0.5%) (8.00 ± 1.28) treated groups. PULC treatment decreased the dermatitis score when compared with CON group (46.21%) C.To evaluate the antipruritic effect of PULC on DNCB-induced NC/Nga mice, scratching behaviors were monitored, and quantified the time spent by every single mouse rubbing their dorsal skin and ears with their hind paws as scratching time. An increased scratching time was observed on the final day of the experiment in DNCB-treated NC/Nga mice (432.21 ± 106.80 s) when compared with that of the normal controls (82.46 ± 18.89 s). The scratching time was significantly reduced in dexamethasone (0.1%) (250.36 ± 64.82 s) and PULC (0.5%) (83.63 ± 39.24 s) treated groups. Also, 0.5% PULC significantly decreased scratching time when compared with that of controls (8.65%) D.To evaluate the serum IgE levels in DNCB induced NC/Nga mice, a blood sample was collected on the last day of the experiment. It was observed that serum IgE levels were increased in DNCB-treated NC/Nga mice (17472.2 ± 1718.86 ng/mL) when compared with that of the control group (2000±961.04 ng/mL). These increased serum IgE levels were significantly decreased in dexamethasone (0.1%) (9527.78 ± 73.49 ng/mL) and PULC (0.5%) (10861.1 ± 1209.85 ng/mL) treated groups E. Also, The skin of DNCB-treated NC/Nga mice became swollen and exhibited epidermal hypertrophy. In contrast, reduced infiltration of inflammatory cells and less severe epidermal hypertrophy were observed in the skin of mice treated with dexamethasone (0.1%) and PULC (0.5%) compared to those in the skin of DNCB-treated NC/Nga mice. H&E staining showed increased epidermal thickness by DNCB treatment (148.6 ± 6.72 µm), whereas a significantly reduced thickness was measured after treatment with dexamethasone (0.1%) (64 ± 4.53 µm) and PULC (0.5%) (45.6 ± 3.76 µm) in DNCB-treated mice Fa,G. IL-1 on protein levels of filaggrin and involucrin in dorsal skin lesions of the DNCB-treated NC/Nga mice  and astragalin  compared to those in the CON2  plays a critical role in the differentiation of skin keratinocytes in the stratum granulosum . DecreasP. ussuriensis Maxim. by bioactivity-guided isolation techniques had good activity in vitro. Topical application of PULC effectively reduced AD-like skin lesions by increasing skin thickening, reducing skin eczema and increasing skin hydration in DNCB-induced AD NC/Nga mice. Thus, PULC and astragalin may be an effective therapeutic agent, which may attenuate AD. Further studies are required to fully elucidate the underlying mechanism of Pyrus ussuriensis Maxim. leaves on AD. Our results indicate that astragalin obtained from the leaves of