In search of novel CH3SeH precursors, we previously synthesized a series of methylselenoesters that were active (GI50 < 10 µM at 72 h) against a panel of cancer cell lines. Herein, we refined the mechanism of action of the two lead compounds with the additional synthesis of new analogs . A novel mechanism for the programmed cell death mechanism for Se-compounds was identified. Both methylseleninic acid and the novel CH3SeH precursors induced entosis by cell detachment through downregulation of cell division control protein 42 homolog (CDC42) and its downstream effector β1-integrin (CD29). To our knowledge, this is the first time that Se compounds have been reported to induce this type of cell death and is of importance in the characterization of the anticancerogenic properties of these compounds.Redox active selenium (Se) compounds have gained substantial attention in the last decade as potential cancer therapeutic agents. Several Se compounds have shown high selectivity and sensitivity against malignant cells. The cytotoxic effects are exerted by their biologically active metabolites, with methylselenol (CH Pancreatic ductal adenocarcinoma is an extremely aggressive neoplasm and one of the cancers with the poorest prognosis, with a five-year survival of only 8% . In addiModulation of redox homeostasis in cancer cells has emerged as a new opportunity for tumor intervention. Induction of reactive oxygen species (ROS) by these compounds may affect all the redox dependent pathways in the cell, which can be detrimental to cells. Antioxidant enzymes are often induced to eliminate elevated ROS production. Due to metabolic transformation, cancer cells have an increased and maximized antioxidant capacity in order to evade the ROS-induced cell death. For instance, the expression of mutant oncogenic KrasG12D is commonly present in pancreatic ductal adenocarcinoma (PDAC), resulting in an elevated basal state of the transcription factor, nuclear factor E2-related factor 2 (NRF2) to mount an antioxidant response ,5. ThereRedox modulating selenium (Se) compounds have gained substantial attention in the last decade as potential cancer therapeutic agents . Several3SeH) is considered a key metabolite in the anticancer activity of Se compounds. However, the in situ production or alternatively, the use of precursors, is required due to the high reactivity and volatility of this molecule.Importantly, along with their active metabolites that execute their biological activity, the dosage and chemical form of Se compounds highly determine their efficacy . Methyls3SeH precursors, we previously synthesized a series of methylselenoesters that were active (GI50 < 10 µM at 72 h) against a panel of cancer cell lines [In search of novel CHll lines . Herein,ll lines . Howeverll lines ,17,18 anll lines  and glucll lines  have beeDuring entosis, the stiffer cell (hereafter target cell) actively participates in its own internalization, via adherent junctions and the actin cytoskeleton that play a pivotal role in this process. Ultimately, the target cell is killed through lysosomal enzyme-mediated degradation, using the autophagy machinery, but independent of autophagosome formation . The dea3SeH precursors induce cell detachment through downregulation of cell division control protein 42 homolog (CDC42) and its downstream effector CD29 [Methylseleninic acid (MSA) and the novel CHtor CD29 . Cell-ce1 or 2 for 72 h. Cell viability was then determined. All three compounds were cytotoxic, with compound 2 being the most potent compound in 2D cultures. The compounds had IC50 values in the low micromolar range in 2D cultures . However, cells grown as spheroids (3D) were consistent with previously reported data [Initial characterization of the compounds was performed through viability assays in 2D and 3D cultures of Panc-1 cells, given that 3D cultures have been demonstrated to mimic tumor behavior more efficiently than traditional monolayer (2D) cultures. Panc-1 cells were treated with increasing concentrations of MSA, and compounds ted data , and morTo further study the induced cell death in 3D cultures, spheroids were stained with Hoechst and propidium iodide (PI) after 72 h treatment. While Hoechst stains the nucleus of all cells, PI only penetrates and stains damaged membranes of dying cells. As shown in 3SeH, which is believed to be a key molecule in Se activity (1 (1’) and 2 (2’) were selectively tested as a proof-of-concept. As seen in The selenoester entity could be easily hydrolyzed by a nucleophile such as water, rendering the corresponding carboxylic acids and releasing CHactivity D. To exc2  polymerase 1 (PARP) and cleaved PARP, wherein MSA and compounds  at 72 h . PARP ha at 72 h . Additio at 72 h , suggestFurthermore, the expression of cathepsins, a structurally and catalytically distinguished class of proteases, was checked. A context-depending role has been described for cathepsins, with either tumor-promoting or suppressing activities. They have not only been reported to function as apoptotic mediators, but also to be related to entosis  and cellBoth cathepsin B (CatB) and cathepsin D (CatD) have been reported to play an important role in entosis ,20,31. I1 and 2 were analyzed. To evaluate if this effect was exclusive to methylated forms of Se or unrestricted to other alkyl or aromatic derivatives, the ethyl derivative for compounds 1 and 2 , the pentyl derivative for compound 1 (1b), and the benzyl analog for compound 2 (2c), were synthesized to cover different alkyl lengths and additional substituents. Methylselenocysteine (MeSeCys) was also selected as another CH3SeH precursor and benzeneselenol (BznSeH) as an additional aromatic selenol for a comparative analysis. In addition, compound 3 was chosen, a previously synthesized selenide in our laboratory, as a proof-of-concept compound without a labile bond between the core of the molecule and the methylseleno residue [In order to distinguish the type of Se compound that could cause this phenomenon, other commercial Se derivatives together with other newly synthesized analogs of compounds  residue , and the2a stopped proliferation at 24 h, while the methylated analog (2) achieved a reduction at 72 h. BznSeH and compound 3 had the highest cytostatic potential, with both of them inhibiting proliferation at 24 h of treatment.First, cell proliferation and cell death were evaluated. As illustrated in 1b, which was not cytotoxic. BznSeH, which was highly cytostatic, did not induce noteworthy cell death, with only 15% of dead cells at 72 h. MeSeCys, which has been reported to have a similar activity to MSA in vivo, required a considerably higher concentration to achieve similar cell death induction in vitro, due to the need of metabolic processing to release CH3SeH, consistent with previous reports [3, on the other hand, was the most potent compound, inducing cell death at 24 h treatment. The methyl derivatives were more cytotoxic at 72 h than analogs with a longer alkyl chain or the benzyl moiety B. Almost reports . Compoun1a and 1b were unable to detach cells. whereas compounds 2c and 3 had detached all the cells at 24 h. MeSeCys detached all the cells at 72 h, and a concomitant increase in cell death was observed at that time point. However, cell detachment potential was not correlated with cell death induction in the case of compound 2c, which was almost innocuous. BznSeH and compound 2a induced mixed populations, with attached and floating cell fractions.  induced similar deadhesive effects. Intriguingly, despite induced cell detachment by this compound, it did not lead to cell death. The decreased cytotoxic effects are consistent with previous reports, showing the impaired cytotoxic activity of selenobenzyl derivatives with respect to their corresponding methylated analogs [Cell detachment could be caused by CHll lines ,48, alon analogs . Hence, In summary, we report a novel mechanism of action for MSA and two methylselenoesters: the induction of cell detachment through CDC42 and CD29 down-regulation leading to cell-in-cell formation (entosis) and death of the inner cell. However, these compounds need to be further evaluated in in vivo studies to gain an in-depth insight into their administration, hepatic metabolism for bioavailability and absorption, distribution, metabolism, and excretion properties. Additionally, the therapeutic potential of these compounds would be governed by the balance between their toxicity and efficacy profiles. Therefore, further research to fully dissect the relationship between structure, detachment abilities, and cell death induction of organic Se derivatives is required in order to understand the complex Se biochemistry.2. The 3D spheroids were cultured following the protocol described by Longati et al. [Panc-1 cells were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM:F12 , 10% FBS , and 1% glutamine (Gibco) at 37 °C under 5% COi et al. . Briefly®, Stockholm, Sweden) assay. 6000 Panc-1 cells were seeded in 96-well plates. Cells were treated with increasing concentrations of the compounds. Dilutions of the compounds in cell medium were freshly prepared from a 0.01 M stock in DMSO. After 72 h treatment, 50 µL of MTT solution in PBS (2 mg/mL) was added and cells were incubated at 37 °C under 5% CO2 for 4 h. Medium was removed and 150 µL of DMSO was added to dissolve the formazan crystals. Absorbance was read at 590 nm in a VersaMax microplate reader . Viability is expressed as the percentage of untreated cells.Cell viability after treatment was assessed by the MTT -2,5-diphenyltetrazolium bromide) . Viability is expressed as the percentage of untreated cells.3D viability after 72 h treatment was analyzed with the acid phosphatase assay, following a previously described protocol . BrieflyTM ULA plate , following the manufacturer’s protocols. Briefly, on day 0, the plate was pre-wetted with 40 µL of medium before seeding 2000 Panc-1 cells in 75 µL phenol red-free DMEM:F12, 10% FBS, and 1% glutamine. The plate was centrifuged for 2 min at 250× g. On day 4, cells were treated, adding 25 µL of the corresponding compound in medium. Dilutions were freshly prepared from a 0.1 M DMSO stock. On day 7, cells were stained with 1 µM Hoechst 33342  for 2 h, and 2 µM PI  for 1 h at 37 °C under 5% CO2. Spheroids were then carefully washed once with PBS and fixed with paraformaldehyde (4%) at RT. Imaging was performed on the Operetta® High-content Imaging System   and processed by the Colombus™  analysis software.Spheroid formation was developed in a Gravity TrapTM, ThermoFisher Scientific, Goteborg, Sweden) and transferred to a nitrocellulose membrane using the iBlot Gel Transfer Device . Incubation with primary antibody (Cathepsin B (D1C7Y), Cell Signaling, Leiden, The Netherlands, Catalog no. 31718; Cathepsin D, BD Biosciences, San Jose, CA, USA, Catalog no. 610800; CDC42, Abcam, Cambridge, UK, Catalog no. ab155940; RhoA (67B89), Cell Signaling, Leiden, The Netherlands, Catalog no. 2117; PARP, Cell Signaling, Leiden, The Netherlands, Catalog no. 9542; Caspase 9, Bioss, Nordic BioSite, Stockholm, Sweden, Catalog no. bs-0049R; beta actin, Sigma-Aldrich, Stockholm, Sweden, Catalog no. A5441) diluted in TBST containing 3.5% bovine serum albumin (BSA) was done overnight at 4 °C. Secondary antibodies, goat anti-rabbit IgG HRP , or goat anti mouse IgG HRP  were incubated for 1 h. Membranes were developed using the AmershamTM ECLTM Start Western Blotting Detection Reagent  and bands were visualized using the Bio-Rad Quantity One imaging system . Images were quantified using ImageJ software.Protein lysate containing 20 µg of proteins was separated on a Bolt 4–12% Bis-Tris Gel . The cells were further stained with 200 µL Coomassie blue staining solution  for 1 h at room temperature. The cells were then washed with PBS and further incubated for 1 h with 0.5 mL elution buffer (0.1 N NaOH and 50% Methanol). Furthermore, 0.5 mL of developing solution containing 10% Trichloroacetic acid (TCA) was added into the wells. Following this, 200 µL of the mix was transferred to a 96-well plate and further absorbance was read at 595 nm using the plate reader Infinite® M200 Pro, Tecan, Mannedorf, Switzerland. 0.5 × 106 cells were seeded in 25 cm2 flasks and allowed to attach for 24 h. After that, medium was replaced and cells were treated with the corresponding compounds or vehicle (DMSO) for 24 h. Cells were collected, washed with PBS-staining buffer , and stained for 30 min at 4 °C and darkness in 50 µL PBS-staining buffer with the corresponding antibody: CD29/integrin 1-β , CD325/N-cadherin . Cells were washed once with PBS-staining buffer and resuspended in fixation buffer  until being read in a BD FACSCalibur™ .0.5 × 106 cells were seeded in 25 cm2 flasks and incubated at 37 °C under 5% CO2 for 24 h. Media was changed and cells were treated with 5 µM of compounds for 24 h, after which floating cells were collected, centrifuged, and seeded at a density of 1000 cells in total volume of 2 mL/well in a six-well plate. The control cells were checked for colony formation for five days. A group of 50 cells were considered as one colony. The plates were later stained with crystal violet according to Franken et al. [0.5 × 101H and 13C) were recorded on a Bruker 400 UltrashieldTM spectrometer  and are provided in the 3 and TMS was used as an internal standard. IR spectra were obtained on a Thermo Nicolet FT-IR Nexus spectrophotometer  using KBr pellets for solids or NaCl plates for oil compounds. The HRMS spectra were recorded on a Thermo Scientific Q Exactive Focus mass spectrometer  by direct infusion. For TLC assays, Alugram SIL G7UV254 sheets  were used. Column chromatography was performed with silica gel 60 . Chemicals were purchased from E. Merck KGaA , Panreac Química S.A. , Sigma-Aldrich Quimica, S.A. , and Acros Organics .The NMR spectra  in water or ethanol (1:1) and N2 atmosphere to form NaHSe. The corresponding acyl chloride dissolved in N,N-dimethylformamide (2 mL) or chloroform (2 mL) was then added and the reaction was stirred at room temperature until the reaction took place (20 min–3.5 h). The reaction was followed by IR or TLC. The mixture was filtered and the intermediate was further alkylated with the corresponding halide until discoloration of the mixture. The product was extracted with methylene chloride and dried over Na2SO4. The solvent was eliminated under rotatory evaporation and the residue was purified through column chromatography. The chemical synthesis was carried out following an already described procedure ,52 with 4 (3 mmol), and ethyl iodide (1.5 mmol). A yellow oil was obtained, which was further purified through column chromatography using methylene chloride as the eluent. Yield: 11%. 1H NMR : δ 1.52 , 3.11 , 7.06 , 7.54 ppm . 13C NMR : δ 15.7 (–CH3), 20.5 (–CH2–), 128.0 (C4), 130.6 (C2), 131.0 (C5), 137.7 (C3), 184.3 ppm (–C=O). IR (KBr): ν 3105 , 2962–2867 , 1649 cm−1 . HRMS calculated for C7H8ClOSSe (M + H): 254.91441, found: 254.91418.From 3-chlorothiophen-2-carboxylic acid (1.5 mmol), Se powder (1.5 mmol), NaBH4 (7.1 mmol), and ethyl iodide (3.48 mmol). Conditions: 45 min reaction with NaHSe and 2 h reaction with ethyl iodide. A yellow solid was obtained, which was purified through column chromatography using ethyl acetate/hexane (1:10) as the eluent. Yield: 10%; m.p.: 35–36 °C. 1H NMR : δ 1.5 , 3.11 , 7.17 ppm . 13C NMR : δ 15.8 (–CH3), 19.2 (–CH2–), 114.7 (C3 + C4), 153.5 (C2 + C5), 183.5 ppm (–C=O). IR (KBr): ν 3143 , 2961–2860 , 1649 cm−1 . HRMS calculated for C10H13O3Se2 (M + H): 340.91896; found: 340.91891.From 2,5-furandicarboxylic acid (1.74 mmol), Se powder (3.48 mmol), NaBH4 (2.15 mmol), and pentyl iodide (2.15 mmol). Under N2 atmosphere, absolute ethanol (10 mL) was added to a mixture of NaBH4 and selenium cooled by an ice bath, with magnetic stirring. After the formation of NaHSe was achieved, the ice bath was removed and the following reactions were carried out at room temperature. Before adding an excess of pentyl iodide, the mixture was filtered. Conditions: 20 min reaction with NaHSe and 20 min reaction with pentyl iodide. The solvent was eliminated under rotatory evaporation. The product was purified through column chromatography using a gradient elution of ethyl acetate: hexane. An orange oil was obtained. Yield: 53%. 1H NMR : δ 0.83 , 1.27–1.35 , 1.66–1.77 , 3.02 , 6.96 , 7.44 ppm . 13C NMR : δ 12.94 (−CH3), 21.19 (δCH2), 25.56 (αCH2), 28.89 (ßCH2), 31.18 (γCH2), 126.82 (C4), 129.50 (C2), 129.76 (C5), 136.56 (C3), 183.18 ppm (−CO). IR (KBr): ν 2922 −2852 , 1669 cm−1 .From 3-chlorothiophen-2-carboxylic acid (2 mmol), Se powder (2 mmol), NABH4 (7.1 mmol), and benzyl bromide (3.48 mmol). Conditions: 1.5 h reaction with NaHSe and 3.5 h reaction with benzyl bromide. The product was extracted with methylene chloride, further washed with water, dried over Na2SO4. The solvent was eliminated under rotatory evaporation. A yellow oil was obtained, which was precipitated and washed with diethyl ether. A yellow solid was obtained. Yield: 25%; m.p.: 114–115 °C. 1H NMR : δ 4.34 , 7.2 , 7.22–7.35 ppm . 13C NMR : δ 28.5 (–CH2–), 115.0 (C3 + C4), 127.3 (C4’), 128.7 (C2’ + C6’), 129.1(C3′ + C5′), 138.21 (C1’), 153.2 (C2 + C5), 182.8 (−C=O). IR (KBr): ν 3123−3088 , 1677 cm−1 . HRMS C20H16O3Se2Na (M + Na+): calculated 486.9322; found 486.9430. From 2,5-furandicarboxylic acid (1.74 mmol), Se powder (3.48 mmol), NABH3 was synthesized in a previous work [3c.Compound ous work , under t6 cells were seeded in 25 cm2 flasks and incubated at 37 °C under 5% CO2 24 h. Media was changed and cells were treated with 5 µM of compounds for 24 h, after which floating cells were collected, centrifuged, and seeded at a density of 50,000 cells in total volume of 100 µL/well in a 96-well plate. Post 48 h of treatment, the cells were imaged live for another 24 h in Operetta and images were captured every 5 min. 0.5 × 105 cells stained with each of the cell trackers were mixed (1:1) and seeded onto 25 cm2 flasks for 24 h, followed by treatment with 5 µM of respective compounds for 18 h. Cells were imaged live using an LSM 800 confocal microscope .Monolayer cultures were stained with CellTracker Red or Green (Invitrogen) for 1 h at 37 °C in the absence of serum. After this, 0.4 × 10p < 0.05, ** p < 0.01, *** p < 0.001). One-way ANOVA followed by Dunnet’s test was performed using GraphPad 6.01 . (*


Fluorescence is not quenched in the presence of other ions such as Cl−, Br−, I−, NO2−, NO3−, or H2PO4−. These compounds can therefore be utilized as practical colorimetric and fluorescent probes for monitoring the presence of F− or CN− anions.Stable zwitterionic spirocyclic Meisenheimer compounds were synthesized using a one-step reaction between picric acid and diisopropyl (ZW1) or dicyclohexyl (ZW3) carbodiimide. A solution of these compounds displays intense orange fluorescence upon UV or visible light excitation, which can be quenched or “turned-off” by adding a mole equivalent amount of F Spirocyclic compounds have found use in the fields of organic optoelectronics ,2,3, phoCompound ZW1 is composed of two main structural fragments: (i) a 1,3,5-trinitrocyclohexadiene anion chromophore displaying intense orange light emission; and (ii) a triazine ring with a labile iminium proton. The relevant features of these two building blocks prompted us to explore the potential use of ZW1 as a colorimetric and fluorescent chemosensor. In a previous work, we demonstrated the reversible fluorescence (on/off) switching of ZW1 upon deprotonation using tetrabutylammonium hydroxide (TBAOH) as a base in acetonitrile (ACN) . This be− and CN− anions. In this sense, there are several promising anion chemosensors based on colorimetric changes or change in fluorescence. These gadolinium (III) organometallic complex-based sensors found promising use for in vivo imaging and fluorescence detection of biologically harmful fluoride ions . ,10. 9,10in water , or as s(406 nm) = 16,600 cm−1 M−1 and for ZW3, ε(406 nm) = 10,810 cm−1 M−1 at the absorption maximum. In addition, a subtle increase in fluorescence quantum yield (ϕF) was encountered: ϕF = 0.5 and 0.6 for ZW1 and ZW3, respectively. This 20% increment in ϕF for ZW3 might be attributed to the bulkier cyclohexyl groups in its triazine ring, which may shield the electron density on the adjacent nitrogen atoms and, as such, increase their oxidation potential and disfavor fluorescence quenching of the 1,3,5-trinitrocyclohexadiene anion chromophore via photoinduced electron transfer. Finally, the presence of three additional methylene groups per substituent in the triazine moiety of ZW3 increases its solubility in non-polar solvents and its miscibility with polymeric matrices such as polyvinyl alchohol and polymethyl methacrylate. These enhanced properties will be useful for future applications involving these derivatives.Replacing the isopropyl groups in ZW1 with cyclohexyls in ZW3 does not affect the overall shape of the absorption spectrum of their 1,3,5-trinitrocyclohexadiene anion chromophore a,b. HoweIn terms of chemical properties, compound ZW3 was found to preserve the colorimetric and fluorescent pH-response previously reported for ZW1 . Adding As shown in − and CN− act as bases under our experimental conditions.When micromolar solutions of ZW1 and ZW3 in a polar aprotic solvent such as acetonitrile were titrated with micromolar solutions of either tetraethylammonium fluoride (TEAF) or tetraethylammonium cyanide (TEACN), a change in the absorption spectrum of the two spirocyclic compounds  was obse0) of the initial ZW1 and ZW3 acetonitrile solutions upon the addition of TEAF or TEACN up to 1 mole equivalent, which resulted in a nearly complete emission “turn-off” of these compounds (~90%). Actually, the addition of a slight excess of cyanide and fluoride anions  totally switched off the fluorescence emission from ZW1 and ZW3 solutions.Moreover, colorimetric changes were accompanied by a decrease in fluorescence emission upon increasing the concentration of fluoride and cyanide anions. As shown in − (from tetrabutylammonium chloride), Br− (from tetrabutylammonium bromide), I− (from potassium iodide), H2PO4− (from tetrabutylammonium dihydrogenphosphate), NO3− (from potassium nitrate), and NO2− (from potassium nitrite). The addition of up to 1.5 equivalents of these anions to solutions of ZW1 and ZW3 in acetonitrile did not result in measurable changes in the absorption spectrum of these zwitterionic compounds, as shown for some selected cases in − and CN− based on colorimetric measurements.The selectivity of the colorimetric and fluorescence detection of fluoride and cyanide anions using ZW1 and ZW3 was investigated by exploring the effect of other anionic analytes of relevance: Cl− did result in appreciable changes in fluorescence emission from the spirocyclic zwitterionic compounds, although no variation was observed in the absorption spectrum .In this work, we have explored the use of zwitterionic spirocyclic Meisenheimer complexes ZW1 and ZW3 as colorimetric and fluorescent chemosensors of fluoride and cyanide anions in solution. We have demonstrated that the absorption spectra of these compounds change upon the addition of Fs of ZW1 , the beh− : 482.2358; experimental: 482.2353. C20H31N7O7 (481.5): calcd. C 49.91, H 6.44, N 20.38; found, C 49.86, H 6.51, N 20.09. UV-Vis (ACN): λabs(max) = 406 nm ; fluorescence  λfl (max) = 563 nm, φfl = 0.5.Characterization of NA1: M.p. 156–158 °C. 1H-NMR : δ (ppm) = 1.15 , 1.17 , 3.92–4.03 , 4.22 , 4.36 , 8.87  ppm. IR (KBr): ν˜ = 723, 912, 1360, 1555, 1611, 1655, 2864, 2940, 3102, 3320 cm−1. C13H17N5O7 (355.3): calcd. 43.95, H 4.82, N 19.71; found C 43.86, H 4.70, N 19.92. Characterization of N,N′-diciclohexylcarbodiimide  instead and catalytic amounts of N,N-dimethylaminopyridine. Compound ZW3 was obtained as red needles . M.p. 183–186 °C with decomposition.This compound was synthesized following a similar procedure to the one used to synthesize ZW1, using ZW3: 1H-NMR : δ (ppm) = 0.92–1.43 , 1.50–2.34 , 2.49 , 2.64 , 3.29 , 3.48 , 3.65 , 4.53 , 9.03 . IR (KBr): ν˜ = 3417, 2938, 2854, 1706, 1586, 1512, 1488, 1430, 1247 cm−1. HRMS (ESI-QTOF): m/z calculated for [C32H47N7O7 + H]: 642.3610; experimental: 642.3601. UV-Vis (ACN): λabs(max) = 406 nm (εabs = 10 810 M−1cm−1); fluorescence  λfl (max) = 563 nm, φfl = 0.6.Characterization of − or CN− anions, which was also accompanied by colorimetric changes. These effects were not observed for other anions such as Cl−, Br−, I−, NO2−, NO3−, or H2PO42−, which makes ZW1 and ZW3 useful optical probes for the fast and direct detection and quantification of F− and CN−.We synthesized two stable zwitterionic spirocyclic Meisenheimer compounds (ZW1 and ZW3) that show intense orange fluorescence in organic solvents such as acetonitrile. Their fluorescence could be “turned-off” upon the addition of c.a. 1 mole equivalent of F


Arabidopsis expressing human anti-rabies mAb (mAbP) SO57 were harvested to isolate the total soluble protein (TSP). An equal amount of TSP solution was applied to five resin groups including commercial protein A resin (GR) as a positive control. The modified 8.5R, 9.5R, 10.5R, and 11.5R showed delayed elution timing compared to the GR control resin. Nano-drop analysis showed that the total amount of purified mAbPSO57 mAbs from 60 g of fresh leaf mass were not significantly different among 8.5R (400 μg), 9.5R (360 μg), 10.5R (380 μg), and GR (350 μg). The 11.5R (25 μg) had the least mAbPSO57. SDS–PAGE analysis showed that the purity of mAbPSO57 was not significantly different among the five groups. Rapid fluorescent focus inhibition tests revealed that virus-neutralizing efficacies of purified mAbPSO57 from all the five different resins including the positive control resin were similar. Taken together, both pH 8.5 and 10.5 coupling conditions with high recovery rate should be optimized for purification of mAbPSO57 from transgenic Arabidopsis plant, which will eventually reduce down-stream cost required for mAb production using the plant system.The main goal of this research was to determine optimum pH conditions for coupling between protein A and epoxy-activated Sepharose beads for purification of monoclonal antibodies (mAbs) expressed in plants. To confirm the effect of pH conditions on purification efficacy, epoxy-activated agarose beads were coupled to protein A under the pH conditions of 8.5, 9.5, 10.5, and 11.5 . A total of 300 g of fresh leaf tissue of transgenic  Plants have enormous potential as bioreactors for the large-scale production of therapeutic reagents such as recombinant vaccines and antibodies . One of Arabidopsis plants. The purified plant-derived mAbs (mAbPs) from four different resins and a commercially available protein A agarose resin as a positive control were compared for purification efficiency, purity, and neutralizing activity.Staphylococcal protein A is one of the first discovered immunoglobulin-binding molecules and has been extensively studied during the past decades. Because of its affinity to immunoglobulins, protein A has been widely used as a tool in the detection and purification of antibodies, and its role has been further developed in one of the most commonly employed affinity purification systems . HoweverAgrobacterium tumefaciens strain GV3101::pMP90 by electroporation , and the pellets were resuspended with infiltration media . Wild type Col-0 Arabidopsis plants were transformed using the floral dip method  in a growth chamber. To induce proliferation of many secondary bolts, the first bolts of Arabidopsis were trimmed off. The plant pots were inverted into the infiltration solution containing 0.02% Silwet L-77 for 5 min at a time, and the infiltrated plants were covered with black plastic bags for 2 days to ensure high humidity. On the following day, the plastic bags were removed, and the plants were maintained in the standard conditions in a growth chamber until seeds were well ripened. Then, the obtained seeds were sown on agar plates containing Murashige and Skoog (MS) medium (pH 5.7) ), containing 50 mg/L kanamycin and 25 mg/L cefotaxime to select true leaf generating shoots. For a further study, all shoots with green leaves were transplanted to a soil pot and maintained in a growth chamber at 22 °C under a 16 h light/8 h dark cycle.Plant expression vector pBI mAb 57 carrying anti-rabies virus mAb light chain (LC) and heavy chain (HC) fused to KDEL ER retention signal was transferred into poration . A. tumep method . Col-0 AArabidopsis plants using a DNA extraction kit , according to the manufacturer’s recommendations. PCR analysis was performed to confirm the presence of HC (281 bp) and LC (227 bp) genes of T1 plants. Primer sets were described as follows: HC forward primer, 5′-CAG ACT CAC CAT TAC CGC-3′; HC reverse primer, 5′-AGT AGT CCT TGA CCA GGC-3′; LC forward primer, 5′-CAC TGG AAC CAG CAG TGA-3′; LC reverse primer, 5′-TGT AGT CGC CTG CAT ATG A-3′. The PCR reaction was subjected to 26 cycles of 94 °C for 20 s, 58 °C for 10 s, and 72 °C for 30 s. The PCR products were analyzed by electrophoresis in a 1.0% agarose gel with ethidium bromide, and visualized under UV illumination. The pBI mAb 57 vector was used as a positive control, and genomic DNA extracted from Col-0 Arabidopsis was used as a negative control.Plant genomic DNA preparation and polymerase chain reaction (PCR) analysis were conducted as described in Arabidopsis, 80 mg of fresh leaf tissue, which had previously been confirmed as having the target mAbPSO57 protein genes (mAb SO57 HC (281 bp), LC (227 bp)) by PCR, was ground in 300 µL of 1× PBS buffer . Homogenates were boiled with sample buffer  and cooled in ice. After centrifugation for 5 min at 10,200×g, 20 μL of supernatant was loaded in 12.5% SDS–PAGE gel and transferred to a nitrocellulose membrane . The membrane was then blocked with 5% skim milk  in 1× PBS-T (1× PBS plus 0.1% (v/v) Tween 20) for 2 h at 25 °C. After blocking treatment, the blots were incubated with peroxidase-conjugated goat anti-human IgG Fcγ  and anti-human IgG F(ab′)2 , which recognize the HC and LC of mAbPSO57, respectively. Protein bands were detected with the SuperSignal West Pico Chemiluminescent Substrate  and visualized using X-ray films . The leaf of Col-0 Arabidopsis was used as a negative control.Western blot analysis was conducted as previously described in PSO57, 350 g of Arabidopsis leaf tissue was homogenized in a HR2094 blender  using extraction buffer  as previously described in g for 30 min at 4 °C, then the supernatant was filtered through a Miracloth , and the pH of the filtrate was reduced to 5.1 by adding acetic acid (pH 2.4). The solution was centrifuged to remove chlorophyll at 10,200×g for 30 min at 4 °C. The pH of the solution was then increased to 7.0 by adding 3M Tris-HCl, and 15% ammonium sulfate was added to the solution at 4 °C. After centrifugation at 9,000×g for 30 min at 4 °C, 45% of ammonium sulfate was added to the collected supernatant at 4 °C. After overnight incubation, the total solution was centrifuged at 10,200×g for 30 min at 4 °C. The pellet was resuspended in an extraction buffer with 1/12 volume of the original extraction buffer, and centrifuged at 10,200×g for 30 min at 4 °C. Final solutions were divided into five groups, and each solution was applied to four different resins and a control GE protein A resin for purification of mAbPSO57 from total soluble protein (TSP) solution  .Eluted fraction F1–F7 of purified mAbVT, USA) . AliquotPs obtained from different resins , rapid fluorescent focus inhibition test (RFFIT) was performed plant-derived mAbps  and anti-rabies immunoglobulin , respectively. The mAbs, at an initial concentration of 20 μg/mL, were diluted in 1:2.5, 1:12.5, 1:62.5, and 1:312.5 with advanced Dulbecco’s modified Eagle’s medium . A 100 μL aliquot of each diluted sample and 100 μL of Challenge Virus Standard (CVS-11) containing 32–100 FFD50 were mixed in an 8-well chamber slide , performed in duplicate. These mixed samples were incubated in a 5% CO2 incubator at 35 °C for 90 min. After incubation, 0.5 × 105 cells of mouse neuroblastoma (N2a) cells with 100 μL total volume were added to each chamber and incubated at 35 °C in a 5% CO2 incubator for 20 h. After incubation, the supernatant was removed, and monolayers were fixed in 80% acetone for 10 min at 23 °C after washing with 1× PBS. The slides were stained with 0.0025% Evans Blue solution , containing anti-rabies virus mAbs conjugated to Alexa Fluor™ 488 , and incubated at 37 °C for 30 min. After incubation, the slides were washed three times for 1 min with 1× PBS and then coverslips were mounted on each chamber slide. The slides were observed under a fluorescence microscope  (200× magnification).To compare neutralizing activities of purified mAb1 seeds obtained after floral dip transformation were plated on MS medium containing kanamycin (50 mg/L) for selection of transformants. Of these seeds, 40 plants had true leaves with green color, whereas almost all the sown seeds failed to develop true leaves and were etiolated with light yellow shoots. Forty transformants with true leaves survived from kanamycin media, and were transplanted to a growth chamber in standard conditions (1Arabidopsis plants (Arabidopsis (data not shown). Repeated kanamycin selections were conducted in successive generations to find homozygous seeds for mass production of transgenic Arabidopsis plants expressing mAbPSO57 , but intensively eluted in Fraction 4 in other resins (pH 8.5 (8.5R), 9.5 (9.5R), 10.5 (10.5R), and 11.5 (11.5R)) ) .PSO57 among seven fractions (F1–F7) of each experimental group were as follows: GR , 8.5R , 9.5R , 10.5R , and 11.5R , respectively (PSO57 in the other fractions compared to the other groups.Each eluted sample (F1–F7) was quantified using a protein concentration analysis . The highest concentration of mAbectively . The totPSO57 purified from different resins were compared to commercial antibodies (Anti-rabies human immunoglobulin). The mean RFFIT values of each group were as follows: GR (7.16 IU/mL), 8.5R (5.50 IU/mL), 9.5R (7.29 IU/mL), and 10.5R (8.29 IU/mL), respectively. The mAbP could not be purified from 11.5R resin  and hydroxyl (OH−) groups, the outward directionality of Protein A is random. Thus, we optimized the cysteine residue at the C terminal of protein A to give a constant directionality. When the protein A binds to the sulfhydryl group (SH−), it is directed outward in a row. It is expected that the constant directionality can deliberately induce protein A to become saturated with the resin by using a large amount of protein A and reacting for a long time. In this research, we manufactured four differently coupled the optimized protein A and epoxy-activated agarose resins to purify plant derived anti-rabies mAbs under different pH conditions. SDS–PAGE results showed that the capacity of pH 11.5 group was too low to effectively purify mAbPSO57 in transgenic Arabidopsis compared to that in the other experimental groups  including protein A Sepharose™ resin . PSO57. Although the 8.5R, 9.5R, and 10.5R showed some non-specific bands below 50 kDa, the purified quantity are more than two times stronger than the GE. The virus-neutralizing efficacies of four mAbPSO57 purified from different resins  were approximately three times higher than that of anti-rabies human immunoglobulin. Among the purified plant-derived antibodies samples, purified sample with the 10.5 resin showed the highest RFFIT value, indicating that the 10.5 resin purified more active mAb form compared to the others.The epoxide groups can be coupled with thiols, primary amines, and hydroxyl HOOC− groups depending on pH 8.5, 9.5, 10.5, and 11.5 conditions, respectively. The immobilized ligand such as protein A on an epoxy-activated agarose matrix can bind to mAbs . The dirArabidopsis expressing mAbPSO57. Overall, our results suggest that both pH 8.5 and 10.5 are optimal for the purification of anti-rabies mAbs from transgenic Arabidopsis plant leaf. This is the first research report on optimization of protein A chromatography resin using manipulation of coupling with pH conditions. Plant expression system has emerged as one of the most promising platforms for the large-scale production of therapeutic reagents , pBI mAb SO57 vector in DH5α competent cells, negative control (−), and non-transgenic tobacco plant (NT).Click here for additional data file.10.7717/peerj.6828/supp-2Supplemental Information 22-specific antibodies, respectively. Lane 3–10, T1 transformants putatively expressing anti-rabies mAbPSO57. Positive control (+); human rabies immunoglobulin(HRIG) and negative control (−); non-transgenic tobacco plant (NT).HC (50 kDa) and LC (25 kDa) were detected with HRP-conjugated goat anti-human IgG Fc- or IgG F(ab′)Click here for additional data file.10.7717/peerj.6828/supp-3Supplemental Information 3P; LC, light chain of mAbP. Epoxy-activated agarose beads were coupled to protein A under the pH conditions of 8.5, 9.5, 10.5, and 11.5 . Commercial protein A resin (GR)  was used as a positive control.Lane 1, protein marker; Lane 2, positive control (+), human rabies immunoglobulin (HRIG); Lane 3–9, eluted fractions of the purification F1–F7, respectively; Lane 11, flow through; HC, heavy chain of mAbClick here for additional data file.10.7717/peerj.6828/supp-4Supplemental Information 4(A) Nano-drop raw data of protein concentration (µg/mL) for GE resin with pH 7.0. (B) Nano-drop raw data of protein concentration (µg/mL) for amicogen resin with pH 8.5. (C) Nano-drop raw data of protein concentration (µg/mL) for amicogen resin with pH 9.5. (D) Nano-drop raw data of protein concentration (µg/mL) for amicogen resin with pH 10.5. (E) Nano-drop raw data of protein concentration (µg/mL) for amicogen resin with pH 11.5. (F) Raw data for mean values of protein concentration ((µg/mL) obtained from GR, 8.5R (AR), 9.5R (BR), 10.5R (CR), 11.5R (DR). The vertical axis values (µg/mL) represent the mean value of triple measurement per each case. Diamond, positive control resin (GE) ; Square, resins under pH 8.5 condition (8.5R); Triangle, resins under pH 9.5 condition (9.5R); Circle, resins under pH 10.5 condition (10.5R), and Cross, resins under pH 11.5 condition(11.5R), respectively.Click here for additional data file.10.7717/peerj.6828/supp-5Supplemental Information 5(A) Raw data of virus-neutralizing activity assay. (B) Mean values of RFFIT (IU/mL) of pH 8.5, 9.5, 10.5, and 11.5 conditions. (C) Graph obtained from “B.” The values (IU/mL) represent the mean value of duplicate measurements. Commercial protein A resin (GR)  was used as a positive control.Click here for additional data file.


Terminalia catappa L. methanolic extract (TCE) in human fibroblasts (Hs68) against hydrogen peroxide (H2O2)-induced oxidative damage.Oxidative stress plays a crucial role in aging-related phenomenon, including skin aging and photoaging. This study investigated the protective role and possible mechanism of Various in vitro antioxidant assays were performed in this study. The effect and mechanisms of TCE on oxidative stress-induced oxidative damage were studied by using western blotting.50 of TCE was 8.2 μg/mL for 1,1-diphenyl-2-picrylhydrazyl radical scavenging, 20.7 μg/mL for superoxide anion radical scavenging, 173.0 μg/mL for H2O2 scavenging, 44.8 μg/mL for hydroxyl radical scavenging, and 427.6 μg/mL for ferrous chelation activities. Moreover, TCE inhibited the H2O2-induced mitogen-activated protein kinase signaling pathway, resulting in the inhibition of c-Jun, c-Fos, matrix metalloproteinase (MMP)-1, MMP-3, MMP-9, and cyclooxygenase-2 expression. TCE also increased hemeoxygenase-1 expression inhibited by H2O2. Finally, TCE was demonstrated reverse type I procollagen expression in fibroblasts after H2O2 treatment.The ICAccording to our findings, TCE is a potent antioxidant and protective agent that can be used in antioxidative stress-induced skin aging.The online version of this article (10.1186/s12906-018-2308-4) contains supplementary material, which is available to authorized users. Oxidative stress plays a crucial role in aging-related disorders, including atherosclerosis, cardiovascular diseases and skin aging . High leisorders .Skin aging is characterized by a sagging appearance, wrinkles, and pigmentary changes, and principally manifests as the degradation of extracellular matrix (ECM) proteins, including type I collagen, elastin, proteoglycans, and fibronectin , 5. TypeTerminalia catappa L. belongs to the family Combretaceae, and in Southeast Asia, it is commonly used as a folk medicine for treating hepatoma and hepatitis [T. catappa have been reported to exhibit chemopreventive, antioxidant, hepatoprotective, and anti-inflammatory activities [T. catappa includes the phytochemicals of flavanoids , tannins , and triterpenoids (asiatic acid and ursolic acid) [T. catappa extract exhibits antifungal and antidepressant activities [T. catappa was shown to promote wound healing in rats [T. catappa L. hydrophilic extract exerts protective effects on UVB-induced photoaging by inhibiting MMPs expression and upregulating type I procollagen expression [T. catappa against oxidative stress-induced skin damaging are unclear. Therefore, this study investigated the effects of T. catappa methanolic extract (TCE) on H2O2-induced skin damage and on the protein expression of MAPKs, which activate protein-1 (AP-1), MMPs, and type I procollagen in human skin fibroblasts (Hs68).epatitis , 15. Thetivities , 17. T. ic acid) , 18. In tivities , 20. Top in rats , and ourpression . However2), DPPH, DL-dithiothreitol, and all other reagents used in this study were purchased from Sigma-Aldrich Chemicals .Fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA, and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Gibco, Invitrogen . The Bradford reagent was supplied by Bio-Rad Laboratories , and Tris and MTT were purchased from USB . Methanol, dimethyl sulfoxide, doxycycline hyclate, calcium chloride , after which TCE was added. Absorbance was measured at 560 nm on the ELISA reader.2O2 was prepared in PBS and mixed with various concentrations of TCE. Then, after incubation, absorption was measured at 230 nm on the ELISA reader.The peroxide scavenging activity of TCE was spectrophotometrically detected using a previously described method , 26. H2O2O2, a monopotassium phosphate–potassium hydroxide buffer, and distilled water; the mixture was then incubated at 100 °C for 15 min and centrifuged. The absorbance of the supernatant was subsequently measured at 532 nm on a microplate reader . Mannitol was used as a positive control, and the hydroxyl radical scavenging activity of TCE was obtained as the percentage inhibition of deoxyribose degradation [The hydroxyl radical scavenging activity assay was performed by mixing TCE, ascorbic acid, deoxyribose, iron (III) chloride, EDTA, Hradation .Various concentrations of TCE were mixed with an iron (II) chloride solution. The reaction was initiated after ferrozine was added. Absorbance was then spectrophotometrically measured at 562 nm on the microplate reader. The results are expressed as the percentage inhibition of the generation of the ferrozine–ferrous complex herein .The reducing power of TCE was determined using a previously described method , 28. VarHs68, HaCaT cells, and B16F0 cells were purchased from the Bioresource Collection and Research Center in Hsinchu, Taiwan. These cells were maintained in DMEM containing 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin in an incubator set at 37 °C.To understand the cytotoxicity of TCE on the skin, Hs68, HaCaT cells, and B16F0 cells were applied to study the cell viability. The cells were seeded in the plate, allowed to attach overnight, and were treated with 1 mL of various concentrations of TCE dissolved in DMEM for 24 h. The cytotoxicity of TCE was then evaluated using the MTT assay, as described elsewhere .2O2 for 1 h. Subsequently, the cells were incubated with 10 μM DCFDA in DMEM for 30 min, after which they were examined under a fluorescence microscope . Fluorescence  was measured on a microplate reader .Intracellular ROS generation was measured using a previously detailed method . In brie2O2 for 1 h. The cells were collected and lysed with protein extraction buffer, as previously described [The cells were incubated with TCE (5–50 μg/mL) for 4 h, followed by incubation with 150 μM Hescribed . An equap < 0.05.Values are presented as the mean ± standard deviation of at least three independent experiments. The results were analyzed using one-way analysis of variance, followed by Scheffe’s test. Statistical significance was set at The extraction yield of TCE from leaves was 11.5%. The total phenolic content of the extract was determined using the Folin–Ciocalteu method, and the regression coefficient of the calibration curve was 0.9995. Specifically, the total phenolic content of TCE was 220.2 ± 0.2 μg/mg gallic acid equivalent (GAE). Additionally, the total flavonoid content of TCE was determined using the aluminum chloride colorimetric method, and the regression coefficient of the calibration curve was 0.9991. The total flavonoid content was 109.0 ± 0.8 μg/mg quercetin equivalent (QE). The content of gallic acid was 74.62 μg/mL by HPLC/UV analysis  and the positive control BHA (250 μg/mL) are shown in Fig. 50 = 166.1 μg/mL). The hydroxyl radical scavenging activities of TCE (50–1000 μg/mL) and the positive control mannitol (15 mM) are shown in Fig. 50 of TCE for hydroxyl radical scavenging was 39.6 μg/mL.The antioxidant activity of TCE was study by using free radical scavenging assay and chelating assay. Figure 50 of TCE was 427.6 μg/mL for metal chelation. The reducing power ranged from 20.1% ± 1.0% to 101.7% ± 1.2% for 50–1000 μg/mL TCE, whereas the reducing power for 100 μg/mL ascorbic acid (positive control) was 62.6% ± 3.5% , human keratinocytes (HaCaT), and mouse melanoma cells (B16F0) were treated with various concentrations of TCE (5–100 μg/mL), and their cell viability was measured using the 3--2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Fig. 2O2 treatment. Cell viability ranged from 72.7% ± 1.8% to 81.9% ± 3.9% for 5–50 μg/mL TCE. These results indicated that TCE protects the skin from oxidative stress-induced cytotoxicity.As shown in Fig. 2O2-exposed fibroblasts than in control cells. Moreover, this increase in ROS generation was attenuated in H2O2-exposed fibroblasts pretreated with various concentrations of TCE (5–50 μg/mL). ROS generation in H2O2-exposed fibroblasts increased to 1.7-fold compared with control cells, and significantly decreased to 1.3-fold compared with control cells. TCE at 50 μg/mL decreased H2O2-induced intracellular ROS generation by 23.1%. Thus, TCE protects the skin from ROS damage.The 2′,7′-dichlorofluorescin diacetate (DCFDA) fluorescence assay was used to qualitatively characterize intracellular ROS generation. As shown in Fig. 2O2 induced the phosphorylation of p38, extracellular signal–regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). TCE (5–50 μg/mL) dose-dependently inhibited the phosphorylation of ERK, and the effect was significant in the cells treated with > 20 μg/mL TCE. Similar to the effect on ERK, TCE inhibited JNK and p38 activation, which were significantly suppressed when TCE concentration was 20 μg/mL.As shown in Fig. 2O2 increased c-Jun and p-c-Jun expression to 1.5- and 2.9-fold that of the control, respectively, whereas > 5 μg/mL TCE significantly reduced the effect. In addition, H2O2 induced c-Fos expression, but TCE reduced the effect. These results further indicated that TCE protects the skin from oxidative stress-induced damage.As shown in Fig. 2O2-exposed Hs68 from oxidative stress-induced damage, the expression of cellular MMP-1, − 3, and − 9 proteins was measured. As depicted in Fig. 2O2 significantly elevated the expression of MMP-1, − 3, and − 9 proteins by 1.3-, 1.2-, and 1.2-fold compared with controls in Hs68, respectively. By contrast, TCE attenuated H2O2-induced MMP expression. Specifically, treatment with > 5 μg/mL TCE significantly reduced H2O2-induced MMP-1, − 3, and − 9 expression. These results indicated that TCE prevents the H2O2-induced elevation of MMP-1, − 3 and − 9 levels, thus protecting the skin from oxidative stress-induced damage.To examine whether TCE protects H2O2 significantly reduces HO-1 protein expression in Hs68, whereas TCE treatment dose-dependently increases HO-1 expression -1 gene and protein play a pivotal role in the modulation of antioxidant, anti-inflammatory, and antiapoptotic activities. This study revealed that Hion Fig. .2O2 than in control cells -2 levels were 1.3-fold higher in fibroblasts exposed to 150 μM Hlls Fig. . In addi2O2, the expression of type I procollagen increased to 1.3-fold compared with that in control cells, whereas TCE inhibited this effect  and Keap1 are redox-sensitive transcription factors and key intracellular modulators of antioxidant defense against environmental stresses. For example, Nrf2 has been reported to protect skin cells from UV- and pollutant-induced oxidative damage and cellular dysfunction . On expo2O2 has been reported to cause skin aging by inducing oxidative stress and MMP expression [2O2 disrupts transforming growth factor beta transduction and subsequently inhibits collagen biosynthesis, inducing skin aging [2O2 was determined to upregulate the phosphorylation of MAPKs, c-Jun, c-Fos, and MMP-1, − 3, and − 9 proteins, whereas TCE inhibited these effects. This finding suggests that TCE activity is dependent on this signaling transduction. MMPs mediate degradation of ECM and play an important role in tissue homeostasis and remodeling including angiogenesis and tissue repair. Over suppression of MMPs may cause abnormal accumulation of ECM.Exposure of the skin to UV induces ROS generation and regulates the expression of genes and proteins, resulting in photodamage and photocarcinogenesis . In addipression , while Upression . In addiin aging . In the T. catappa water extract protected skin from photodamage by inhibiting the MAPK/AP-1/MMP pathway [T. catappa water extract elevated the collagen content in Hs68 [2O2 was found to increase the collagen content in Hs68, whereas TCE reversed the effect. One previous study showed that H2O2 also reduces mRNA expression of type I collagen (COL1A1) in fibroblasts [2O2 induces oxidative stress damage to cells and the body, which can trigger the repair process of the skin, thereby increasing the collagen content. However, excessive collagen synthesis may cause collagen fibrosis and scleroderma [The results are consistent with those of our previous study, in which the  pathway . UVB ray in Hs68 . Similarroblasts , althougeroderma . Overall2O2-induced skin damage by inhibiting the protein expression of MMP, AP-1, MAPKs, and COX-2 . (DOCX 134 kb)


Cyclocybe aegerita (synonyms Agrocybe aegerita and A. cylindracea) is one of the most praised cultivated edible mushrooms and is being cultivated at large scale for food production. Furthermore, the fungus serves as a model organism to study fruiting body formation and the production of secondary metabolites during the life cycle of Basidiomycota. By studying the secondary metabolite profiles of C. aegerita, we found several terpenoids in submerged cultures. Aside from the main metabolite, bovistol (1), two new bovistol derivatives B and C  and pasteurestin C as a new protoilludane (4) were isolated by preparative HPLC. Their structures were elucidated by mass spectrometry and NMR spectroscopy. The relative configurations of 2–4 were assigned by ROESY correlations, and 3JH,H coupling constants in the case of 4. Applying quantitative PCR for gene expression validation, we linked the production of bovistol and its derivatives to the respective biosynthesis gene clusters.The strophariaceous basidiomycete  Agrocybe aegerita (synonym: A. cylindracea) was traditionally accommodated in the genus Agrocybe (family Bolbitiaceae) until a recent phylogenetic study based on comparisons of rDNA sequence data has resulted in its placement in the Strophariaceae family and it was accordingly moved into the resurrected genus Cyclocybe Velenovsky + peak at m/z 519.2718 in the HRESIMS spectrum. 1H and 1H,13C-HSQC NMR data . This methoxy was connected to C-3 due to its HMBC correlation to this carbon atom, along to the ones from 1-H2, 10-H2 and 12-H3 to C-3. Compared to 1, the Δ2,3 double bond is shifted to Δ2,9, explaining the high field shift of the conjugated ketone C-8 (δC 197.2). A ROESY correlation between 3-OCH3 and 13-Ha revealed a 3S configuration. The closest known structural relative of 3 is the 3-demethoxy-3-hydroxy derivative of 3, which has been described as a spontaneous dimerization product of psathyrellon B indene-3-carboxylic acid.In addition to disesquiterpenoids NMR data  revealed1) showed weak cytotoxic effects , but was inactive against all test organisms in our standard test panel, comprising selected Gram-positive and Gram-negative bacteria as well as fungi .Optical rotations were measured on a Perkin-Elmer 241 spectrometer, the UV spectra on a Shimadzu UV–vis spectrophotometer UV-2450. NMR spectra were recorded with a Bruker Avance III 700 spectrometer, equipped with 5 mm TCI cryoprobe  5.00 g/L, oatmeal  5.00 g/L, D(+)-sucrose  4.00 g/L, D-mannitol  4.00 g/L, D-glucose monohydrate  1.50 g/L, CaCO3  1.50 g/L, lactalbumin hydrolysate  0.50 g/L, (NH4)2SO4 0.50 g/L]. The pH value of the medium was set to 7.2. To inoculate the medium, 5 mL of a preculture were used. The inoculated shaking flasks were incubated on a lab shaker  at 23 °C. After one week of incubation the glucose concentration in the medium was tested daily. If no more free glucose could be found in the medium, the cultivation was extracted.A culture of the strain 2O) in a separating funnel (two times) and dried with water-free sodium sulfate . The sodium sulfate was filtered off and the extract was narrowed up to the dry with a rotary evaporator. The crude extract was dissolved in 4 mL methanol, evaporated under a nitrogen evaporator  and stored in a freezer at −20 °C. The crude extract of the fermentation broth was obtained the same way.The mycelium was separated from the fermentation broth with the help of a fluted filter. The filtered mycelium was overlaid with actone (1:1 ratio) and digested in an ultrasonic bath  for 30 min (two times). After the digest the mycelium was separated from the filtrate. The filtrate was narrowed up to the aqueous phase in vacuo. The aqueous phase was shaken out in a 1:1 ratio with ethyl acetate  and methanol  by a Gilson RP-HPLC system  equipped with a GX-271 Liquid handler, a diode array detector (DAD) 172 and a 305 and 306 pump. The separation was performed with a VP Nucleodur C18ec  column and a flow rate of 20 mL/min. The gradient was set from 30 to 70% of solvent B in 45 min, with an increase to 100% B in 15 min, followed by isocratic conditions at 100% B for 15 min. All LC fractions were collected according to the UV absorption at 210 nm. Methanol was evaporated in vacuo. The aqueous residues were frozen and then removed by using an Alpha 1-4 LSC freeze dryer . 12.6 mg of fraction V were obtained as a mixture of compounds 1, 2.2 mg of compound 2 and 0.4 mg of compound 3.Subsequently fraction V was separated via another RP-LC under different conditions. The separation was performed with the same Gilson RP-HPLC system and flow by using a Gemini 10u C18  column. Solvent B was changed to acetonitrile and also no TFA was added. The gradient was set from 50% to 75% of solvent B in 45 min with an increase to 100% B in 15 min, followed by isocratic conditions at 100% B for 15 min. Collection and work-up of the obtained fractions was performed as described above. This separation yielded 4 mg of compound Bovistol A (1): Colourless oil; 1H and 13C NMR data  in CDCl3: see m/z): 1015.52 [2M + Na]+, 479.30 [M + H − H2O]+, 991.64 [2M − H]−, 541.39 [M + HCOO]−; HRESIMS (m/z): [M + Na]+ calcd for C30H40O6Na, 519.2717; found, 519.2718.Bovistol B (2): colourless oil; [α]D25 +3 ; 1H and 13C NMR data  in CDCl3: see m/z): 979.57 [2M + Na]+, 957.61 [2M + H]+, 479.35 [M + H]+, 523.30 [M + HCOO]−, 477.29 [M − H]−; HRESIMS (m/z): [M + Na]+ calcd for C30H38O5Na, 501.2585; found, 501.2606.Bovistol C (3): Colourless oil; [α]D25 +106 ; 1H and 13C NMR data  in CDCl3: see m/z): 1043.65 [2M + Na]+, 533.37 [M + Na]+, 493.31 [M + H − H2O]+, 509.30 [M − H]−, 555.32 [M + HCOO]−; HRESIMS (m/z): [M + Na]+ calcd for C31H42O6Na, 533.2874; found, 533.2859; [M + H]+ calcd for C31H43O6, 511.3054; found, 511.3044.Pasteurestin C (4): Colourless oil; [α]D25 +21 ; 1H and 13C NMR data  in acetone-d6: see m/z): 523.32 [2M + Na]+, 233.13 [M + H − H2O]+, 249.04 [M − H]−; HRESIMS (m/z): [M + Na]+ calcd for C15H22O3Na, 273.1461; found, 273.1460; [M + H]+ calcd for C15H23O3, 251.1642; found, 251.1637.C. aegerita AAE-3 with a total volume of 500 mL was prepared in ZM ½ medium. After incubation for 11 days the seed culture was homogenized with an ULTRA-TURRAX under sterile conditions and used for inoculation of a 15 L bioreactor (xCUBIO in-situ bbi biotech) filled with 10 L ZM ½ medium. The pH value was set to 7.2 (unregulated); the DO was also not regulated. The temperature was regulated at 23 °C. Furthermore the submerged aeration rate and stirrer speed was fixed to 0.15 vvm and 200 rpm (rushton turbine). For foam destruction into the process Tego Antifoam D2310 (Evonik Nutrition & Care GmbH) was used. After 29 days of cultivation the fermentation broth was harvested. The biomass and suspended substrates was separated by centrifugation.A seed culture of the strain ® Pearl . Reverse transcription was performed with the Invitrogen M-MLV Reverse Transcriptase kit  according to the manufacturer’s protocol. 10 µL of extracted RNA and 1 µL of 10 µM oligo-(dT)30 primer  were used for cDNA synthesis. For removal of RNA in the transcribed cDNA sample, 1 µL of AMRESCO RNase A  was added and the mixture was incubated at 37 °C for 20 min. Primers for qPCR analysis were designed using Geneious 11.0.4. . Primer pairs for C. aegerita housekeeping genes going by the gene IDs AAE3_02268 and AAE3_07669 (http://www.thines-lab.senckenberg.de/agrocybe_genome) have been identified and validated by NormFinder and geNorm algorithm to be the best combination for qPCR-based transcription analyses of C. aegerita by means of qPCR (data not published). Briefly, KAPA SYBR® FAST qPCR Master Mix , 900 nM forward primer, 900 nM reverse Primer  until further use. Fungal mycelium was freeze-dried and ground with liquid nitrogen. RNA was extracted from ground mycelium using Ambion TRIzol™ Reagent  according to the manufacturer’s instructions with minor changes according to the method of Chomczynski and Sacchi . RNA cone Primer , 10 ng oFile 11H and 13C NMR spectra of compound 1 and 1H, 13C, COSY, ROESY, HSQC and HMBC NMR spectra of compounds 2–4.


We hypothesized that acute exposure to ambient fine particles  throughout the exposure window of the preceding 1–14 days. No significant difference was observed for the changes in pulmonary biomarkers between the two groups.All the subjects exhibited significant acute elevation of exhaled nitric oxide, white blood cells, neutrophils, interleukin-1α, and glycated haemoglobin with increased exposure to PMPreDM individuals are more susceptible to the acute cardiometabolic effect of air pollution than the healthy individuals. A considerable public health burden can be inferred, given the high prevalence of prediabetes and the ubiquity of air pollution in China and worldwide.The online version of this article (10.1186/s12940-019-0502-6) contains supplementary material, which is available to authorized users. The moving average of ambient PM2.5 concentration during the 1–14 days prior to the clinical visit was linked with the health outcome measurement for each subject as an indicator of cumulative exposure. These moving average concentrations are referred to as “average 1- to 14-day exposure to PM2.5” in the following context: e.g. “average 7–day exposure to PM2.5” refers to the mean concentration of ambient PM2.5 in the preceding 1–7 days before measurement of health outcomes. This time window was chosen because previous studies have suggested that most acute to sub-acute effects of PM2.5 on study outcomes can be observed during this time period  to 56.3% [CI: 37.8–77.2%] throughout the exposure window of the preceding 1–14 days. A significant increase was also observed in inflammatory cells and cytokines, with maximum elevation of 3.0% [CI: 1.5–4.6%], 3.9% [CI: 1.6–6.2%], 15.2% [CI: 7.5–23.4%], 5.7% [CI: 1.9–9.7%], 6.2% [CI: 1.2–11.5%] for WBCs, neutrophils, IL-1α, IL-2, and IL-8 respectively in different exposure time windows. No significant changes were observed for BP, endothelial function, and glucose metabolism, except for elevated fasting glucose and HbA1c with maximum increase of 0.9% [CI: 0.1–1.6%] and 1.6% [CI: 0.9–2.3%] respectively.Figure 2.5 associated adverse effect between healthy and preDM subjects for the selective 12 biomarkers  between the two groups.Figure 2.5-associated effect on the biomarkers among all the subjects were basically unchanged with similar lag pattern , elevated systolic and diastolic BP, impaired endothelial function (increased AP75), and elevated fasting glucose levels.In this community-based panel study with nested case-control design, we observed significant differences between preDM and healthy subjects in biological changes after short-term exposure to ambient PM2.5 concentration of 32 μg m− 3 also observed a significant positive association between daily mortality and 3-day mean ambient PM2.5 in diabetic patients [Early evidence of the susceptibility of diabetic patients to air pollution was mostly based on time series studies, and collectively argued for a potential increased short-term impact of PM on CVD-related morbidity , 10 and patients . To explpatients , vasculapatients , 20, elepatients . These ppatients , which pNO, WBCs and neutrophils in both groups after acute exposure to ambient PM2.5 observed in this study confirm this classic mechanism of immune activation [2.5-associated elevations in some pro-inflammatory cytokines , but we were surprised to observe significant decreases in TNF-α and IL-6  vs. non-diabetic (n = 36) subjects differed in inflammatory responses to air pollution, and they reported a significant enhancement in CRP and IL-6 levels in diabetic patients. The differences between Dubowsky’s study [Inflammation is known as a key pathway in both PM-induced adverse health effects  and diabtivation  in both tivation . However’s study  and ours− 3 increase in ambient PM2.5 (average 1–7 days). Our study further confirmed that pattern, as we observed a − 0.2–0.8% increase in SBP  per 56 μg m− 3 increase in PM2.5 in preDM subjects, and a statistically nonsignificant change in healthy subjects. Considering the potential confounding effect of hypertensive status and medication, we had further run the examinations excluding subjects with high blood pressure . The differences in the PM2.5-associated effect on BP elevation between the two preDM and healthy subjects remained significant with similar lag pattern


Stenotrophomonas acidaminiphila. The strain BJ1 uses chlorothalonil as a sole carbon source. At initial concentrations of 50, 100, 200 and 300 mg l−1, it degraded 91.5%, 89.4%, 86.5% and 83.5% of chlorothalonil after 96 h of inoculation under optimum conditions (30°C and pH 7.0). Two metabolites, methyl-2,5,6-trichloro-3-cyano-4-methoxy-benzoate and methyl-3-cyano-2,4,5,6-tetrachlorobenzoate, were detected and identified based on HPLC–MS analysis, which suggests that the strain BJ1 metabolized chlorothalonil through the hydroxylation of chloro-group and hydration of cyano-group. The results of this study highlight the great potential for this bacterium to be used in chlorothalonil pollution remediation.Chlorothalonil is a widely used fungicide, but the contamination of soil and water environments by this chemical causes potential threats to biodiversity. Given the metabolic potential of soil microorganisms, there is a need for developing microbiological approaches to degrade persistent compounds, such as chlorothalonil, in contaminated sites. Here in this study, we isolated a bacterial strain  capable of degrading chlorothalonil from a chlorothalonil-contaminated farmland soil in the Shandong Province, China. Using 16S rDNA gene sequencing, morphological and biological characteristics, we identified the strain BJ1 as  HoweverEnterobacter cloacae TUAH-1, Paracoccus sp. XF-3, Bacillus subtilis WB800, Ochrobactrum lupini TP-D1, Pseudomonas CDS-8, etc. are reported to metabolize chlorothalonil [Several methods are suggested to remove chlorothalonil from contaminated sites that include but are not limited to these: dechlorination, hydrolysis, photocatalysis and biodegradation ,16,17. Athalonil –23, therthalonil .We isolated strain BJ1 from a chlorothalonil-contaminated farmland soil. It can colonize chlorothalonil-contaminated soil and accelerate the degradation of chlorothalonil, as reported in our previous study . However2.2.1.−1 tryptone, 5.0 g l−1 yeast extract, 1.0 g l−1 NaCl (pH 7.0). The mineral salt medium (MSM) contained 1.0 g l−1 NH4NO3, 1.5 g l−1 K2HPO4, 0.5 g l−1 KH2PO4, 0.5 g l−1 MgSO4, 0.5 g l−1 (NH4)2SO4, 0.5 g l−1 NaCl (pH 7.0). The solid agar medium was prepared by adding 16.0 g l−1 agar. The medium was autoclaved at 121°C. The chlorothalonil was dissolved in acetone as a stock solution (1000 g l−1). To avoid the possible utilization of acetone as carbon by strain BJ1, the different quantity of chlorothalonil stock solution was filter-sterilized and evaporated before adding the MSM.Chlorothalonil  was obtained from Qingdao Hansen Biologic Science Co., Ltd, Qingdao, China. Unless otherwise stated, all reagents used in this study were of analytical grade. The bacterial growth medium  contained 10.0 g l2.2.−1 chlorothalonil. Then, the mixture-containing Erlenmeyer flask was incubated in a rotary shaker at 160 r.p.m. and 30°C for 7 days. The final enrichment culture was diluted with sterile water and then spread onto the MSM agar plate containing 100 mg kg−1 chlorothalonil. The plates were incubated at 30°C for 48 h. We picked individual colonies based on their morphologies and tested them for chlorothalonil-degrading capabilities. Among these, one strain (named as BJ1) demonstrated the highest degradation ability, and it used chlorothalonil as the sole carbon for its growth.To isolate chlorothalonil-degrading bacteria, we collected soil from chlorothalonil-contaminated farmland located in Yantai city, Shandong province, China. The enrichment culture technique was employed to isolate chlorothalonil-degrading bacterial strains. Briefly, 10 g of fresh soil was added to 90 ml MSM solution containing 100 mg lBergey'sManual of Determinative Bacteriology [−1), 0.5 µl of each primer, 0.5 µl of DNA template and sterile filtered milli-Q water to 25 µl. The PCR conditions were: denaturation at 94°C for 5 min, followed by 30 cycles at 94°C for 30 s, 55°C for 30 s, 72°C for 90 s, and a final extension at 72°C for 10 min [E. coli DH5a. The PCR product was sequenced by Sangon Biotech Co., Ltd . We deposited the gene sequence of 1464 bp in the NCBI GenBank under the accession number JQ247581.1. The relative sequences were analysed by using the basic local alignment search tool (BLAST) (www.ncbi.nlm.nih.gov/BLAST). A phylogenetic tree was built by the neighbour-joining method using MEGA7 software.We determined the taxonomic identity of the stain BJ1 using its morphological, physiological and biochemical properties, as well as 16S rDNA gene sequencing analysis. The morphological features and physiological and biochemical properties of strain BJ1 were analysed according to eriology . The genr 10 min . The PCR2.3.600 nm) of cells was adjusted to approximately 1.0. Then, we added chlorothalonil into the new MSM as sole carbon and energy source. Unless otherwise stated, we inoculated cells suspension at a 5% (v/v) level into a 10 ml glass tube containing 5 ml MSM with 100 mg l−1 of chlorothalonil. We also set non-inoculated controls following the same protocol. The mixture-containing tubes were incubated at 30°C and 160 r.p.m. in a rotary shaker. During incubation, samples of the cultures were collected every 12 h and the residual chlorothalonil was determined. The bacterial growth was determined by measuring the optical density (OD600 nm).The strain BJ1 was cultured in LB medium at 30°C and 160 r.p.m. in a rotary shaker until it reached the stationary stage. The bacterial cell pellet was collected by centrifuging cultures at 5000 r.p.m. for 10 min. Then, the cells were washed twice and resuspended in the MSM. The optical density (OD−1) were also studied under optimum pH and temperature. All samples were set up in triplicate.To determine the optimum conditions, we investigated the impact of different pH  and temperature levels  on the biodegradation of chlorothalonil by the strain BJ1 after 4 days of incubation. In addition, the effects of different initial chlorothalonil concentrations  system equipped with an electron capture detector and an HP-5 capillary column (30 m × 0.32 mm × 0.25 µm) was used to determine the content of chlorothalonil. The operating conditions of GC were as follows: injector temperature, 250°C; detector temperature, 300°C; oven temperature was initially set at 80°C for 1 min, ramping at 25°C min−1 to 260°C and held for 4 min. Nitrogen was used as carrier gas and the injection volume was 1 µl. The recovery rate of chlorothalonil from MSM was 85.6–107.4%.The chlorothalonil residue in MSM was extracted with 2.4.−1 of chlorothalonil as a sole carbon source. The 2 ml of sample was extracted after 4 days of incubation with an equal volume of acetonitrile with 5% NaCl and analysed by high-performance liquid chromatography–electrospray ionization–mass spectrometry (HPLC–ESI–MS). The HPLC–ESI–MS (Agilent) conditions were set up as described by Shi et al. [−1. The MS conditions were: mass scanning range (m/z), 80–600; mass detector, quadrupole mass spectrometer; source temperature, 100°C; cone voltage, 30 V; capillary voltage, 3000 V; desolvation gas (N2) flow rate, 500 l h−1; and desolvation temperature, 300°C.To detect the metabolites, the strain BJ1 was cultured in 10 ml MSM containing 100 mg li et al. : column,2.5.All statistical analyses were performed in SPSS 18.0 software . All data are presented as mean ± s.d. One-way ANOVA and Duncan's multiple range tests were used for comparing the differences between treatments at a confidence level of 0.05.3.3.1.Stenotrophomonas acidaminiphila strain CCUG54933, etc. (S. acidaminiphila BJ1 (GenBank accession no. JQ247581.1).According to the degradation capacity of chlorothalonil, the strain BJ1 was selected for this study. The colonies of strain BJ1 on LB plate were round with edge neat, uplifted surface, opaque and yellow in colour. This strain was a Gram-negative and asporulate bacterium with a coryneform morphology (0.4–0.5 × 0.2–0.3 µm). Biochemically, the strain was negative for oxidase, urease, nitrate reduction, hydrolysis starch, citrate utilization test and was positive for catalase and gelatin hydrolysis. It did not produce hydrogen sulfide and indole while it was able to use glucose, propionate, malic acid, pyruvic acid and acidic amino acid as sole carbon sources. The strain BJ1 was unable to tolerate 7% sodium chloride. The BLAST results showed that the sequence of 1464 bp of the 16s rDNA gene from the strain BJ1 exhibited 99% similarity with 33, etc. ; the str3.2.−1) in the MSM followed the corresponding patterns with time (600), indicating that this strain could use chlorothalonil as the sole carbon source for its growth. Both temperature and pH influenced the activity of strain BJ1. For instance, the efficiencies of chlorothalonil removal and cell densities at pH 6 and 7 were significantly higher than those of pH 5, 8 and 9. Approximately 93.2% and 92.7% of chlorothalonil were removed at pH 6 and 7, respectively (a). The relatively lower temperature (20°C and 25°C) did not accelerate the strain BJ1 growth and thus chlorothalonil degradation. At 30°C, 35°C and 40°C, the strain BJ1 grew well and demonstrated its ability to remove chlorothalonil efficiently. Among the three temperature treatments, the degradation ability of strain BJI on chlorothalonil was best at 30°C, and the degradation efficiency reached up to 92.8% (b). Thus, the results of this study indicated that the optimal conditions for BJ1 degradation were pH 6.0–7.0 and 30°C. Under the optimal conditions, strain BJ1 has excellent ability to remove chlorothalonil at the initial concentrations of 50, 100, 200 and 300 mg l−1 in MSM, with the degradation efficiencies up to 91.5%, 89.4%, 86.5% and 83.5% at 96 h, respectively . According to the mass spectrum, characteristics of fragments ion peaks and previous reports [We detected two chlorothalonil metabolites (peaks A and B) by HPLC after a 4-day incubation . Peaks A and B were then detected by the LC–ESI–MS at positive mode a,b. Acco reports ,28, prod4.−1 chlorothalonil in MSM after 96 h. The degradation ability of strain BJ1 on chlorothalonil is comparable to those of previously reported strains; such as, E. cloacae TUAH-1 could remove 97.4% of chlorothalonil (20 mg l−1) in aqueous solution after 48 h [Stenotrophomonas H4 could remove 82.2% of chlorothalonil (20 mg l−1) after 7 days incubation in liquid culture [Ochrobactrum lupini TP-D1 could degrade 90.4% of chlorothalonil (50 mg l−1) after 4 days of incubation in mineral salt broth [S. acidaminiphila based on its morphological and biological characteristics as well as the 16S rDNA gene sequence. Previously, bacterial species belonging to the Stenotrophomonas genus have been reported to degrade pesticides in the soil and water environments. For example, Dwivedi et al. [S. acidaminiphila strain JS-1 that was capable of using butachlor as a sole carbon source, the half-life of butachlor shortened to 4 days in the soil treated with strain JS-1. Moreover, Deng et al. [S. acidaminiphila strain G1 efficiently degraded eight organophosphorus pesticides, such as chlorpyrifos, methyl parathion, methyl paraoxon, diazinon, phoxim, parathion, profenofos and triazophos. According to our knowledge, this is probably the first report that provides detailed information on chlorothalonil degradation by S. acidaminiphila.Microbial-mediated removal of pesticides has received significant research attention in the last few decades ,28,29. Wthalonil  mg l−1 i culture , and Ochlt broth . The stri et al.  reportedg et al.  reportedb). It is very likely that low temperatures reduced the bacterial growth and metabolic activity [Both temperature and pH are two important environmental factors that significantly influence the microbial-driven degradation of pesticides in water and soil environments ,33. Thisfigure 3activity . Contraractivity . We sugga), thus suggesting the negative effect of extreme pH conditions on bacterial growth and activity. Our results also suggested that the extreme pH conditions might have negatively influenced the activities of enzymes responsible for the degradation of chlorothalonil [−1). Compared with strain BJ1, the other reported degrading bacteria usually only degrade below 200 mg l−1 of chlorothalonil [−1 chlorothalonil  accelerated the biodegradation of chlorothalonil by strain BJ1, whereas both highly acidic (5) and basic  pH conditions were not conducive for chlorothalonil biodegradation a, thus sthalonil ; howeverthalonil ,20,23,27thalonil . A similbacteria ,36.3 group might be esterified in the presence of formic acid during the analysis process [et al. [O. lupini TP-D1) degradation of chlorothalonil. In this regard, some key enzymes in bacteria, such as dehalogenase and glutathione S-transferase, play important roles in the degradation of chlorothalonil [Vicia faba root tips and found that the genotoxicity of chlorothalonil in the soil was reduced when it was degraded by the strain BJ1 [Until now, about a dozen of microbial metabolites of chlorothalonil have been reported in previous studies. The main metabolites mainly include hydroxy chlorohydrin, 3-methoxycarbonyl-2,4,5,6-tetrachlorobenzeneacetamide, methyl-2,5,6-trichloro-3-cyano-4-methoxy-benzoate, methyl-3-cyano-2,4,5,6-tetrachlorobenzoate, 4-sulfydryl-2,5,6-trichloroisophthalonitrile, 2-chloro-4,6-disulfydryl-5-chloromethylisophthalonitrile, 2-chloro-4-sulfydryl-5-chloromethylisophthalonitril, etc. ,23,37. H process ,39, thusthalonil ,41. Therrain BJ1 . Thus, orain BJ1 .S. acidaminiphila BJ1. The strain efficiently removed chlorothalonil at a wide range of temperature, pH and initial concentration. One of the important aspects of this study is that the strain BJ1 was capable of removing chlorothalonil at 40°C, thus suggesting the performance of this bacteria under climate warming. Although we identified two degradation products and two possible chlorothalonil-degrading pathways, more work is required to underpin the toxicity and metabolic pathway of chlorothalonil by the strain BJ1.In conclusion, we report the isolation and characterization of a novel chlorothalonil-degrading strain


Compound 5 showed the best docking score  with the least RMSD value (< 1.5) obtained by molecular dynamic simulations over 20 ns. All compounds showed promising anticancer activities especially compound 5 that achieved the highest inhibitory activities with IC50; 0.013, 0.031, 0.064, and 0.048 mmol/L against breast, colon, prostate, and lung cell lines respectively. The discovery of this lead compound confirmed the rational design. Further investigations may be required for optimization of this compound.Recent studies have suggested that aldose reductase inhibition preferentially inhibits the growth of cancer cells. However, the investigations of this issue are not many. Novel nine substituted- 4′-iminospiro thiadiazolidinyl]-2-one 1′,1′-dioxide derivatives were designed by both isosteric replacement of the imidazolidine-2,5-dione moiety in spirohydantoin scaffold and conformational rigidification approaches. A QSAR with high predictive power (r Aldose reductase (AR) is an enzyme that is found in mammalian tissues , 2. Its Recently, the AR inhibition was reported to prevent the growth factors’ activation in colon cancer . The sel50 = 2.5 × 10−6 M) however, it was orally inactive. In 1978, Pfizer discovered some spirohydantoin AR inhibitors with imidazolidine-2,5-dione scaffold like Sorbinil (IC50 = 0.5 μM) . Screening of other series of carboxylic acids-containing compounds showed that phthaloylglycine was a potent AR inhibitor  acetic acid scaffold (scaffold A) were discovered . These iThe first step of our work involved the building of a QSAR model for AR inhibitors by using the most active series that has (scaffold A). The main aim of this step was to derive all-important descriptors that contribute to the AR inhibition. The QSAR model can be used for prediction of the biological activity of newly designed compounds with unknown activity.pIC50 values for all derivatives were calculated , and a r2 coefficient was 0.99 and the Z-score was < 2.5 . The analysis of the ligand-binding pocket of AR crystal structure showed that it has three pockets namely; specificity pocket, anion pocket and hydrophobic pocket Fig. .Fig. 4LiScaffold A was studied for its predicted mode of orientation within these pockets. It was oriented between the specificity and anion pockets Fig.  24]. Th. Th24]. A pre-molecular docking study was performed in order to compare the free energy of binding ΔG and the orientation of both spirohydantoin (scaffold B) and the (scaffold A). The results showed that the later showed higher ΔG  while, spirohydantoin showed ΔG . The imidazolidine-2,5-dione ring of the spirohydantoin showed only two hydrogen bonds when compared to the (scaffold A) Fig. .Fig. 6ThWe tried to keep the 2-oxo-2,3-dihydroindol-3-yl ring with the high affinity and to apply an isosteric replacement of the imidazolidine-2,5-dione with imino-thiadiazolidine-1,1-dioxo scaffold to increase the pharmacodynamic interactions. The selection of this scaffold features more predicted interactions with the strong electron accepting sulfonyl group. In addition, the imino group that acts as an electron accepting that mimics the nitrile group in scaffold A. The imino-thiadiazolidine-1,1-dioxo ring is a rigid scaffold that will fit in a stable orientation because of its conformational restriction where it will have less number of proposed conformations when compared to the acyclic cyano acetic acid moiety that is more flexible and has many conformations which can lead to less binding stability. This proposed design is an optimization of both the acetic acid derivatives and sorbinil scaffold aiming to get a highly potent novel agents Fig. .Fig. 7ThNine compounds were designed that feature different substitutions at the indol ring to study the structure activity relationship of these substituents. Molecular docking study was conducted for 9 proposed compounds with indole—imino-thiadiazolidine-1,1-dioxo scaffold that share the proposed design in order to predict their mode of binding and compare it to the well-known AR inhibitors Fig. . Two sofThe docking results of compound 2 showed a H–B formed between the C=O of indole with Trp111 (NH). The sulfonyl group formed a strong H–B with the hydroxyl group of Tyr48. The imino-thiadiazolidine-1,1-dioxo scaffold contributed by one of its –NH to form a H–B with His110  among the other docked compounds when was docked with Leadit and it retained the same top rank with the second docking with MOE 2016.08  Table e–h.The main aim of molecular dynamic simulations was to evaluate the stability of binding of the proposed docked compounds inside the active side of AR during simulation time. The time period was 20,000 picoseconds (20 ns). The trajectory was analyzed each 1000 picoseconds. The root mean square deviation (RMSD) for each compound-protein complex was computed. According to the results, compound 5 with 5-nitro substitution achieved the lowest RMSD ranged from 1.00 to 1.50 which confirmed its strength of binding. The nitro group of this compound may contribute to the π–π hydrophobic interactions between the electron deficient environment of the phenyl moiety of indole ring that resulted from the nitro group substitution and the hydrophobic residues in the specificity pocket like; Leu300, Phe115 and Trp111. The second two compounds were compound 8 and 7 with the 5,7-dichloro and 4,7-dichloro substitution that showed RMSD values of 2.00 and 2.50 respectively. It seems that the electron deficiency environment that was created either by the electron withdrawing groups or halo atoms can support the stability of binding. The 5-chloro derivative showed RMSD of 3.00 that started to decrease gradually by time till reach a steady state at 2.00. The 7-chloro derivative (compound 9) and 5-bromo (compound 3) shared a common oscillation that appeared at RMSD of 2.70. Then the 5-iodo derivative appeared to have RMSD at 3.50. The absence of any substitution as in compound 1 was very clear to affect its RMSD that was 4.50. Also, the electron donating methyl group in compound 4 was high at RMSD of 4.00. Generally, the binding at both the specificity pocket and the hydrophobicity pocket may require a kind of π–π hydrophobic interactions that may be supported by either electron withdrawing groups or halo substitution  cm−1 for the sulfonyl group. The imino stretching band was found at 1650 cm−1.The chemical synthesis of the new compounds was achieved according to the reported method . The ketThe mechanism by which the reaction is performed is a modified Strecker synthesis, which was used for hydantoin synthesis Scheme a. The re50 of 0.013, 0.031, 0.064, and 0.048 mmol/L against MCF-7, HCT-116, PC-3, and A-549 respectively  which are already available in MOE 2016.08 package in the QSAR study were computed. The validation of the generated models was done by using the training set. Two types of descriptors were tested; 2D descriptors that consist of physical properties, atom and bond counts, subdivided surface area, pharmacophore feature descriptors and partial charges, Kier and Hall connectivity and Kapper shape indices. The second type includes 3D descriptors as; potential energy descriptors, surface area, volume, MOPAC, shape descriptors and conformation dependent charge descriptors. The significance of the models was obtained statistically by partial least square analysis (PLS). 2) and root mean square error (RMSE) values were calculated for all descriptors. MSE was computed by applying the following equation:To validate the model power, the correlation coefficient . X-ray crystallography method was used to obtain this protein with resolution of 2.8 Å and R-value free of 0.289. All coordinates were derived from protein data bank and all interactions were visualized between the conserved residues; Trp111, His110, Tyr48, Trp29, Trp219, Phe122, Phe115, Cys298, Leu300 and the complexed ligand. The MOE 2016.08 docking protocol was applied, in which the triangle method was used as a placement method with timeout of 300 s, and number of return poses as 1000. London dG was used as a rescoring method. Force field was selected as a refinement method by applying MMFF94x. While in case of leadit, the docking protocol involved loading the protein into Leadit 2.1.2 and the receptor components were chosen by selection of chain A as a main chain. Fidarestat was chosen as a reference ligand to which all coordinates were computed and this enabled the definition of the binding site. Amino acids within radius 6.5 Å were selected in the binding site. All chemical ambiguities of residues left as default. Ligand binding was computed by the classic Triangle matching method . The scoring default settings were kept. Intra-ligand clashes were computed by using clash factor = 0.6. Maximum number of solutions per iteration = 200. Maximum of solution per fragmentation = 200. The docking strategy was applied by using the base placement method.3. Amber 10:EHT was selected as a force field in the potential setup step. All Van der Waals forces, electrostatics, and restraints were enabled. The temperature of the system was increased from 0 to 300 K by adjusting the heat followed by equilibration and production for 300 ps; cooling was then initiated until 0 K was reached. The simulation was conducted over 20 ns period of time .MOE 2016.08 was used for computing the molecular dynamic simulations. The best pose from each docking process for each compound was kept inside the active site in order to be used for MD. The quality of the temperature-related factors, electron density, and protein geometries was tested. Energy minimization was computed after adding all the hydrogens. All solvent molecules within the system were deleted before solvation; salt atoms (NaCl) were added in 0.1 mol/L concentration to get a neutral biomolecular system. The solvent atoms (water) were added to surround the biomolecular system (protein–ligand complex) in a cube shape with cell dimensions; 81.9 × 81.9 × 81.9. The total number of molecules within the system was 16,475, 1.01 g/cmTo a 70% aqueous ethanolic solution (20 mL) contains an appropriate isatin derivatives (10 mmol), we added sulfamide (20 mmol) and sodium cyanide (11 mmol). The resulted mixture was refluxed for 6 h and then evaporated to dryness using rotavapor. An aqueous solution of 1 N sodium hydroxide (10 mL) was added to the residue. The solution was washed with ethyl acetate (2 × 10 mL) and diethyl ether (10 mL). The aqueous layer was then acidified with 1 N hydrochloric acid solution, leading to the precipitation of a solid that was filtered, dried and recrystallized from ethanol.max cm−1 (KBr): 1115, 1335, 1630, 1700, 1550, 1600. 1H NMR  δ: 6.45 , 6.60 , 6.92 , 7.25 , 7.35 , 7.40 , 8.10 , 11.01 .Yellowish brown solid, Yield (68%). mp; 263 °C. IR γmax cm−1 (KBr): 1120, 1350, 1638, 1700, 1550, 1600. 1H NMR  δ: 6.48 , 6.60 , 7.26 , 7.46 , 7.58 , 8.07 , 10.97 .Dark red solid, Yield (62%). mp; 271 °C. IR γmax cm−1 (KBr): 1122, 1348, 1650, 1710, 1550, 1650. 1H NMR  δ: 6.50 , 6.68 , 6.82 , 7.30 , 7.54 , 8.25 , 11.00 .Brown solid, Yield (55%). mp; 268 °C. IR γmax cm−1 (KBr): 1117, 1336, 1640, 1705, 1550, 1600. 1H NMR  δ: 2.30 , 6.50 , 6.54 , 6.87 , 7.23 , 7.40 , 7.66 , 11.10 .Red solid, Yield (70%). mp; 280 °C. IR γmax cm−1 (KBr): 1125, 1355, 1642, 1700, 1565, 1336. 1H NMR  δ: 6.62 , 6.70 , 7.05 , 7.35 , 8.22 , 8.75 , 11.53 .Reddish brown solid, Yield (73%). mp; 278 °C. IR γmax cm−1 (KBr): 1123, 1345, 1635, 1700, 1550, 1600. 1H NMR  δ: 6.60 , 6.65 , 6.72 , 7.55 , 7.40 , 7.71 , 10.53 .Yellowish white solid, Yield (69%). mp; 282 °C. IR γmax cm−1 (KBr): 1115, 1335, 1632, 1702, 1550, 1600. 1H NMR  δ: 6.60 , 6.68 , 6.99 , 7.25 , 7.33 , 11.97 .Orange solid, Yield (58%). mp; 265 °C. IR γmax cm−1 (KBr): 1118, 1342, 1638, 1710, 1550, 1600. 1H NMR  δ: 6.60 , 6.62 , 7.23 , 7.47 , 7.91 , 11.37 .Dark orange solid, Yield (64%). mp; 272 °C. IR γmax cm−1 (KBr): 1120, 1350, 1638, 1700, 1550, 1600. 1H NMR  δ: 6.45 , 6.58 , 6.90 , 7.20 , 7.24 , 7.34 , 10.99 .Dark yellow solid, Yield (70%). mp; 257 °C. IR γ4 cells per well in 100 µL of growth medium. The next step involved the addition of different concentrations of the tested sample. A multichannel pipette was used to dispense two-fold dilutions of the tested synthesized compound to a confluent cell monolayers distributed into 96-well . Then incubated at 37 °C in a humidified incubator with 5% CO2 for 48 h. Three wells were used for each concentration of the tested sample. Control cells were performed without test sample and with or without DMSO (0.1%). After incubation. Various concentrations of sample were added, and incubated for 24 h. A colorimetric method was used to determine the viable cells yield. The media were aspirated and the crystal violet solution (1%) was added. Tap water was used to rinse the plates after removing the stain. Glacial acetic acid (30%) was then added to all wells and mixed. The plates were gently shaken on Microplate reader , and then the absorbance of the plates were measured at a wavelength of 490 nm. All results were corrected for background absorbance detected in wells without added stain. Treated samples were compared with the cell control in the absence of the tested compounds. All experiments were carried out in triplicate. The cell cytotoxic effect of each tested concentration was calculated. A microplate reader  measured the optical density to determine the number of viable cells. The percentage of viability was calculated as [1 − (ODt/ODc)] × 100% where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells. The survival curve of each tumor cell line after treatment was obtained by plotting the relation between surviving cells and drug concentration. The 50% inhibitory concentration (IC50), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose response curve for each conc. using Graphpad Prism software . The biological evaluation was done at the Regional Center for Mycology & Biotechnology, Al-Azhar University.The cytotoxicity assay was performed against breast cancer MCF-cell line and Lung cancer A-549 cell lines. This was done by seeding the cells in 96-well plate at a cell concentration of 1 × 10