Aspergillus fumigatus (AF), A. niger (AN), A. ustus (AU), A. japonicus (AJ), A. terreus (AT), Fusarium oxysporum and Botrytis cinerea isolated from grapevine in the province of San Juan, Argentina. Three of the synthesised compounds (1-(Biphenyl-4-yloxy)-3,3-dimethyl-1-butan-2-one, 1; 2-(Biphenyl-4-yl)-3-cyclopropyl-1-butan-2-ol, 3; 3-Cyclopropyl-2-(4’-fluorobiphenyl-4-yl)-1-butan-2-ol, 4) presented remarkable in vitro fungicidal properties, with better effects than TDM and CPZ on some of the target fungi. Cytotoxicity was assessed using human lung fibroblasts MRC5. Derivative 1, with IC50 values of 389.4 µM, was less toxic towards MRC-5 human lung fibroblasts than commercial TDM (248.5 µM) and CPZ (267.4 µM). Docking analysis and molecular dynamics simulations suggest that the compounds present the same interaction in the binding pocket of the CYP51B enzyme and with the same amino acids as CPZ. The derivatives investigated could be considered broad-spectrum but with some selectivity towards imperfect fungi.Triadimefon (TDM) and cyproconazole (CPZ) are two triazoles widely used as fungicides. Several azoles were synthesised starting from commercial TDM and CPZ. The compounds were evaluated against phytopathogenic filamentous fungi, including The online version contains supplementary material available at 10.1007/s13659-022-00329-0. Vitis vinifera L.) as well as other relevant crops, including tomatoes (Solanum lycopersicum), apples, pears and citrus fruits, are very susceptible to a large number of pathogenic microorganisms such as Botrytis cinerea, Aspergillus spp., and Fusarium spp., among others. Fungal diseases cause economic losses during pre- and/or post-harvest, affecting production, processing and export, together with the quality of fruit. Argentina is the sixth wine producer worldwide + exact mass calcd for C22H26N3O2: 364.2025, found: 364.20300.3-Cyclopropyl-2--1-butan-2-ol (6). The product was isolated as a colourless oil after preparative thin layer chromatography using dichloromethane/ethyl acetate (50/50). 1H NMR , δ (ppm): 0.85–1.37 ; 1.58–1.71 ; 1.74–1.83 ; 4.50 ; 4.57 ; 4.91 ; 7.13–7.50 ; 7.78 ; 7.84 .13C NMR , δ (ppm): 3.1; 3.2; 7.8; 13.1; 30.0; 57.3; 79.6; 113.2; 114.5; 127.0; 128.0; 128.1; 128.3; 128.7; 131.4; 131.8; 133.4; 140.4; 141.8; 152.0, 162.2. MS (EI+) m/z (%):312 (3), 311 (17), 310 (100), 264 (27), 257 (4), 256 (11), 198 (45), 82 (19), 77 (21). HRMS [M+Na]+ exact mass calcd for C21H22 NaN4O3: 401,1584, found: 401,1586.1H-1,2,4-Triazole (8).1HNMR , δ (ppm): 8.32 , 13.42  [6 147.0, 147.0.35 MS, m/z: 69 (M+); Anal calc. (%) for C2H3N3:C, 34.78; H, 4.38; N, 60.84; Found C, 29.63; H, 4.34; N, 60.62.2  . DMSO-d6Botrytis cinerea isolated from grapevine samples, Aspergillus fumigatus (ATTC 26934), A. niger (ATCC 9029), A. ustus (PN-S4), A. japonicus (M15C), A. terreus (M16C) and Fusarium oxysporum (M15-Pa) isolated from soil samples from the province of San Juan, Argentina. The microorganisms were grown on Czapek-yeast extract agar (CYA) prepared with yeast extract, K2HPO4, saccharose, agar, distilled water and enriched with a solution of mineral salts . Inocula were adjusted to 1–5 × 104 colony forming units (CFU/ mL).The imperfect fungi used for the assays included 4 CFU/mL in accordance with the Clinical and Laboratory Standards Institute   [The minimum inhibitory concentration (MIC) of each compound was evaluated using broth microdilution techniques in 96-well microtiter plates according to the guidelines of the National Committee for Clinical Laboratory Standards for filamentous fungi M 38 A. The starting inoculum was 1–5 × 10s fungi) .Aspergillus and Fusarium, and 22 °C for B. cinerea in a moist and dark chamber. MIC values were recorded after 48 h for Aspergillus and Fusarium spp., and after 72 h for B. cinerea, according to the fungal growth control. TDM and CPZ were used as positive controls (12.5–50 μg/mL). Stock solutions were two-fold diluted with RPMI from 1000 to 0.98 μg/mL , and a final DMSO concentration ≤ 1%. An inoculum suspension (100 μL) was added to each well with the exception of the sterility control. Endpoints were defined as the lowest compound concentration resulting in total growth inhibition (MIC100) compared to the growth in control wells. MICs values ≤ 50 μg/mL were considered of interest. The assay was carried out in triplicate.Microtiter plates were incubated at 30 °C for Vitis vinifera grapes inoculated with a Botrytis cinerea conidia suspension [. Appropriate amounts of the tested compounds were dissolved in DMSO and then suspended in the distilled water (DMSO concentration < 2%).The in vivo activity was determined on the spension . Appropr4 (concentration of 12.5 ⎧g/mL and 2.5 ⎧g/mL) and evaporated at room temperature (~ 25 °C) during 24 h to favour the penetration of the compound. The same procedure was carried out with CPZ and the control blank (< 2% DMSO solution). The grapes epidermis was punctured with a sterile inoculation needle , 200 µL of the pathogen was inoculated at a concentration of 5.104 UFC/mL. The fruits were incubated at 20 °C to favour fungal growth, and fungal growth was assessed 7 days later. CPZ was used as positive control under the same conditions. The control effectiveness of the target compound was calculated as (1 − c/d) × 100, where c is the diameter of the treatment and d is the diameter of the blank control (˂ 2% DMSO solution).Healthy grapes, previously washed with a diluted solution of sodium hypochlorite, were punctured with 100 ⎧L of a solution of compound 4 on the morphology of the hyphae with the mycelia after 7 days of culture of B. cinerea treated with the compound was assessed by optical microscopy (40x). The same procedure was carried out with CPZ and blank control (< 2% DMSO solution).The effect of the most active compound 2 in the air at 37 °C. Cell passage was maintained between 10 and 16. The medium was changed every 2 days.The cytotoxic effect of the compounds, expressed as cell viability, was assessed on a permanent fibroblast cell line derived from human lung (MRC-5) (ATCC CCL-171). MRC-5 fibroblasts were grown as monolayers in minimum essential Eagle medium (MEM) with Earle’s salts, 2 mM l-glutamine and 2.2 g/L sodium bicarbonate, supplemented with 10% heat inactivated fetal bovine serum (FBS), 100 IU/mL penicillin and 100 µg/mL streptomycin in a humidified incubator with 5% CO50 values , the results were transformed to percentage of controls and the IC50 values were graphically obtained from the dose–response curves.Confluent cultures of MRC-5 fibroblasts were treated with medium containing the compounds at concentrations ranging from 0 to 1000 µM. For comparison purposes, the commercial antifungals CPZ and TDM were used as references, the products were first dissolved in DMSO and then in MEM, supplemented with 2% FBS. The final content of DMSO in the test medium and controls was 1%. Cells were exposed for 24 h to the test medium with or without the compound (control). Each concentration was tested in quadruplicate together with the control, and repeated three times in separate experiments. At the end of the incubation, the neutral red uptake (NRU) assay was carried out . To calcThe lipophilicity of the compounds was estimated using the ChemBioDraw Ultra 12.0. The parameter is presented as log P , and when the F value was significant, post hoc differences were determined by the Dunnett’s multiple comparison test. The level of significance was set at p < 0.05. All statistical analyses were performed using the software Statistica 5.1 .B. cinerea CYP51 (Accession Number: AAF85983) was taken from the NCBI protein database (http://www.ncbi.nlm.nih.gov/ protein). Two crystal structures of A. fumigatus CYP51 (PDB codes 4UYL and 4UYM) were used as the crystallographic coordinate templates [B. cinerea was obtained using Swiss-model [The amino acid sequence of emplates . The strss-model . The docAutomatic docking was performed using the Autodock Vina 1.1.1. During the docking procedures, water molecules and ligands were removed from the protein. The receptor structure was defined as rigid, and the grid dimensions were 25, 25, and 25 for the X, Y, and Z axes, respectively, taking the active site of the BcCYP51 as the centre of coordinates. Gasteiger charges were assigned to all the compounds, and non-polar hydrogen atoms were merged. All torsions of the ligand were allowed to rotate during docking. The value for the exhaustiveness of the search was 400, whereas the number of poses collected was 10. All graphic manipulations and visualisations were performed by means of the AutoDock Tools 1.5.4 and ligand docking with Autodock Vina 1.1.1.34.The geometries of the complexes obtained from docking were soaked in boxes of explicit water using the TIP3P model and subjected to MD simulation. All MD simulations were performed with the Amber 14 software package using periodic boundary conditions and cubic simulation cells. The particle mesh Ewald method (PME) was applied using a grid spacing of 1.2 Å, a spline interpolation order of 4, and a real space direct sum cut off of 10 Å. The SHAKE algorithm was applied, allowing for an integration time step of 2 fs. MD simulations were carried out at a 310 K temperature. Three MD simulations of 5 ns were conducted for each system under different starting velocity distribution functions; thus, in total, 15 ns were simulated for each complex. The NPT ensemble was employed using Berendsen coupling to a baro/thermostat . Post MD analysis was carried out with the program CPPTRAJ.Additional file 1: Histograms of interaction energies partitioned with respect to the amino acids of 14α-demethylase complexed with compounds: a) 7 and b) 8. The x-axis denotes the residue number of 14α-demethylase, and the y-axis denotes the interaction energy between the compounds and specific residue. Negative values and positive values are favorable or unfavorable to binding, respectively.


Cordyline terminalis leaf extract (aqCT) possesses abundant polyphenols and other bioactive compounds, which are encapsulated in gelatin–polyethylene glycol–tyramine (GPT)/alpha-cyclodextrin (α-CD) gels to form the additional functional materials for biomedical applications. In this study, the gel compositions are optimized, and the GPT/α-CD ratios equal to or less than one half for solidification are found. The gelation time varies from 40.7 min to 5.0 h depending on the increase in GPT/α-CD ratios and aqCT amount. The aqCT extract disturbs the hydrogen bonding and host–guest inclusion of GPT/α-CD gel networks, postponing the gelation. Scanning electron microscope observation shows that all gels with or without aqCT possess a microarchitecture and porosity. GPT/α-CD/aqCT gels could release polyphenols from 110 to 350 nmol/mL at the first hour and sustainably from 5.5 to 20.2 nmol/mL for the following hours, which is controlled by feeding the aqCT amount and gel properties. GPT/α-CD/aqCT gels achieved significant antioxidant activity through a 100% scavenging DPPH radical. In addition, all gels are non-cytotoxic with a cell viability more than 85%. Especially, the GPT3.75α-CD10.5aqCT gels with aqCT amount of 3.1–12.5 mg/mL immensely enhanced the cell proliferation of GPT3.75α-CD10.5 gel without extract. These results suggest that the inherent bioactivities of aqCT endowed the resulting GPT/α-CD/aqCT gels with effective antioxidant and high biocompatibility, and natural polyphenols sustainably release a unique platform for a drug delivery system or other biomedical applications. Van der Waals interactions, hydrogen bonds, and inclusion complex formation of the biocompatible modified polymers [Hydrogels are a promising candidate for biomedical applications, especially for drug/cell encapsulation applied in regenerative medicine and tissue engineering. Hydrogels possess a three-dimensional structure, high porosity, high water content, biocompatible properties, and biodegradable ability ,2,3. CheCordyline terminalis L. Kunth belongs to the Asparagaceae family and is one of the tropical plants grown popularly in Asia, Australia, and the Pacific Islands [Cordyline terminalis L. Kunth leaves [Cordyline terminalis Kunth leaves was chosen instead of isolated compounds. Islands . It is w Islands . Until nh leaves , but furh leaves . The alth leaves . Howeverh leaves . These dh leaves ,12. Indih leaves . They coh leaves , even ash leaves . MotivatCordyline terminalis L. Kunth leaf extract (aqCT) to release polyphenols and investigate their antioxidant and cell biocompatible capacity. Firstly, the GPT precursor polymers were prepared with suitable PEG substitution, and the aqCT was primarily tested on the phytochemical compositions. The optimal GPT/α-CD ratios were investigated to form the supramolecular gels and incorporate the various amounts of aqCT. The intra-morphology of gels was observed by SEM. The polyphenol release profile was determined as a function of time. DPPH radical scavenging capacity was carried out for GPT/α-CD/aqCT gels to demonstrate their effective antioxidant. The non-cytotoxicity of GPT/α-CD/aqCT gels was confirmed by two-dimensional culture of human dermal fibroblast viability.In this study, we structured the non-covalently crosslinking gels from gelatin–polyethylene glycol–tyramine (GPT) and alpha-cyclodextrin (α-CD) for encapsulation of an aqueous Qualitative analysis of aqueous Cordyline terminalis Kunth leaf extract (aqCT) was performed to recognize the presence of alkaloids, glycosides (cardiac and coumarin glycosides), saponins, phenolic compounds, steroids, and phytosterols. As shown in 1H NMR spectra and ultraviolet-visible spectrophotometry as previously reported [Gelatin–polyethylene glycol–tyramine polymers (GPT) were synthesized by a two-step reaction; then its structure was confirmed by reported ,21. The reported ; thus, αreported . These tTo explore the effect of the GPT:α-CD ratio on hydrogel formation, four mixtures with different ratios  were prepared. As shown in 10.2 min . This chTo explore the effect of PEG content on gel formation, GPT’ containing a low PEG substitution was used to fabricate the GPT’:α-CD gels with the GPT’:α-CD ratios of 1:2 and 1:3. However, these two gels were not formed, while the GPT with high PEG content could be gelled at those same ratios. Therefore, the PEG substitution indirectly recognized by tyramine content immensely affected gel formation. Next, we fixed the solid concentration and tyramine/PEG content but replaced a part of α-CD with β-CD. Specifically, the final mixture named GPT3.75α-CD9.45β-CD1.05 was composed of 3.75 wt% of GPT, 9.45 wt% of α-CD, and 1.05 wt% of β-CD. The gelation time of GPT3.75α-CD9.45β-CD1.05 gel was 78.8 ± 13.5 min, which was 1.4-fold longer than GPT3.75α-CD10.5 gels. This observation revealed that α-CD played an essential role in GPT/CD gel formation. It could be explained by the host–guest formation between PEG chains and α-CD being more stable than β-CD . BesidesCordyline terminalis Kunth leaf extract  were utilized to incorporate the natural compounds of aqueous  extract c. GPT anectively . These rCordyline terminalis Kunth leaf extract (aqCT), the GPT3.75α-CD15 hydrogels with an increased α-CD amount were applied. The gelation time of GPT3.75α-CD15aqCT9.4, GPT3.75α-CD15aqCT12.5, and GPT3.75α-CD15aqCT15.6 hydrogels were 51.1 In the case of encapsulating high content of aqueous ectively . The preTo explore the inner structure, six gel samples, including GPT3.75α-CD10.5, GPT3.75α-CD10.5aqCT3.1, GPT3.75α-CD10.5aqCT9.4, GPT3.75α-CD10.5aqCT12.5, GPT3.75α-CD15aqCT9.4, and GPT3.75α-CD15aqCT15.6 gels, were observed their cross-section by SEM. Cordyline terminalis Kunth leaf extract (aqCT) contained 1587.13 ± 94.32 μmol/g of polyphenols, which was determined by the Folin–Ciocalteu assay. Besides, the different solvents, including hexane, ethyl acetate, and ethanol, were also used to extract the Cordyline terminalis Kunth leaves. However, the polyphenol contents in those extracts were 227.78 ± 9.42, 500.07 ± 31.47, 930.49 ± 78.50 μmol/g, which were significantly less than that in aqCT. Thus, aqCT was selected to encapsulate inside GPT/α-CD gels to create the polyphenol release scaffold for biomedical applications. Six formulations with various aqCT amounts and GPT/α-CD ratios were prepared to record the polyphenol releasing profile  for 24 h. The control samples were set up for hDFBs contacting pure DMEM and aqCT solutions of 0.1 mg/mL. The WST-1 assay was performed to determine the cell viability percentage in those conditions. As shown in Gelatin , 1-ethyl-3-(3-dimethylamino propyl)-carbodiimide (EDC), 3-(4-hydroxyphenyl)propionic acid (HPA), N-hydroxysuccinimide (NHS), poly(ethylene glycol) , 4-dimethylamino pyridine (DMAP), alpha-cyclodextrin (α-CD), beta-cyclodextrin (β-CD), p-nitrophenyl chloroformate (PNC), and Folin and Ciocalteu’s reagent were bought from Sigma–Aldrich . Tyramine (TA) was supplied by ACROS Organic . Triethylamine (TEA) was purchased by Kanto Chemical Co. .2, ACS reagent, 99%), acetic anhydride , iron chloride reagent grade , sodium carbonate , and 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl  were purchased from Merck KGaA . Hydrochloride acid , glacial acetic acid , concentrated sulfuric acid , and ethanol  were obtained from Xilong Scientific Co., Ltd. . Cordyline terminalis Kunth leaves were harvested from Dak Lak province, Vietnam.Potassium iodide , potassium hydroxide , iodine  were purchased from Lonza Inc. . Dulbecco’s modified Eagle medium (DMEM), trypsin/ethylenediaminetetraacetic acid (EDTA), and penicillin−streptomycin (P/S) were obtained from Gibco BRL . Dulbecco’s phosphate buffered saline (DPBS) and fetal bovine serum (FBS) were supplied by Wisent . WST-1 assay kit (cell proliferation) was obtained from Merck KGaA .The collected Cordyline terminalis Kunth leaves without diseases were washed carefully with distilled water (DIW) and sundried until humidity less than 5 weight percentage (wt%). Dried leaves were ground into fine powder. The leaf powder was immersed in DIW at a weight/volume ratio of 1/1. After boiling for 3 h, the mixture was cooled and filtrated with a Buchner funnel and vacuum pump. The filtrated solution was freeze-dried to obtain the aqueous crude extract of Cordyline terminalis Kunth leaves, which was named aqCT.The aqCT solution was prepared at 1 wt% in DIW for phytochemical screening.2 and 6 g of KI in 100 mL of DIW [Alkaloid identification: Wagner’s reagent was prepared by dissolving 2 g of IL of DIW . The aqC3 solution 5 wt%, and concentrated sulfuric acid at a volume ratio of 1:1:1:1. If the interface of two layers appeared a reddish-brown color, there was the presence of cardiac glycosides [Cardiac glycoside identification (Keller–Kiliani test): The aqCT solution 1 wt% was mixed with glacial acetic acid, FeClycosides .Saponin identification: A foam test was performed to determine the presence of saponins . The aqCCoumarin glycoside identification: The aqCT solution 1 wt% (1 mL) was mixed with 1 mL of ethanol. This mixture was adjusted to basic pH by a few drops of potassium hydroxide solution (0.5 N in ethanol). Coumarin glycosides were present if the blue/violet/brown/green or yellow fluorescence appeared.3 solution (5 wt% in ethanol) [Phenolic compound identification: The aqCT solution 1 wt% (1 mL) was reacted with FeClethanol) . The reaSteroid identification: The aqCT solution 1 wt% (1 mL) was mixed with chloroform (1 mL), anhydric acetic (1 mL), and two drops of concentrated sulfuric acid. If there were steroids in aqCT, the mixture would turn to red, blue, and green color respectively.Phytosterol identification: Few drops of glacial acetic acid were added into the aqCT solution 1 wt% (1 mL). Then anhydride acetic (3 mL) and a few drops of concentrated sulfuric acid were added to the mixture. If the bluish-green color appeared, the presence of phytosterols was confirmed.Gelatin–polyethylene glycol–tyramine (GPT) was synthesized according to a previous study . In brieGPT solutions of 20 wt%, α-CD solutions of 14 wt% and 20 wt% were prepared in DIW. AqCT solutions were prepared in DIW at various concentrations, including 0.05 g/mL, 0.10 g/mL, 0.15 g/mL, 0.20 g/mL, and 0.25 g/mL. In the microtube, GPT of 20 wt% (75 μL), DIW (25 μL), and α-CD solutions of 14 wt% 300 μL) were mixed and sonicated for 15 min to fabricate the GPT3.75α-CD10.5 hydrogels. This mixture was kept at RT until no flow was observed. In the case of GPT/α-CD hydrogels encapsulating aqCT extract, the fabrication procedure was carried out as GPT3.75α-CD10.5 hydrogels, but various aqCT extract solutions replaced DIW (25 μL). For example, to form GPT3.75α-CD10.5aqCT3.1 hydrogels, GPT of 20 wt% (75 μL), aqCT solution of 0.05 g/mL (25 μL) and α-CD solutions of 14 wt% (300 μL) were mixed and sonicated for 15 min. The numerical part in hydrogel code indicated the final concentration of GPT and α-CD in wt%, and the final aqCT concentration in mg/mL. Similarly, other hydrogels were formed; the specific formulations and corresponding code are presented in 0 μL wereFor the scanning electron microscopy (SEM), all hydrogels in The release of polyphenols was carried out by direct contact between the gels and the media ,33. Six Similarly, the polyphenol release experiment of GPT3.75α-CD10.5aqCT5 gel samples was performed in different pH media. The pH 5.5 buffer was prepared from 272 g of sodium acetate and 6 mL of glacial acetic acid in 1000 mL of DIW. The pH 8.3 solution was prepared from 6.18 g of boric acid, 9.54 g of sodium tetraborate, and 4.38 g of sodium chloride in 1000 mL of DIW.2CO3 solution 700 mM (1.5 mL), the reaction was incubated at RT for 2 h. The optical density of reacted solutions was read at 765 nm using a UV–Vis spectrophotometer . The polyphenolic content in samples was calculated by the gallic acid standard curve.Folin and Ciocalteu’s method was utilized to measure the polyphenolic content . Gallic sample, and ODDPPH was of the DPPH solution and DIW. The scavenging percentage of each sample was calculated as the following formula:DPPH solution was prepared in ethanol at 0.5 mM , stored 4 cells/well. After 3 days of incubation at 37 °C under 5% CO2 atmosphere, all hDFBs attached and fully spread on the bottom surface of the well plate. To prepare the hydrogel samples for cell study, 400 μL of each hydrogel including GPT3.75α-CD10.5, GPT3.75α-CD10.5aqCT3.1, GPT3.75α-CD10.5aqCT6.2, GPT3.75α-CD10.5aqCT12.5, GPT3.75α-CD15aqCT9.4, GPT3.75α-CD15aqCT12.5, and GPT3.75α-CD15aqCT15.6 was respectively fabricated in Eppendorf tubes. DMEM (1 mL) was added to each hydrogel and incubated for 24 h. These hydrogel extracts were utilized to treat the hDFBs proliferated in a 48-well plate. The hDFBs were cultured with pure DMEM that was a control sample. The aqCT solution of 0.1 mg/mL was utilized to test the cytotoxicity of crude extract. After 24 h, all hDFBs were washed with DPBS carefully. WST-1 reagent and DMEM (1:1 volume ratio) were added into each hDFBs well and incubated for 2 h at 37 °C under 5% CO2 atmosphere. After, the WST-1-reacted media (100 μL) was transferred into the 96-well plate. Optical density (OD) was recorded at 450 nm by a microplate reader . A blank solution was prepared from DMEM and WST-1 reagent (1/1 volume ratio) without contacting cells. The viability percentage was calculated as the following formula:Human dermal fibroblasts (hDFBs) were seeded on a 48-well plate at a density of 10t-test [p-value was less than 0.05. Vice versa, p-value was more extensive than 0.05, and the difference between the two values was nonstatistical significant (NS).All experiments were carried out in triplicate. The obtained results were expressed as the mean ± standard deviation (SD). Statistical analyses were performed by the Student’s t-test . StatistCordyline terminalis Kunth leaf extract (aqCT). It was determined that GPT with a phenol content of 151.5 μmol/g and GPT/α-CD ratios of less than or equal to 0.5 could form the gel with α-CD through inclusion complex and hydrogen bonding. The gelation time was around one hour or longer depending on a decrease of α-CD amount. The aqCT solutions containing bioactive compounds  disturbed the physical gel networks due to interacting competition. Thus, GPT/α-CD/aqCT gels were formed more slowly than GPT/α-CD ones. By SEM observation, GPT/α-CD and GPT/α-CD/aqCT gels possessed microarchitecture and high porosity, but GPT/α-CD/aqCT had more irregular cavities than another. GPT/α-CD/aqCT gels could sustainably release natural polyphenols from a few nmol/mL to several μmol/mL depending on initial aqCT encapsulation and the used GPT/α-CD ratios. All GPT/α-CD/aqCT formulations have demonstrated to have high antioxidant activity through 100% scavenging DPPH radicals. Besides, those gels with or without aqCT extract were non-cytotoxicity. Their cell viability gained more than 85% versus DMEM media. Significantly, the GPT3.75α-CD10.5aqCT gels with aqCT amount of 3.1–12.5 mg/mL enhanced the cell proliferation more so than GPT3.75α-CD10.5 gel without extract. These findings would contribute to developing a biocompatible scaffold for drug delivery systems or other biomedical applications. However, these GPT/α-CD/aqCT systems were still gelled slowly, which should be improved further. Along with this, the GPT/α-CD/aqCT gels might be studied to integrate multi-properties, such as tunable mechanical strength, self-healing, adhesive, stimuli-responsive and antibacterial characteristics satisfying the requirements for advanced biomaterials.In summary, we presented an interesting supramolecular gel fabricated from gelatin–polyethylene glycol–tyramine (GPT) and α-cyclodextrin (α-CD) for encapsulating an aqueous


Mycoplasma mobile, a fish pathogen, exhibits gliding motility using ATP hydrolysis on solid surfaces, including animal cells. The gliding machinery can be divided into surface and internal structures. The internal structure of the motor is composed of 28 so-called “chains” that are each composed of 17 repeating protein units called “particles.” These proteins include homologs of the catalytic α and β subunits of F1-ATPase. In this study, we isolated the particles and determined their structures using negative-staining electron microscopy and high-speed atomic force microscopy. The isolated particles were composed of five proteins, MMOB1660 (α-subunit homolog), -1670 (β-subunit homolog), -1630, -1620, and -4530, and showed ATP hydrolyzing activity. The two-dimensional (2D) structure, with dimensions of 35 and 26 nm, showed a dimer of hexameric ring approximately 12 nm in diameter, resembling F1-ATPase catalytic (αβ)3. We isolated the F1-like ATPase unit, which is composed of MMOB1660, -1670, and -1630. Furthermore, we isolated the chain and analyzed the three-dimensional (3D) structure, showing that dimers of mushroom-like structures resembling F1-ATPase were connected and aligned along the dimer axis at 31-nm intervals. An atomic model of F1-ATPase catalytic (αβ)3 from Bacillus PS3 was successfully fitted to each hexameric ring of the mushroom-like structure. These results suggest that the motor for M. mobile gliding shares an evolutionary origin with F1-ATPase. Based on the obtained structure, we propose possible force transmission processes in the gliding mechanism. Mycoplasma species exhibit a unique gliding motility, which is necessary for their infection  and trajectories of gliding cells (right). All cells are gliding in the direction of the tapered end as indicated by a black arrow. For trajectories, video frames of every 0.2 s were colored differently from red to blue, and stacked for 4 s. Download FIG S1, PDF file, 0.1 MB.Gliding of Copyright © 2021 Toyonaga et al.2021Toyonaga et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 1-ATPase, respectively. MMOB1670 has an extra N-terminal region (amino acids 1 to 299), which is not present in the β subunit. The chains most likely contain the motor for gliding, because the gliding motility is coupled to ATP hydrolysis .–26.23–26–1Fo-ATPases, found in most organisms, are rotary motors that perform biological energy conversion 3 that rotates the central stalk penetrating the ring using ATP hydrolysis. Phylogenetic studies have shown that Mycoplasma cells have three F1-like ATPase clusters, which are referred to as type 1 to 3 ATPases  fraction; lane 4, supernatant after incubation in suspension buffer containing 137 mM NaCl; lane 5, peak fraction of Superdex 200 gel filtration chromatography. The bands of dimer components are marked by black triangles. (C) Gel filtration profile in monomer isolation using a Sephacryl S-400 HR column. The small peak is marked by a green triangle. (D) The F1 to F13 fractions in the gel filtration indicated by the green line in panel C were subjected to SDS-12.5% PAGE and stained with CBB. F6, which corresponds to the small peak in the gel filtration, was used for further analyses as the monomer fraction. Bands of dimer components are marked by black triangles. For panels A, B, and D, focused proteins were identified by PMF, and molecular masses are shown on the left. Download FIG S3, PDF file, 0.7 MB.Isolation profile of dimer (unit complex) and monomer fractions. (A) Solubility of the chain components. Pellet-1 fraction was treated with buffers containing the specified concentrations of NaCl and centrifuged. Pellet-1 fraction, the supernatants (S), and the pellets (P) were analyzed by SDS-12.5% PAGE. See Materials and Methods for details. (B) Isolation of dimer (unit complex). The fractions of each step were subjected to SDS-12.5% PAGE gel and stained with CBB. Lane 1, lysate of Copyright © 2021 Toyonaga et al.2021Toyonaga et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. mobile cells with 1% Triton X-100 and recovered the insoluble fraction by centrifugation .–26.M. moTo examine the assembly of these proteins, we applied the isolated fraction to gel filtration chromatography using a Sephacryl S-400 HR column, which can fractionate up to 8,000-kDa globular proteins . The promK of 74 μM at 25°C 3. Considering that the amino acid sequences of MMOB1660 and -1670 have high identity to the α and β subunits of F1-ATPase, respectively, the dimeric complex is likely an evolutionary related F1-ATPase. The distance between the centers of the two hexamers was 11.0 nm. The complex had 10 filamentous structures around the two hexamers, four of which appeared to form bridges across the two hexamers. The filamentous structures are unlikely artifact, because they showed common features in four independent averaged images  3. In pattern II, the two central peaks were positioned 11.2 nm apart  . In pattr frame) . The posr frame) , similarr frame) . These onm apart , bottom,nm apart , top, sunm apart , left. Tnm apart . We concnm apart . To visuglobules . These pglobules .10.1128/mBio.01414-21.7MOVIE S12 with 56 by 56 pixels. The video was played at 10 frames per s (fps). Download Movie S1, AVI file, 0.4 MB.HS-AFM movie showing pattern I particles. The particles were scanned at 10 fps. The scanning field was 70 by 70 nmCopyright © 2021 Toyonaga et al.2021Toyonaga et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01414-21.8MOVIE S22 with 56 by 56 pixels. The video was played at 10 fps. Download Movie S2, AVI file, 0.4 MB.HS-AFM movie showing pattern II particles. The particles were scanned at 10 fps. The scanning field was 70 by 70 nmCopyright © 2021 Toyonaga et al.2021Toyonaga et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01414-21.9MOVIE S32 with 56 by 56 pixels. The video was played at 10 fps. The peaks are indicated by the red triangles. Download Movie S3, AVI file, 1.4 MB.HS-AFM movie showing the shedding process of the peaks. The particles were scanned at 10 fps. The scanning field was 70 by 70 nmCopyright © 2021 Toyonaga et al.2021Toyonaga et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01414-21.10MOVIE S42 with 120 by 120 pixels. The video was played at 1 fps. The protrusions are indicated by the red triangles. Download Movie S4, AVI file, 0.2 MB.HS-AFM movie showing fluctuations in protrusions. The particles were scanned at 2 fps. The scanning field was 120 by 120 nmCopyright © 2021 Toyonaga et al.2021Toyonaga et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 1-ATPase, which is characterized by a 12-nm-diameter umbrella and a 3-nm-long stalk 3 from Bacillus PS3 (PDB identifier [ID] 6N2Y)  . The disd HS-AFM  and H. Td HS-AFM . The crod HS-AFM . A cavitd HS-AFM . Next, wd HS-AFM . Two shod HS-AFM , left wed HS-AFM . Previoumembrane , left  and the corresponding reprojection images (bottom) calculated from the 3D map of a chain are compared. Download Copyright © 2021 Toyonaga et al.2021Toyonaga et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. mobile gliding machinery evolved from F1-ATPase 3. Integrating available information, we can now describe the outline of the internal structure of the M. mobile gliding machinery  and the β-subunit homolog MMOB1670 (88.4 kDa) likely correspond to the smaller and larger subunits, respectively, of the hexameric ring of an Fhe dimer  and 4C. 8.7 kDa aric ring . In the ric ring  and 5F, 1-ATPase, in which the γ subunit is removed from the hexameric ring by optical tweezers  The affinity for ATP estimated by the saturation extent is comparable between the ATPase activity of the internal structure and the speed of the gliding head  Subing head . (c) Theing head , 53. (d)ing head , 24. (e)ing head .1-ATPase, the three catalytic sites in the hexameric ring cooperatively hydrolyze ATP, and each catalytic β subunit undergoes a conformational change that drives the rotation of the central stalk .The cultured cells were inserted into a tunnel chamber assembled with two coverslips and double-sided tape and observed by phase-contrast microscopy using an inverted microscope  , 19. MovM. mobile cells from 60 ml of culture medium were collected by centrifugation at 14,000 × g for 20 min and washed twice with phosphate-buffered saline (PBS) consisting of 8.1 mM Na2HPO4, 1.5 mM KH2PO4 (pH 7.3), 2.7 mM KCl, and 137 mM NaCl. Cells were resuspended in PBS to a 12-fold higher concentration than the culture and sonicated for 1 min at 24 to 27°C to be dispersed in microtubes using an ultrasonic generator . The cells were then treated with Triton solution  in a total volume of 10 ml. After gentle shaking for 1 h, the suspensions were centrifuged at 20,000 × g for 20 min, and pellets were collected and washed once with suspension buffer (PBS including 5 mM MgCl2). This fraction was pellet-1. Pellet-1 was then resuspended separately in suspension buffer adjusted to contain different concentrations of NaCl  by pipetting several times. After treatment for 8 h, suspensions were centrifuged at 20,000 × g for 20 min, and supernatants and pellets were collected for SDS-PAGE analysis.All procedures for fractionation were performed at 4°C unless otherwise noted, and focused protein bands were identified by peptide mass fingerprinting (PMF), as previously reported , 57. To g for 20 min and loaded onto a HiLoad 16/600 Superdex 200 column  equilibrated with 1 mM MgCl2 in PBS at a flow rate of 0.8 ml/min. The fractions were analyzed by SDS-PAGE and Coomassie brilliant blue (CBB) staining.For isolation of the dimer, pellet-1 from a 1.2-liter culture was resuspended in 5 ml suspension buffer by pipetting up and down and allowed to dissolve for 8 h. The soluble fraction was collected by centrifugation at 20,000 × 2 by pipetting and allowed to dissolve for 8 h. The soluble fraction was collected by centrifugation at 20,000 × g for 20 min and mixed with 1.5% (wt/vol) sodium cholate. After 7 h of incubation, the complexes were loaded onto a Sephacryl S-400 HR column (Cytiva) equilibrated with 0.7% sodium cholate, 20 mM Tris-HCl (pH 7.5), 250 mM NaCl, and 1 mM MgCl2 at a flow rate of 0.5 ml/min. The fractions were analyzed by SDS-PAGE and CBB and reverse staining , 250 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 1 mM MgClstaining , 59. Theg for 5 min.For isolation of the chain, pellet-1 from a 15-ml culture was resuspended in 60 μl suspension buffer. The suspension was then gently mixed with an equal volume of suspension buffer adjusted to contain 637 mM NaCl. The chain was recovered as the supernatant by centrifugation at 5,000 × 2 at a flow rate of 0.5 ml/min at 4°C. Thyroglobulin  was dissolved in gel filtration buffer and loaded onto the column as a size standard at a flow rate of 0.5 ml/min. The stoichiometry of protein complexes was estimated by densitometry of SDS-PAGE gels stained with CBB, using a scanner  and ImageJ (The dimer solution was loaded onto a Sephacryl S-400 HR column equilibrated with gel filtration buffer containing 20 mM Tris-HCl (pH 7.5), 200 mM NaCl, and 1 mM MgCl4, and 0.2% (wt/vol) Pb(NO3)2. The gels were rinsed once with water, and images were taken using an ImageQuant LAS 4000 mini (Cytiva). White precipitates were then dissolved by gentle shaking for 8 h at 24 to 27°C with 50% (vol/vol) methanol and 10% (vol/vol) acetic acid in water. The gels were restained with 0.025% (wt/vol) CBB G-250 and 10% acetic acid in water for 80 min at 24 to 27°C with gentle shaking and destained with 10% (vol/vol) ethanol and 10% acetic acid in water for 180 min at 24 to 27°C with gentle shaking. The gels were rinsed once with water, and images were taken using an ImageQuant LAS 4000 mini.BN-PAGE was performed according to the user manual of the NativePAGE Novex bis-Tris gel system . For BN-PAGE of sodium cholate (SC)-treated dimer, a dimer fraction was mixed with sodium cholate (1.5%) and incubated at 4°C for 9 h. When this sample was mixed with a sample buffer, NativePAGE 5% (wt/vol) G-250 sample additive was also added at 0.5% (wt/vol) to prevent protein aggregation. Thyroglobulin was dissolved in water and used as a negative control for the in-gel ATPase activity assay. For the in-gel ATPase activity assay , 61, nat2. ATPase activity was assayed by a continuous spectrophotometric method using a 2-amino-6-mercapto-7-methylpurine ribonucleoside–purine nucleoside phosphorylase reaction to detect released inorganic phosphate  , 150 mM NaCl, and 2 mM MgClentific) . The reaDimer solution was placed on a glow-discharged  carbon-coated grid  and incubated for 1 min at 24 to 27°C. The solution was then removed, and the grid was stained with 2% uranyl acetate (wt/vol) for 30 s. The stain was then removed, and the grid was air dried. To observe the monomer and chain, the grids were washed with water after 1 min of incubation and then treated as described for the dimer solution. Samples were observed using a transmission EM  at 80 kV, equipped with a FastScan-F214 (T) charge-coupled-device (CCD) camera , and images were captured at 2.58 Å/pixel.The contrast transfer function parameters for electron micrographs were estimated using Gctf . Further1-ATPase catalytic (αβ)3 from Bacillus PS3 (PDB ID 6N2Y)   were fitA0) and set-point amplitude were set at ∼2.5 nm and ∼0.8 × A0, respectively. The probe tip was grown on the original tip end of a cantilever through electron beam deposition and further sharpened using a radio frequency plasma etcher  under an argon gas atmosphere . The sample was deposited on a freshly cleaved mica disc glued to a glass stage beforehand. After 3.5 min, the stage surface was immersed in a liquid cell containing an observation buffer consisting of 20 mM Tris-HCl (pH 7.5), 50 mM KCl, and 2 mM MgCl2. Imaging was performed at 24 to 27°C. AFM images were processed using a low-pass filter to remove spike noise and make the xy plane flat and analyzed using Kodec software (version 4.4.7.39)  . SurfaceThe density map has been deposited in the Electron Microscopy Data Bank with accession code EMD-31520.


The crucial role of the residue C467 in the recognition and binding of the ASCT2 substrate glutamine, has been highlighted by structure/function relationship studies. The reconstitution in proteoliposomes of the human ASCT2 produced in P. pastoris is here employed to unveil another role of the C467 residue in the transport reaction. Indeed, the site-directed mutant C467A displayed a novel property of the transporter, i.e., the ability of mediating a low but measurable unidirectional transport of [3H]-glutamine. This reaction conforms to the main features of the ASCT2-mediated transport, namely the Na+-dependence, the pH dependence, the stimulation by cholesterol included in the proteoliposome membrane, and the specific inhibition by other common substrates of the reconstituted human ASCT2. Interestingly, the WT protein cannot catalyze the unidirectional transport of [3H]-glutamine, demonstrating an unspecific phenomenon. This difference is in favor of a structural conformational change between a WT and C467A mutant that triggers the appearance of the unidirectional flux; this feature has been investigated by comparing the available 3D structures in two different conformations, and two homology models built on the basis of hEAAT1 and GLTPh.The plasma membrane transporter ASCT2 is a well-known Na Then, a specific conformation of ASCT2 has been assigned to each step of the transport cycle , was further studied by analyzing the structures of ASCT2 in the various conformation of the transport cycle . The 3D rt cycle , as prevrt cycle A; only irt cycle B. +-dependent  iependent ; (iii) iependent A; and , the pPICZB vector, zeocin, Ni-NTA agarose resin were from Invitrogen ; PD-10 columns were from Cytiva Europe Gmbh ; L-[3H]-glutamine and L-[3H]-serine were from Perkin Elmer ; C12E8 was from TCI Europe ; cholesterol, Amberlite XAD-4, egg yolk phospholipids (3-sn-phosphatidylcholine from egg yolk), Sephadex G-75, L-glutamine, L-serine, and all the other reagents were from Merck Life Science . The P. pastoris wild type strain X-33 by electroporation [P. pastoris cells were selected using YPDS agar plate containing 2000 µg/mL zeocin. For large scale protein production, transformed P. pastoris cells were inoculated in the BMGY medium and grown at 30 °C under rotatory stirring. P. pastoris cells were centrifuged and resuspended at final OD of 1 in 250 mL BMMY medium containing 0.5% methanol. The cells were placed in a 2 L conical flask and grown in the same medium at 30 °C for 3 days under rotatory stirring. Fresh methanol was added every 24 h. To isolate the P. pastoris membrane fraction, 30 g of cells were resuspended in 300 mL buffer containing 50 mM Tris HCl pH 7.4, 150 mM NaCl, 2 mM β-mercaptoethanol, and 0.5 mM PMSF. This cell suspension was loaded in the chamber of the bead beater  for disruption with glass beads (0.5 mm) for 5 min cycle, reaching 90% of cell disruption. Then, the broken cell suspension was centrifuged in a JA10 rotor at 10,000× g for 30 min at 4 °C to remove cell debris and unbroken cells. The supernatant, containing membrane and cytosolic fraction, was centrifuged again in a JA 30.50 rotor at 45,000× g for 90 min at 4 °C. The resulting membrane pellet was washed with urea buffer  and subjected to another centrifugation cycle, as described above. To reach a concentration of about 400 mg/mL, the washed membrane fraction was resuspended in a buffer composed of 25 mM Tris HCl pH 7.4, 250 mM NaCl, 2 mM β-mercaptoethanol, and 10% glycerol and homogenized using a potter homogenizer. Membrane fractions were stored at −80 °C before solubilization.The C467 residue was mutated to Ala by PCR overlap extension method, as previously described . Then, tporation ,31. Tran12E8 (w/w). The membranes were incubated with the solubilization buffer for 3 h at 4 °C under rotatory stirring. The solubilized membranes were centrifuged at 18,000× g for 45 min at 4 °C and the supernatant was applied to 2 mL Ni-nitrilotriacetic acid (NTA) agarose resin pre-equilibrated with a buffer containing 20 mM Tris HCl pH 7.4, 300 mM NaCl, 10% glycerol, 6 mM β-mercaptoethanol, 0.03% C12E8, 1 mM L-glutamine, and 50 mM imidazole. The resin was incubated, with gentle agitation, at 4 °C over-night to allow specific binding of recombinant hASCT2. After incubation, the Ni-NTA resin was packed by gravity into a glass-column and washed with 30 mL of the above described equilibration buffer. Protein was eluted in a buffer containing 20 mM Tris HCl pH 7.4, 300 mM NaCl, 10% glycerol, 6 mM β-mercaptoethanol, 0.03% C12E8, 1 mM L-glutamine, and 500 mM imidazole. Fractions of 1 mL were collected, and 2.5 mL of the purified protein was desalted on a PD-10 desalting column, pre-equilibrated with a buffer composed of 20 mM Tris HCl pH 7.4, 100 mM NaCl, 10% glycerol, 6 mM β-mercaptoethanol, 0.03% C12E8, and eluted in 3.5 mL of the same buffer.For large-scale solubilization and purification of hASCT2 WT protein and mutant C467A, about 1.2 g of washed membranes (400 mg/mL) were solubilized using a buffer containing 25 mM Tris HCl pH 7.4, 250 mM NaCl, 6 mM β-mercaptoethanol, 10% glycerol, 1 mM L-glutamine, and 2% CThen, 7.5 mg of cholesterol (unless differently indicated in the figure legends) were added to 100 mg of egg yolk phospholipids and solubilized with 1 mL chloroform. After incubation under rotatory stirring (30 °C 5 min 1200 rpm) solution is dried using rotavapor. The dried lipid film was resuspended in 1 mL water to a 10% final concentration. Unilamellar liposomes were prepared by two sonication cycles of 1 min  with a Vibracell VCX-130 sonifier, as previously suggested , and use12E8; 100 µL of 10% (w/v) egg yolk phospholipids , together with 50 mM Na-Gluconate, at room temperature. The transport reaction was stopped at the indicated times adding 1 mM HgCl2. In control sample, the same inhibitor was added at time zero, according to the inhibitor stop method previously described [−1⋅mg protein−1, was measured by stopping the reaction after 20 min, i.e., within the initial linear range of radiolabeled substrate uptake. To remove the external compounds, 600 µL of proteoliposomes was passed through a Sephadex G-75 column (0.7 cm diameter × 15 cm height) pre-equilibrated with a buffer containing 20 mM HEPES Tris pH 7.0. The eluate was collected and divided in 100 µL samples used for transport assay. The uptake was started by adding 500 µM [escribed . Then, 1p < 0.05 and p <0.001 as specified in the figure legends.Results are expressed as means ± SD. The software GraFit 5.0.13 was used to plot data in Michaelis–Menten and in first-order rate equations. Then, kinetic parameters and initial transport rate were calculated from plotted data. The statistical significance was assessed by Student’s test for Protein amount was measured by densitometry of SDS-PAGE stained by Coomassie Blue, using the ChemiDoc imaging system equipped with Quantity One software (Bio-Rad Laboratories). Molecular visualization of 3D structures was performed with the UCSF Chimera 1.13.1 software  (Resourc


Frankliniella occidentalis is an invasive insect pest that incites damage to ornamental and agronomic crops on a global scale. In this study, the effects of temperature on gene expression and enzyme activity were studied for superoxide dismutase (SOD), peroxidase (POD), and glutathione-S-transferase (GST) in F. occidentalis. SOD, POD and GST enzyme activity increased significantly at 35–37 °C but declined as the temperature increased to 41 °C. In a time course study at 35 °C, SOD, POD and GST activities were significantly elevated at 0.5, 1 and 2 h in comparison to the control at 26 °C. Expression patterns were evaluated for the three antioxidant genes under high and low temperature stress. In a time course study at –4 °C, SOD, POD and GST expression peaked at 1 h and declined at 2 h of exposure. In contrast, when transcription was monitored at 35 °C, expression was lowest at 1 h and increased at 2 h. The results provide data that will be useful in deciphering the role of antioxidant enzymes in the adaptation of F. occidentalis to climate change. In mainland China, F. occidentalis was initially found in Beijing in 2003 (The western flower thrips (WFT), enhouses . In addienhouses . WFT is enhouses . Accordi in 2003  and has  in 2003 .F. occidentalis  or low  temperatures for 1 h in glass tubes as described . Following thermal stress, larvae were incubated at 26 °C for 30 min and used a brush to touch it gently, thrips would be identified as surviving if it respond to the stimulus. Survivors were frozen in liquid nitrogen and stored at –80 °C for future use. Four replicate pools were used for each temperature and time period.escribed . ThroughThe assay kit used for protein extraction was from Nanjing Jiancheng Bioengineering Institute, Jiangsu, China. Treated samples were homogenized in 0.9% saline and then centrifuged at 2,500 × rpm for 10 min . SupernaA340 with a microplate spectrophotometer , and results are shown as μmol GSH-CDNB/min/mg protein.POD and SOD activities were assessed with commercially available kits . AbsorbaF. occidentalis as recommended by the manufacturer . RNA quality and concentration were determined, and cDNA was generated from total RNA with the First Strand cDNA Synthesis Kit  as outlined previously , high temperature (40 °C) and normal temperature control (26 °C) at the Shanghai Biotechnology corporation by Illumina sequencing platform. The RNA-seq data were deposited with the Sequence Read Archives PRJNA73493 at NCBI. To remove adapter contamination, low-quality bases and bases artificially introduced during library construction, we trimmed all raw reads using Trimmomatic 0.32 (http://www.usadellab.org/cms/index.php?page=trimmomatic) before transcript assembly, while the unpaired reads were discarded . According to the obtained gene fragments, the corresponding primers  was conducted using the protocols described by –ΔΔCt method (–ΔΔCt. The lg(X) method was used to transform the expression level data for normality and homogeneity of variance. Significant differences were detected by one-way analysis of variance (ANOVA) and Duncan’s multiple comparisons test. Data were analyzed with SPSS v. 16.0 and considered significant at P < 0.05.qRT-PCR data analyzing was conducted in Bio-Rad CFX Manager 3.1 software. The average Ct values of biological replicates were used to calculate the relative expression levels. The results of qPCR were analyzed with the 2t method . FirstlyF6,19 = 4.245, P < 0.05)  .SOD activity increased with rising temperature from 31 to 37 °C and was highest at 37 °C. The activity of SOD activity began to decline at 39 °C, and the level at 41 °C was significantly lower than 37 °C ( < 0.05) . A simil < 0.05) . GST act < 0.05)  and begaF3,10 = 10.005, P < 0.05; POD: F3,12 = 8.037, P < 0.05; GSTs: F3,10 = 5.815, P < 0.05). No significant differences in antioxidant activity were detected between 0.5, 1 and 2 h of exposure  when insects were exposed to 35 °C for 0.5, 1 and 2  . In contrast, POD expression levels at 33, 37 and 39 °C were significantly higher than the control at 26 °C; however, except that the expression level was significantly decreased at 35 °C, there was no significant difference in the expression level of 31 °C compared with the control    . With th < 0.05) . GST1 ex < 0.05) . All thrSOD expression showed a significant decline at all temperatures relative to the control at 26 °C; although the expression of –4 °C was higher than other temperatures, it was also inhibited by low temperature . POD expression was also strongly inhibited, with the lowest expression appeared at –6 °C.    . Like SO < 0.05) .SOD expression decreased significantly when 2nd instar larvae were exposed to 35 °C for 0.5, 1 and 2 h  and was lowest at the 1 h exposure period (POD expression was significantly upregulated at 0.5 and 2 h and was higher than expression levels at 0 (control) and 1 h, with the peak appeared at 2 h.  .Compared to the control , e period . POD exp < 0.05) . GST1 exr to POD . AlthougSOD: F3,10 = 201.898, P < 0.05; POD: F3,11 = 204.420, P < 0.05; GST1: F3,10 = 72.835, P < 0.05). Interestingly, all three genes showed a peak in expression after a 1 h exposure to −4 °C; however, it should be noted that expression at 1 h was lower than the control (After exposure to −4 °C, the expression levels of the three antioxidant genes decreased significantly when compared to the control ( control .Bactrocera dorsalis, Bombyx mori, Mononychellus mcgregori, Diaphorina citri and Neoseiulus cucumeris (F. occidentalis (2O2. Although increased levels of antioxidant enzymes suggests a defensive function of these enzymes in counteracting the negative effect of ROS, there were no significant differences in SOD, POD or GST activity at 0.5, 1.0 and 2.0 h of exposure to 35 °C (Insects are poikilotherms that are greatly impacted by temperature fluctuations . When exucumeris . In a prdentalis . The incto 35 °C . This miSOD, POD, GST1 and related enzymes in Mythimna separate, Apis cerana cerana and Helicoverpa armigera (CAT expression in F. occidentalis (POD in this study might be attributed to increased expression of CAT. In the case of SOD and GST1, thermal stress may induce the synthesis of unknown substances that could inhibit transcription. Further research is needed to validate or disprove these conjectures.Many researchers have shown that temperature stress can lead to changes in antioxidant gene expression in insects . Previouarmigera . These rarmigera . A previdentalis ; therefoPOD, CAT and SOD expression patterns were not necessarily correlated with enzyme activity during high temperature stress in Mononychellus mcgregori (Bombyx mori, carboxylesterase activity was not correlated with gene expression (F. occidentalis to thermal stress.Differential regulation of antioxidant genes and enzymes has been reported in insects; for example, cgregori . In larvpression . ElevateF. occidentalis, and provide new avenues for further study of antioxidant mechanisms and physiological responses of F. occidentalis. The inconsistencies between gene expression and enzyme activity further illustrate the complexity of thermal adaptation in F. occidentalis. Future multidisciplinary research in genomics, transcriptomics, proteomics, and metabolomics will help explain the underlying mechanisms of thermal adaptation in F. occidentalis.This study reveals differential regulation of antioxidant gene expression and enzyme production in response to thermal stress. The results confirm the importance of antioxidant enzymes in modulating the response to thermal stress in 10.7717/peerj.12089/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.12089/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.12089/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.12089/supp-4Supplemental Information 4Click here for additional data file.


X-ray-induced photodynamic therapy is based on the energy transfer from a nanoscintillator to a photosensitizer molecule, whose activation leads to singlet oxygen and radical species generation, triggering cancer cells to cell death. Herein, we synthesized ultra-small nanoparticle chelated with Terbium (Tb) as a nanoscintillator and 5-(4-carboxyphenyl succinimide ester)-10,15,20-triphenyl porphyrin (P1) as a photosensitizer (AGuIX@Tb-P1). The synthesis was based on the AGuIX@ platform design. AGuIX@Tb-P1 was characterised for its photo-physical and physico-chemical properties. The effect of the nanoparticles was studied using human glioblastoma U-251 MG cells and was compared to treatment with AGuIX@ nanoparticles doped with Gadolinium (Gd) and P1 (AGuIX@Gd-P1). We demonstrated that the AGuIX@Tb-P1 design was consistent with X-ray photon energy transfer from Terbium to P1. Both nanoparticles had similar dark cytotoxicity and they were absorbed in a similar rate within the cells. Pre-treated cells exposure to X-rays was related to reactive species production. Using clonogenic assays, establishment of survival curves allowed discrimination of the impact of radiation treatment from X-ray-induced photodynamic effect. We showed that cell growth arrest was increased (35%-increase) when cells were treated with AGuIX@Tb-P1 compared to the nanoparticle doped with Gd. Glioblastoma multiforme (GBM) is one of the main incurable brain tumors, mainly due to the presence of infiltrated cells within the parenchyma, responsible for GBM recurrence into the surrounding brain tissue . The con®), 5-aminolevulinic acid , m-tetrahydroxyphenylchlorin  and benzoporphyrin derivative monoacids ring A .Alternative therapeutic strategies have been developed, notably photodynamic therapy (PDT). PDT appears as an innovative technology being investigated to fulfil the need for a targeted cancer treatment that may reduce recurrence and extend survival with minimal side effects ,4. It ai2O3 coated with a polysiloxane layer or silica-doped with lanthamide [Compared to radiotherapy, the light irradiation used in PDT is less energetic and it cannot penetrate deeply enough into the tumor, as most tissue chromophores absorb visible light commonly used in clinical practice ,6. The pnthamide ,29,30,31®, were developed [Among them, ultra-small Gadolinium (Gd) based nanoparticles, namely AGuIXeveloped . The naneveloped . Moreoveeveloped . The radeveloped ,36; or aeveloped ,37. In Xeveloped . The syn−1 dose rate. Moreover, cell exposure to AGuIX@Tb-P1 improved the effect on cell growth arrest when it was compared to similar treatment with AGuIX@Gd or AGuIX@Gd-P1.Therefore, in order to use the AGuIX platform for X-PDT, we suggested the replacement of Gd in the Gd-based AGuIX nanoparticle by Tb as a nanoscintillator and the grafting of P1 . In thes14 M−1 nm4 cm−1. The corresponding Förster radius was found to be 2.5 nm (from 1 up to 10 nm). Moreover, Tb luminescence lifetime was 390 µs, long enough to allow energy transfer to P1. Thus, Tb luminescence in presence of P1 showed an exponential decay of its fluorescence intensity  were detected at 485, 545, 590, and 620 nm, respectively. For these different Tb cation emission peaks, the correlation coefficients between tube currents and peak intensities were 0.97, 0.99, 0.99, and 0.84, respectively , a singlet oxygen quencher, confirmed that the type II-PDT reaction (singlet oxygen generation) was mainly involved in X-PDT as SOSG and APF fluorescence signals were mostly inhibited.We highlighted reactive species and singlet oxygen production using fluorescent probes, respectively, SOSG a and APF50 was estimated at 34.8 ± 9.9 µM after 24 h and 10.4 ± 3.4 µM after 72 h-treatment duration. In addition, IC50 was similar when GBM cells were exposed to AGuIX@Tb with IC50 estimated at 1.73 ± 0.3 and 1.56 ± 0.1 mM, after 24 and 72 h exposition, respectively. We finally tested the effect t of increasing concentration  of AGuIX@Tb-P1 and AGuIX@Gd-P1  to a fluence range of 2.5 to 10.0 J cm−2, corresponding to an exposition duration of 2 min to 38 min). At the dose of 1 µM, cell growth inhibition was obtained with a fluence applied at 10.0 J cm−2 (−2 (90%-inhibition) (−2 (20%-inhibition). Cell growth arrest increased with fluence up to 10.0 J cm−2 (80% inhibition) , and a fluence higher than 2.5 J cm−2  b. Similaibition) a. Since ibition) c, when c5 J cm−2 b, the apSimilarly, we studied whether X-ray irradiation promoted U-251 MG cell growth arrest by photodynamic-mediated effect. Reactive species generation was quantified immediately after cell exposure to X-ray irradiation a. X-ray −1, respectively. These conditions were chosen since cell clone formation was mainly inhibited when cells were pre-treated with 2.5 µM AGuIX@Tb-P1 and irradiated for X-ray doses higher than 2.0 Gy at 320 kVp (p = 0.0018) was found at 2.0 Gy between scintillating (AGuIX@Tb-P1) and non-scintillating (AGuIX@Gd-P1) nanoparticles, when X-ray energies were set to 320 kVp corresponding to a mean 116 keV X-ray energy; DMF was estimated of 0.650 versus 0.876. The DER at 2.0 Gy was estimated at 1.54. Finally, for the same Gy dose, the enhanced factor was 35% when cells were treated with AGuIX@Tb-P1. Moreover, we did not find any significant change between results from cells treated with AguIX@Gd-P1 and those obtained of the cells treated with AguIX@Gd for the same experimental conditions porphyrin with low irradiation source; or with SrAl2O4:Eu2+ nanoparticles; collectively, allowing to the conclusion that tumors cells undergo cell death by cumulative/synergistic effects of irradiation treatment and X-ray induced photo-treatment [We assessed whether AGuIX@Tb-P1 altered U-251 MG cell survival by PDT and X-PDT. We used U-251 MG cells whose behavior is close to that of GBM in situ . Collectreatment ,56,57,58reatment , whereas−1 dose rate. However, it has to be noted that such a strategy should not be considered with external radiotherapy but with low-energy devices (corresponding to a few hundred of keV), such as radiograph tubes, to ensure high energy conversion and singlet oxygen production while lowering exposure dose. Such investigations are currently being conducted in our group.In conclusion, we showed that replacement of Gd by Tb in the initial AGuIX design leads to a promising nanoparticle for X-PDT in in vivo experimental. Moreover, we demonstrated that chelated Tb nanoparticles react linearly to X-ray energy (at least up-to 320 kVp) and flow, and they were able to activate P1 to produce singlet oxygen. The constructed nanoparticle was not toxic, while remaining unexposed to light. In vitro experiments confirmed the interest of these AGuIX design, notably at a 3.0 Gy.minFluorescent probes, 3′-(p-aminophenyl) fluorescein (APF), 2′,5′(di-acetate) dichlorofluorescein (DCF2-DA), Singlet Oxygen Sensor Green™ (SOSG) and propidium iodide were from Molecular Probe . 3--2,5-diphenyl tetrazolium (MTT) was purchased from Acros Organics . 5-(4-carboxyphenyl succinimide ester)-10,15,20-triphenylporphyrin (P1) and zinc(II) 5-(4-carboxyphenyl)-10,15,20-(tri-N-methyl-4-pyridyl) porphyrin trichloride (ZnPy3P1) were purchased from Porphychem . Other reagents were of analytical grade.® polysiloxane core surrounded by 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)/metal cation (3+) complexes, covalently grafted to the inorganic matrix. The cations used were Terbium  and Gadolinium . P1 was covalently grafted as a photosensitizer (AGuIX@Tb-P1 or AGuIX@Gd-P1).We used four different nanoparticles, based on the same AGuIX® membranes with a cut-off at 5 kDa . The final volume was 400 mL with the purification factor of 1000. The pH of the solution was adjusted to 7.4 with 1 M NaOH solution. The final amount of free DOTA was measured by europium titration, as described previously [® membranes . The volume obtained was 15 mL and the purification factor was 100. The Tb chelation yield was determined by Inductively Coupled Plasma–Mass Spectroscopy and was estimated at 60%. Finally, the pH was adjusted at 7.4, before freeze-drying.Ultra-small siloxane particles were obtained in a two-step synthesis procedure as described previously . In brieeviously . Free DO2O/DEG/DMSO was 13/53/34. The mixture was left stirring at 40 °C for 12 h in the dark and was filtrated through VivaSpin® membranes . The purification factor was 100 and the final volume was 15 mL. The Tb chelating yield, determined by Inductively Coupled Plasma–Mass Spectroscopy, was 47%. The P1 coupling yield was estimated at 29% by recording the absorbance at 520, 550 nm, 590 and 650 nm Q bands. The final Tb:P1 ratio was 16 moles Tb for 1 mole P1. All concentrations of nanoparticle containing P1 will be referred to thereafter as the concentration of P1. A stock AGuIX@Tb-P1 suspension was prepared in water and was 3 mM P1 equivalent; the solution of AGuIX@Tb was 37.5 mM  in water.P1 was grafted following the protocol described previously ,39. In sDirect measurements of the size distribution of the nanoparticles suspended in any medium were performed via Zetasizer NanoS DLS . Prior to the experiment, the nanoparticles were diluted in 0.01 M NaCl (pH 7.4).TM Buffer Solution, ThermoFischer Scientific, Ilkirch, France).Potential ζ measurements were carried out with a Zetasizer Nano-Z  equipped with a He-Ne laser at 633 nm. Before measurement, the nanoparticles were dispersed in 0.01 M NaCl and buffer solutions . Fluorescence spectra were recorded on a Fluorolog FL3-222 spectrofluorometer  equipped with a 450 W Xenon lamp and thermostated cell compartment (25 °C), a UV-visible photomultiplier R928 , and an InGaAs infrared detector . The excitation beam was diffracted by a double ruled grating SPEX monochromator (1200 grooves/mm blazed at 330 nm). The emission beam was diffracted by a double ruled grating SPEX monochromator (1200 grooves/mm blazed at 500 nm). Singlet oxygen emission was detected through a double ruled grating SPEX monochromator (600 grooves/mm blazed at 1 µm) and a long-wave pass (780 nm). All spectra were measured in four-face quartz vials. All the emission spectra (fluorescence and singlet oxygen luminescence) have been displayed with the same absorbance (less than 0.2) with the lamp and photomultiplier correction.3+) to P1. Moreover, Tb luminescence decay profile was recorded using a Fluorolog spectrofluorometer; the excitation wavelength was set at 351 nm and the emission peaks were scanned in the 400–690 nm region. The luminescence lifetime of Tb alone or in mixture with P1 was recorded using lifetime Fluorolog. We assessed the 545 nm peak decay as it is the highest Tb fluorescence peak. If relevant, we computed the quenching constant (expressed as L.mol−1 s−1) as Kq = K/τ0, where K is the Stern-Volmer constant which was graphically determined; τ0 is the Tb fluorescence lifetime without photosensitizer.Spectral overlap, as well as Förster radius, was computed to characterize the energy transfer from the Tb cation  with a tungsten anode. Photons were produced by X-ray tubes and produced continuous energy distribution. The tube parameters were set from 25 up to 320 kVp (id est mean photon energies of 8 and 116 keV) with the current set from 0.5 up to 12.5 mA . A 2 mm ® 220–1600 nm; cat.no. 952010051, Eppendorf, Hamburg, Germany). Emission spectra were recorded at different times to assess photonic density configurations on the Tb scintillation performance. Each measurement was repeated 7 times and all spectra were subtracted with the same solution spectrum obtained without irradiation. When varying X ray energy, we set the tube current to maximum and we set the voltage to 320 kVp when we assessed the tube current on the AGuIX@Tb response. A linear correlation coefficient was computed to characterize the relation between AGuIX@Tb peaks intensities, exciting photons energy (X ray kVp) and X ray flow (X ray mA), respectively. The energy transfer from the nanoparticles to a photo-agent was assessed with setting irradiation parameters to 320 kVp and 12.5 mA .An optical fiber was inserted inside the irradiation chamber, in front of the vial containing AGuIX@Tb solutions to gather emission fluorescence photons. Emission spectra were recorded with an USB2000 spectrometer . This versatile high-resolution spectrometer (FWHM = 3.5 nm) is an optical instrument based on a diffraction grating and a one-dimensional CCD detector array. Integration time was set to 5 s, the spectrum bandwidth ranged from 340 to 820 nm and the optical fiber was placed across from a transparent vial  prepared in the same buffer, to a final concentration at 10 mM. Irradiation was set to 320 kVp, 12.5 mA, and source-surface distance adjusted to yield a 3.0 Gy min−1 dose rate. Fluorescence emission was detected spectroscopically at 515 and 525 nm for APF and SOSG, respectively. Home-made software allowed long acquisition times and synchronization between laser illumination and signal recording. Integration time was set to 100 ms and time points were acquired every 5 min during 20 min. Moreover, P1 at 100 µM was irradiated without a nanoscintillator with the same parameters to validate the absence of the direct excitation by X-rays.The reaction mixture was prepared in 30 mM Tris/HCl (pH 7.4) containing 400 µM AGuIX@Tb-P1 or 45 mM AGuIX@Tb and 5 µM APF or 10 µM SOSG probe. Singlet oxygen quenching was achieved by addition of sodium azide (NaNv/v) heat-inactivated (30 min at 56 °C) fetal calf serum , 1% (v/v) non-essential amino acid (Invitrogen), 0.5% (v/v) essential amino acid (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 1% (v/v) vitamin (Invitrogen) 0.1 mg.mL−1 of L-serine, 0.02 mg.mL−1 L-asparagine (Merck-Sigma), and 1% (v/v) antibiotics  (Merck-Sigma). The cells were seeded routinely at 105 cells mL−1 and cultivated at 37 °C in a 5% CO2 humidified atmosphere .Human U-251 MG  glioblastoma-derived cells were cultivated in Roswell Park Memorial Institute medium (RPMI) without phenol red, containing 10% (4 cells/well and left growing for 24 h. Cells were treated with increasing concentrations of ZnPy3P1 (up to 400 µM) and AGuIX@Tb (0.1 to 2 mM) over three days. In addition, glioblastoma cells were exposed to increasing concentrations of AGuIX@Tb-P1 or AGuIX@Gd-P1 (1 to 10 µM) for 72 h. At the time, the medium was discarded and replaced by 100 µL of complete medium containing 0.5 mg mL−1 MTT. The plates were incubated for 2 h at 37 °C and formazan crystals obtained were dissolved by adding 100 µL of DMSO. The plates were read at 540 nm . Results are expressed relative to those obtained from untreated cells (control), taken as 100. They represented quadruplicate determinations from two independent experiments (n = 8).Impact of AGuIX@Tb or ZnPy3P1 on U-251 MG cell survival was assessed by the MTT procedure, based on the measurement of mitochondrial succinate dehydrogenase activity (EC 1.3.5.1) . Cells w5 cells/well and left to grow over 48 h. Cells were treated with increasing concentrations of nanoparticles (0.5 to 5.0 µM AGuIX@Tb-P1 or AGuIX@Gd-P1) over 48 h. After incubation, cell layers were washed with 2 mL of Dulbecco’s Phosphate buffer saline  and cells were suspended with 0.5 mL of 0.05% (w/v) Trypsin/0.02% (w/v) EDTA solution (Invitrogen) per well for 5 min at 37 °C. Complete medium (0.5 mL RPMI containing 10% (v/v) fetal calf serum) was then added. The cell suspension obtained was centrifuged at 1000 g for 5 min at 4 °C. Cell pellets were washed with 1 mL of DPBS and centrifuged again. Cells were suspended finally in 0.5 mL of DPBS and left on ice. Fluorescence of P1 was measured in 5000 cells/sample by flow cytometry , with excitation/emission settings at 638 nm and 660/30 nm. Results obtained from nanoparticle uptake were expressed relative to those obtained reaching the maximum of nanoparticle absorption taken as 100. Results are expressed as mean ± SD from triplicate determinations from 3 independent experiments.U-251 MG cells were seeded in 6 well-plates at 10−2. Cell layers were exposed to 0.5 to 10.0 J cm−2, corresponding to an exposition duration of 2 min to 38 min. X-ray irradiation was performed using the X-ray Irradiator X-RAD 320 , using tube parameters set at 160 and 320 kV (mean photons energies of 58 and 116 keV) with current set at 10 mA. A 2 mm Al filter was used to remove low energy photons. The dose rates were 1.5 and 3.0 Gy min−1 for 160 and 320 kVp respectively. The doses delivered were 0.5, 2.0, 4.0, and 5.0 Gy for both dose rates.Cell exposure to a red light was performed at 630 nm with a Laser diode  at 0.7 W, corresponding to an irradiance at 4.54 mW.cm5 cells/well. Cells were left to grow over 48 h, then, treated in the presence of 1 µM AGuIX@-complexes for 24 h. Cells were washed with 2 mL of DPBS. Each well was filled again with complete medium before light exposition or X-ray irradiation. Reactive species generation measurements were achieved post treatment over 1 h. At each time point, cell medium was changed by 2 mL of pre-warmed medium containing 50 µM DCF2-DA for 30 min at 37 °C. Cells were then successively harvested by trypsination, washed with DPBS, and suspended in 0.5 mL DPBS before flow cytometry analysis. Reactive species generation was measured in 5000 cells/sample by flow cytometry with excitation/emission settings at 488 nm and 520/30 nm. Cell death was quantified also using propidium iodide in kinetic uptake experiments. Cells were treated with 20 µM propidium iodide (diluted in DMSO) added to the medium. Propidium iodide-positive cells were numbered by flow cytometric analysis with excitation/emission settings at 488 nm and 620/630 nm (FL3), respectively. Results represent the median of fluorescent peak. Results are expressed relative to those obtained from untreated cells, taken as 100. Results are expressed as mean ± SD of triplicate determinations from 3 independent experiments.U-251 MG cells were seeded in 6 well-plates at 2 × 10v/v) formol (pH 7.4) at room temperature for 15 min, washed with 1 mL of DPBS, and stained for 30 min with 0.05% (w/v) crystal Violet solution prepared in DPBS and containing 25% (v/v) methanol. Finally, cells were washed three times with 2 mL of bi-distilled water. Cell clones obtained were analysed after picture capture  and ImageJ quantification . Image analysis was performed with well area taken as 862 mm2. Cell clone counting was improved by background subtraction. Data from untreated and treated cell conditions were compared and expressed as the mean ± SD (n = 12).The clonogenic assay was performed in 6 well-plates with U-251 MG cells seeded at 500 cells/well. Cells were then treated in the presence of 16.6 µM of AGuIX@Tb, 25 µM AGuIX@Gd, 1 µM AGuIX@Tb-P1 or 1 µM AGuIX@Gd-P1  for 24 h at 37 °C. After incubation, cells were washed with 2 mL of DPBS. 2 mL of complete medium were added in each well before X-ray radiation or red-light exposition. Cells were left to grow at 37 °C for 7 days. At time, cell clones were successively washed with 2 mL DPBS, fixed with 1 mL of 4% (D = exp (−(αD + βD2)), where survival fraction is defined as SFD = (plating efficiency at the dose D)/(platting efficiency at 0 Gy); D corresponds to the Gy dose; α and β, are determined from the established semi log curve, as SFD = f (Gy dose). The effects of radiation alone or X-PDT, related to untreated cells or exposed to each AGuIX@NP, were compared by calculating the dose modifying factor (DMF) and the survival fraction at 2.0 Gy (SF2). We also determined the Dose Enhanced Ratio (DER) at 2.0 Gy. DER is defined as the ratio of SF2.0 Gy calculated for untreated cells to that calculated of treated cells after irradiation (DER = SF2.0 Gy (control cells)/SF2.0 Gy (treated cells)). We finally calculated the enhanced factor, expressed in percentage, as EF (%) = 100 × (SF2.0 Gy (control cells) − SF2.0 Gy (treated cells))/SF2.0 Gy (control cells)).Survival fraction (SF) was calculated using the linear quadratic (LQ) model, based on the equation: SFp < 0.05. Results obtained from clonogenic assays were analyzed using the Kruskal-Wallis test (with α = 0.05), and post-hoc by the Mann-Whitney test (α = 0.05) for unpaired groups.Results obtained were analyzed using the Kruskal-Wallis test (α = 0.05) and post-hoc Dunn’s test (α = 0.05) for paired groups. Any difference was considered significant at


Immunohistochemistry was used to detect the protein expression of MMP-9, VEGF, and VEGFR2. The contents of HIF-1α, NLRP3, caspase 1, IL-1β, IL-6, and TNF-α in the serum were determined by ELISA. Our study showed that HQCFT and HQCFT-SR could improve the pathological condition and reduce the infarcted area of the brain tissue in a rat model. In addition, HQCFT and HQCFT-SR significantly decreased the expression levels and serum contents of NLRP3, caspase 1, IL-1β, IL-6, and TNF-α; increased the expression levels of the VEGF and VEGFR2 proteins; and obviously reduced the serum content of HIF-1α. Importantly, the cytokines in brain tissue and serum from the HQCFT group exhibited better efficacy than those from the HQCFT-SR group. HQCFT exerted significant angiogenic and anti-inflammatory effects in rats subjected to middle cerebral artery occlusion (MCAO); these effects can be attributed to the guiding and enhancing effect of SR.Huangqi Chifeng Tang (HQCFT), a traditional Chinese formula of three herbs, has been used to treat cerebral infarction (CI). Saposhnikoviae Radix (SR) was designed as a guiding drug for HQCFT to improve its angiogenic and anti-inflammatory effects. In this study, TTC staining was used to detect the area of CI. H&E staining was used to detect the histopathologic changes in the cerebral tissue. Western blotting was performed to detect the protein expression of NLRP3, caspase 1, IL-1 Huangqi Chifeng Tang (HQCFT), a traditional Chinese herbal formula, was documented in “Yi Lin Gai Cuo”  by Wang Qingren in 1830 AD –4, whichSaposhnikovia divaricata (Turcz.) Schischk., is commonly used to treat colds, headaches, rheumatic arthralgia, and rubella-associated itching. It has been reported that SR can increase the distribution of atractylenolide I, paeoniflorin, and hesperidin in the liver, spleen, brain, and small intestine; reduce the distribution of these components in the blood and lungs; and improve drug efficacy by promoting entry into the brain [SR, derived from the root of he brain . The abshe brain . Moreovehe brain , 8.SR often serves as a guiding drug, which improves the therapeutic effects of other drugs or components, especially in the respiratory tract and brain diseases. Nevertheless, little is known about the guiding effect of SR. In the present study, we investigated the pharmacodynamic effect of the SR in HQCFT in the treatment of cerebral infarction (CI) by investigating its angiogenic and anti-inflammatory effects.α (HIF-1α), NLR family pyrin domain-containing 3 (NLRP3), caspase 1, interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and matrix metalloproteinase-9 (MMP-9) were purchased from Nanjing Jiancheng Bioengineering Institute .2,3,5-Triphenyltetrazolium chloride (TTC) solution was purchased from Beijing Solarbio Science & Technology Co., Ltd. . ELISA kits for hypoxia-inducible factor 1Astragalus membranaceus (Fisch.) Bge., No. 20190401), Radix Paeoniae Rubra , and Saposhnikoviae Radix (Saposhnikovia divaricata (Turcz.) Schischk., No. 20190402) were all obtained from Heilongjiang Xiushengtang Medicinal Materials Co., Ltd.  and authenticated by Prof. Wang Zhen Yue . The voucher specimens  were stored in the Herbarium of Heilongjiang University of Chinese Medicine . All herbs in HQCFT were soaked in distilled water  for 1 h before extraction and then extracted three times for 1.5 h each. The extraction solutions were collected and concentrated to a suitable volume by decompression, and the residue was deposited in a vacuum freeze dryer for 48 hours. In addition, the extraction method for HQCFT without SR (HQCFT-SR) was the same as that used for HQCFT. The extraction ratios of HQCFT and HQCFT-SR were 38.53% and 37.31% (w/w), respectively.Astragali Radix  were supplied by Shenyang Changsheng Biotechnology Co., Ltd. (China). The rats were housed six per cage in the animal house of the Institute of Chinese Medicine, Heilongjiang University of Chinese Medicine, with a controlled room temperature (24 ± 2°C) and relative humidity (55 ± 5%). The rats were allowed to acclimate for 7 days prior to the experiments, with a 12 h light and dark cycle (lights on from 08:00 a.m. to 08:00 p.m.) and given food and water ad libitum. The animal studies received ethical approval in accordance with the Legislation on the Protection of Animals Used for Experimental Purposes (Directive 86/609/EEC), and the experiments were approved by the Institutional Animal Care Committee .The MCAO rat model was established according to previous studies , 10. BriForty-eight SD rats were randomly divided into 4 groups: (1) the model group: rats subjected to MCAO were administered normal saline; (2) the sham operation group: rats subjected to MCAO were administered normal saline; (3) the HQCFT group (1.6 g/kg): rats subjected to MCAO were administered HQCFT, that is, AR : RPR : SR = 10 : 3 : 2; and (4) the HQCFT-SR group (1.31 g/kg): rats subjected to MCAO were administered HQCFT without SR, that is, AR : RPR : SR = 10 : 3 : 0. All treatments were administered every 24 h for 14 days.After 14 days of treatment, all rats were fasted with free access to water for 12 h. Then, the rats were deeply anesthetized with 3% pentobarbital sodium (45 mg/kg) by intraperitoneal injection. Whole blood samples were collected from the abdominal artery prior to rat sacrifice. Fresh blood was allowed to stand for approximately 30 min at ambient temperature (22–25°C), and serum was obtained after centrifugation at 3500 g for 15 minutes at 4°C. Simultaneously, one portion of the whole brain tissue was rapidly separated and frozen at −20°C for 30 min, and the other portions were stored in 4% paraformaldehyde and liquid nitrogen.After blood collection, the auricula dextra of the rats was dissected, and prechilled normal saline was perfused from the left ventricle. Then, the fresh brain tissue was eviscerated and frozen at −20°C for 30 min. The brain was carefully cut into 2.0 mm thick serial cross sections from front to back and quickly incubated in 2% TTC solution at 37°C for 30 min, protected from exposure to light. Finally, the brain slices were fixed in 4% paraformaldehyde at room temperature for 12 h. The rat CI area was measured with Image-Pro Plus software, and the equation used for calculations was as follows:T represents the thickness of each slice.Ai represents the CI area, At represents the whole cerebral area, and μm paraffin slices. Then, the sections were dewaxed with xylene and dehydrated in an alcohol gradient. Hematoxylin and eosin were used to stain the sections, and pathological features of the brain tissue were examined under a 400× optical microscope .H&E staining was performed to observe the histopathological changes in rat cerebral tissues. Briefly, rat brain tissue was fixed in 4% paraformaldehyde for 48 h and prepared into 5 β , IL-6 , TNF-α , MMP-9 , vascular endothelial growth factor (VEGF) , vascular endothelial growth factor receptor 2 (VEGFR2) , and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) . After rinsing with TBST three times, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies  for 1 h at 37°C. Chemiluminescence (ECL) kits were used to visualize the membranes, and a Syngene Tanon 5200 imaging system  was used for imaging. The optical density of the bands was quantified using ImageJ software and corrected according to the corresponding GAPDH level.Briefly, the brain tissues from the left hemisphere were washed twice with PBS and disrupted with RIPA lysis buffer. Then, the tissue suspension was centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was used to determine the concentration of the sample with a BCA protein assay kit . Equal amounts of samples were separated by SDS-PAGE and electrotransfered to polyvinylidene difluoride membranes. Subsequently, the membranes were blocked with 5% skim milk for 1 h at room temperature and incubated overnight at 4°C with the indicated primary antibodies against NLRP3 , caspase 1 , IL-1μm paraffin sections for immunohistochemical analysis. In brief, the endogenous peroxidase activity of the slices was quenched by incubation with 3% H2O2 for 10 min and blocking with goat serum for 30 min. Subsequently, MMP-9, VEGF, and VEGFR2 antibodies were added to the slices at 4°C overnight. The slices were incubated at room temperature for 40 min, rinsed with PBS three times, incubated with the secondary antibodies at 37°C for 60 min, and visualized with 3,3-diaminobenzidine. The images were observed under high magnification (400×), and the optical density of positive cells was analyzed with ImageJ software.The brain tissue was fixed in 4% paraformaldehyde for 48 h and prepared into 5 α, NLRP3, caspase 1, IL-1β, IL-6, and TNF-α were detected using ELISA kits following the manufacturer's protocol. The sample absorbance was assessed at 450 nm using a Multifunctional Microplate Scanner . The contents of HIF-1α, NLRP3, caspase 1, IL-1β, IL-6, and TNF-α were calculated by using a standard curve.The serum contents of HIF-1t-tests and one-way analysis of variance (ANOVA) followed by Fisher's LSD multiple comparisons test. A value of p < 0.05 was considered to indicate statistical significance.Experimental data are expressed as the mean ± standard deviation (SD). Statistical significance was determined using Student's p < 0.05), indicating that the SR in HQCFT could attenuate infarction in the brain tissue of rats subjected to MCAO.TTC can be reduced to 1,3,5-triphenylformazan by dehydrogenase in normal brain tissue and is red, while infarcted cells lose vitality and are white or ivory . FiguresThe histopathological changes of rats were assessed after 14 days of treatment, and H&E staining of slices was performed to evaluate the severity of CI. As shown in β have been reported to be involved in inflammatory responses [β in rat brain tissue by Western blot analysis. As shown in p > 0.05).As NLRP3, caspase 1, and IL-1esponses , 13, thip > 0.05), and no significant differences were noted between the HQCFT and HQCFT-SR groups (p > 0.05).As shown in p > 0.05).As shown in α and IL-6 are important inflammatory factors in the pathogenesis of CI. As shown in p > 0.05).TNF-α protein contents were 0.663 ± 0.144, 1.077 ± 0.029, 0.960 ± 0.053, and 0.997 ± 0.035 in the sham, model, HQCFT, and HQCFT-SR groups, respectively. Compared with that in the sham group, the TNF-α protein content increased by 62.44% (p < 0.001) in the model group. However, there was no statistically significant difference compared with the model group, and no significant difference was observed between the HQCFT and HQCFT-SR groups (p > 0.05).As shown in p > 0.05).The expression of MMP-9, VEGF, and VEGFR2 plays an important role in neuroinflammation and angiogenesis , 15. As p > 0.05) in the model group. Compared with that in the model group, VEGF protein expression was increased by 33.68% (p < 0.001) and 23.92% (p < 0.01) in the HQCFT and HQCFT-SR groups, respectively. The level of VEGF in the HQCFT-SR group was not significantly different from that in the HQCFT group (p > 0.05).As shown in p > 0.05) in the model group. Compared with that in the model group, VEGFR2 protein expression was increased by 48.94% (p < 0.01) and 31.36% (p < 0.05) in the HQCFT and HQCFT-SR groups, respectively, and there was no significant difference between these two groups (p > 0.05).As shown in p < 0.001).The MMP-9-, VEGF-, and VEGFR2-positive cells in normal brain tissue are brown, dark brown, or gray. As shown in Figures p > 0.05). Compared with that in the model group, the average optical density of VEGF-positive cells was increased by 31.01% (p < 0.001) and 26.23% (p < 0.001) in the HQCFT and HQCFT-SR groups, respectively, and the average optical density of VEGFR2-positive cells was increased by 54.99% (p < 0.001) and 44.42% (p < 0.01) in the HQCFT and HQCFT-SR groups, respectively. The average optical densities of positive cells in the HQCFT group were significantly higher than those in the HQCFT-SR group (p < 0.01). The above results indicated that the SR in HQCFT could promote angiogenesis in the brain tissue of rats subjected to MCAO.As shown in Figures α, NLRP3, caspase 1, IL-1β, IL-6, and TNF-α in rat serum with ELISA kits. As shown in Figures α, NLRP3, caspase 1, IL-1β, IL-6, and TNF-α in the sham group were 5336.56 ± 1808.12, 108.35 ± 7.72, 1.62 ± 0.36, 32.61 ± 5.80, 22.03 ± 6.35, and 156.83 ± 34.64, respectively. There were significant differences between the sham group and the model group. Compared with those in the sham group, the HIF-1α, NLRP3, caspase 1, IL-1β, IL-6, and TNF-α contents were 6037.98 ± 852.61, 112.61 ± 11.37, 1.62 ± 0.40, 38.12 ± 9.67, 23.38 ± 2.21, and 140.09 ± 31.42, respectively, in the HQCFT group and 6685.32 ± 1781.75, 126.61 ± 6.88, 2.33 ± 0.58, 47.42 ± 4.11, 26.44 ± 2.08, and 192.13 ± 19.56, respectively, in the HQCFT-SR group. The level of these inflammatory factors in the HQCFT group differed significantly from with those in the HQCFT-SR group. These findings indicated that the SR in HQCFT could inhibit the inflammatory response in the serum of rats subjected to MCAO.The inflammatory response is the main feature of the pathological mechanism of CI . To furtCI is a local ischemic condition characterized by decreased cerebral blood flow and vascular embolism . In the α, IL-1β, MMP-9, and NF-κB p65 activity and expression, protect against tight junction and BBB degradation, and prevent nerve cell apoptosis in an MCAO mouse model [β and increase the expression of VEGF/VEGF-R in an MCAO rat model [Astragaloside IV (AS-IV) is the main active component of AR. Many studies have proven that AS-IV can promote neurogenesis and angiogenesis and improve neurological function , 25. In se model . Paeonifat model , 30. Howat model . TherefoMCAO is a typical model for studying CI . In thisLesions of neuronal cells, a feature of ischemic diseases, are usually used to evaluate the extent of CI . Our resα is involved in the pathological process and inflammatory reactions associated with ischemic disease [α in the HQCFT and HQCFT-SR groups was reduced. As an important inflammatory regulator of CI, HIF-1α can stimulate the activation of the NLRP3 inflammasome [β and IL-18, leading to the formation of a vicious cycle and aggravating CI [α play a crucial role in the inflammatory responses induced by CI [HIF-1 disease . In the ammasome . Howeverating CI –35. Addied by CI .β, and IL-6 in brain tissues from rats subjected to MCAO. However, TNF-α protein expression levels in the treatment groups were not significantly different from those in the model group. Moreover, HQCFT and HQCFT-SR reduced serum HIF-1α, NLRP3, caspase 1, IL-1β, IL-6, and TNF-α levels. Simultaneously, the contents of these inflammatory factors in the HQCFT-SR group obviously differed from those in the HQCFT group. In summary, these findings suggested that SR can improve the overall anti-inflammatory effect of HQCFT and confirmed that HQCFT-mediated inhibition of inflammation after CI exerts beneficial effects in the cerebrum.HQCFT and HQCFT-SR attenuate the abnormal expression of NLRP3, caspase 1, IL-1MMP-9 is a matrix metalloproteinase that participates in neuroinflammatory responses and angiogenesis . In the VEGF is the most important regulatory factor involved in vascular system development and differentiation . VEGF caThe present study has several limitations. Although we verified that SR could guide or enhance the overall effect of HQCFT on CI in a rat model, whether it has the same effect in other animal models needs further study. In addition, traditional Chinese herbal formulas typically have multiple components, multiple pathways, and multiple targets, but which component of SR exerts a guiding effect on HQCFT needs to be clarified.In summary, HQCFT and HQCFT-SR are potentially useful in the treatment of CI because they promote angiogenesis and exert anti-inflammatory activity. Importantly, the cytokines in brain tissue and serum from the HQCFT group exhibited better efficacy than those from the HQCFT-SR group. Therefore, SR can improve the effect of HQCFT on CI, and the mechanism by which SR guides and enhances the effect of HQCFT warrants further study.