Parkia biglobosa (Fabaceae) roots and barks by Liquid Chromatography - Electrospray Ionization and Direct Injection Tandem Mass Spectrometry analysis. Mass spectral data indicated that B-type oligomers are present, namely procyanidins and prodelphinidins, with their gallate and glucuronide derivatives, some of them in different isomeric forms. The analysis evidenced the presence of up to 40 proanthocyanidins, some of which are reported for the first time. In this study, the antiradical activity of extracts of roots and barks from Parkia biglobosa was evaluated using DPPH method and they showed satisfactory activities.The present study investigates the chemical composition of the African plant  Parkia biglobosa) −, 289 [M−H−152−176]− and 305 [M−H−152−176]−, which suggested the presence of (epi)gallocatechin-O-glucuronide (5) −, assigned to the loss of one (epi)gallocatechin unit through a QM fission. The MS3 spectrum  plus the loss of an BFF fragment (138 Da) [m/z 617 ion. The important ion at m/z 423 [M−H−168−194]− was assigned to a sequential loss of an RDA fragment (168 Da), a water molecule (18 Da) and a loss of a glucuronide moiety (176 Da). These data confirm the (epi)gallocatechin-O-glucuronide as the lower unit and the (epi)gallocatechin as the upper unit. Finally, the molecule was characterized as an O-glucuronide of (epi)gallocatechin-(epi)gallocatechin (27–28) and this is the first report about this species. The MS spectrum of the m/z 785 A showed yl group . The ion(176 Da)  followedspectrum B of the M−H−168−1− could rm/z 881, 897, 913 and 937 [M−H]− which indicate that the corresponding compounds are mixed dimers consisting of (epi)gallocatechin and/or (epi)catechin units, with galloyl residues to three first m/z and galloyl and glucuronide residues to m/z 937.Another series are represented by the signals at m/z 881 produced ions at m/z 729 [M−H−152]− corresponding to the loss of one gallate moiety and at m/z 577 [M−H−304]−, that can corresponds to the loss of two gallate moieties or a gallocatechin residue. Thus, we suggested that this compound could be a dimer constituted of two (epi)catechin-O-gallate units [epi)catechin-O-gallate–(epi)gallocatechin (29–31).Fragmentation of the precursor ion at te units  or (epi)2 fragmentation of the precursor ion at m/z 897 showed fragment ions at m/z 745 [M−H−152]−, assigned to the loss of a galloyl group, at m/z 727 [M−H−170]−, due to the loss of a gallic acid molecule. The products ions at m/z 287 and 305 Da observed in the MS2 spectra after a QM fission suggest that the (epi)catechin core is the upper unit −, due to the loss of one galloyl group, and at m/z 743 [M−H−170]−, due to the loss of a gallic acid molecule. The ion observed at m/z 787 [M−H−126]− can result to the loss of an HRF fragment. Thus, this metabolite was tentatively identified as a dimer constituted of (epi)gallocatechin-O-gallate moieties (34–35) − which could result from the loss of a gallic acid (170 Da), plus a BFF at the lower unit of the m/z 767 ion. Hence this dimer was characterize as an (epi)gallocatechin-O-gallate-(epi)gallocatechin-O-glucuronide (36–37) and it is reported for the first time.The MS m/z 937 A shows t spectra B of the  spectra C of the 38) (m/z 865 [M−H]−) were analyzed in the same way. First investigations by ESI in the negative ion mode showed that the fragmentation was similar to that observed to the dimers. The MS2 shows a sequence of the fragmentation characteristics of these compounds: at m/z 739 [M−H−126]− from the loss of an HRF fragment (126 Da), at m/z 713 [M−H−152]− from the loss of an RDA fragment, the neutral loss of 152 Da indicates that the B ring of the upper unit has a catechol group [m/z 577 [M−H−288]−, was assigned to interflavanic bond cleavage and at m/z 451 [M−H−288−126]−, which could be formed after the interflavanic fission plus the loss of an HRF fragment (126 Da). Also, similar to the behavior in the positive ion mode described [m/z 575 [M−H−290]−, assigned to interflavanic bond cleavage following the QM mechanism. This precursor ion was then identified as a trimer of (epi)catechin.The trimeric proanthocyanidin  (m/z 865escribed  a prominepi)catechin, probably due to stereoselectivity of the enzymes involved in the biosynthesis of these substances , at tR = 55.12 min [m/z 457 (4)], at tR = 55.77 min [m/z 481 (5)], at tR = 55.74 min [m/z 617 (6)], and at tR = 55.40 min [m/z 633 (7)]. Noteworthy, two isomers at m/z 305 (2–3) were identified in the extracted ion chromatogram of roots and barks at the retention time of 24.98 and 43.33 min.Deprotonated molecules corresponding to proanthocyanidins monomers were detected at tm/z 577 (8–11), 593 (12–14) and 609 (15–16). Another series, with isomeric forms, were identified as esterified catechin-gallate and glucuronide dimers for ions at m/z 729 (17–18), 745 (19–22), 761 (23–26), 785 (27–28), 881 (29–31), 897 (32–33), 913 (34–35) and 937 (36–37). The retention time of these isomeric compounds were listed in Compounds isomeric with catechin dimers were detected for precursor ions at 38) at m/z 865 [M−H]− was confirmed at tR = 55.54 min.The trimeric proanthocyanidin  (tR = 54.95 min) and m/z 1,049 (40–43)  were assigned to trimers of (epi)catechin and (epi)gallocatechin esterified with a gallic acid unit respectively, as postulated by Lazarus [m/z 1,089 (44–45)  was tentatively assigned to a glucuronide trimer namely a trimer of (epi)gallocatechin-O-glucuronide present in the extract with its stereoisomer. The m/z values corresponding to tetramer molecular weights at m/z 1,153 (46) (tR = 55.25 min), m/z 1,169 (47) (tR = 55.23 min), m/z 1,185 (48) (tR = 55.32 min), m/z 1,201 (49) (tR = 54.83 min) and m/z 1,217 (50) (tR = 54.90 min) were assigned to tetramers containing from zero to four trihydroxylated units, respectively [Using the same analytical approach, it was possible to detect from the total ion current chromatogram (TIC)  Lazarus . On the ectively .m/z values that could be detected by electrospray in these fractions of roots and barks of P. biglobosa. Thus, proanthocyanidins present in this plant are constituted, of procyanidins, with mixed procyanidin-prodelphinidin structures and (epi)gallocatechin oligomers. In order to identify higher polymers, these were concentrated in the aqueous fraction [Finally, tetramers were the highest fraction   extracts and the ethyl acetate and aqueous fractions of the barks and roots was evaluated using a DPPH assay, with gallic acid and quercetin as antiradical reference compounds.The antiradical activity of the CH50 20.6 ± 0.1 μg·mL−1) and gallic acid (IC50 16.2 ± 0.1 μg·mL−1). The aqueous fraction of the roots was the one that had the highest free radical sequestration capacity (IC50 31.5 ± 0.1 μg·mL−1), followed by the aqueous fraction of the barks (IC50 = 37.1 ± 0.1 μg·mL−1), the EtOAc fractions of the barks (IC50 = 37.8 ± 0.3 μg·mL−1), the CH2Cl2/MeOH  extract of the roots (IC50 = 41.7 ± 0.1 μg·mL−1), the CH2Cl2/MeOH  extract of the barks (IC50 = 44.6 ± 0.1 μg·mL−1) and finally the EtOAc fraction of the roots (IC50 = 53.1 ± 0.1 μg·mL−1). The aqueous fraction showed more activity due the presence of oligomeric proanthocyanidins.The results showed that all the extracts and fractions of roots and barks exhibited good free radical scavenging activity compared to the references quercetin (ICP. biglobosa extracts and fractions tested could be associated with the presence of (epi)catechin derivatives, particularly (epi)gallocatechin-O-gallate [The remarkable antioxidant activities exhibited by all the -gallate  and cond-gallate . Other p-gallate .2Cl2 and EtOAc were purchased from Tedia . HPLC-grade water was prepared using a Millipore  Milli-Q purification system. The RP18 cartridge is a Phenomenex Strata C18-E, 55 µm, 70 Å, 500 mg·3 mL−1. The filter membrane (0.45 µm) was of nylon.HPLC-grade methanol and acetic acid were purchased from J.T. Baker . CHP. biglobosa were collected in October 2008 at Ngaoundere-Cameroon, Africa, and authenticated in loco by Professor Dr. Pierre Marie Mapongmetsem and confirmed in the National Herbarium through the voucher sample (n° 58980 HNC) collected by Dr. Francois Villiers from National Museum of Natural History of Paris. Barks and roots of P. biglobosa were macerated separately for a week, at room temperature, with 100 mL of a 1:1 (v/v) mixture of CH2Cl2/MeOH. The solutions were evaporated to dryness under vacuum to give 100 mg (10%) of crude bark and root extracts. In order to minimize the interference of very high order polymeric compounds, a combination of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) was employed [2O (50 mL of each solvent). After drying, the EtOAc fractions afforded 30 mg of each sample. An aliquot (10 mg) of the EtOAc fraction of each extract were submitted to the SPE using RP18 cartridge, eluted with H2O/MeOH 8:2 (v/v) (5 mL). The eluate was filtered through the nylon membrane and directly analyzed by ESI-IT-MSn as well as by HPLC/ESI-MS. Dried and powdered barks (1 g) and roots (1 g) of employed . In the −1 in methanol) at a flow rate of 5 µL·min−1 and working under the following conditions: capillary voltage −31 V, spray voltage 5 kV, tube lens offset 75 V, capillary temperature 300 °C, sheath gas (N2) flow rate 8 (arbitrary units). Negative ion mass spectra were recorded in the range m/z 100–2000 Da. The first event was a full - scan mass spectrum to acquire data on ions in the m/z range. The second scan event was an MS/MS experiment performed by using data-dependent scan that was carried out on deprotonated molecules from the compounds at collision energy of 30% and activation time of 30 ms. Data acquisition and processing were performed using the Xcalibur software.Flow injection analysis (FIA) was performed using a ThermoFisher Scientific ion trap mass spectrometer  equipped with an electrospray ionization source. The MS and MS/MS analysis in negative ion mode were selected after calibration infusing a standard solution of (+)-catechin (1 µg·mL−1. Gradient elution was performed by using H2O (A) and MeOH (B), both added of 0.1% acetic acid, as mobile phases. After a 5 min hold at 5% B, elution was performed according to the following conditions: from 5% B to 25% B in 50 min; 25% to 100% B in 60 min. The column effluent was split into two by means of a “T junction” placed after the chromatographic column and analyzed “on-line” both by ESI/MS and UV-DAD; 80% of the effluent sent to the UV-DAD detector; 20% of the effluent was analyzed by ESI/MS in negative ion mode. The mass spectra were acquired and processed using the Xcalibur software (version 1.3) provided by the manufacturer. Capillary voltage 20 V, capillary temperature 275 °C, nitrogen as drying gas at a flow rate 4 (arbitrary units) and as nebulizing gas, ion spray voltage 5 kV, tube lens offset 80 V, sweep gas at a flow rate 15 (arbitrary units). Data were acquired in MS1 scanning modes in the range of m/z 100–2,000 Da. The ESI interface and MS parameters were optimized to obtain maximum sensitivity. PAs were identified based on observation of the m/z values of their deprotonated molecules.Analysis were performed by HPLC/ESI-MS using a SURVEYOR MS micro HPLC system coupled on-line with an LCQ Fleet ion trap mass spectrometer (Thermo Fisher Scientific). HPLC separation was conducted on a Synergi Hydro RP-18 column , at a flow rate of 800 µL·min2Cl2/MeOH  extracts and ethyl acetate and aqueous fractions of the barks and roots, separately, were obtained by partition using 2.5 mg of each in 10 mL of the H2O/MeOH  and them dilutions were made until the concentrations of 6.25, 12.5, 25, 50, 100 and 200 mg·mL−1. Solutions of 0.004% of DPPH in H2O/MeOH  were used, which were mixed with the sample solution being examined. Aliquots (20 μL) of the sample were added to the DPPH solutions (200 μL). After 30 min of reaction, absorbance was immediately recorded at 517 nm. Quercetin and gallic acid  standard solutions were prepared and analyzed under the same conditions. The results were expressed as 50% inhibitory concentration (IC50 in μg·mL−1). All analyses were the mean of triplicate measurements ± standard deviation. Lower IC50 value indicates higher antioxidant activity.DPPH assays were used to measure the free radical scavenging potential of the extracts . The CH2Parkia biglobosa. Thereby, with the aim of providing further scientific contributions to support and improve African economy, we decided to carry out an analytical study to define the metabolic fingerprinting of this plant.To the best of our knowledge no study had previously described the tannins profile of n methods were developed for characterizing the oligomeric constituents in the plants. The work established the profile of proanthocyanidins in the extract of roots and barks of P. biglobosa. Mass spectral data indicated that B-type oligomers are present, namely procyanidins and prodelphinidins, with their gallate and glucuronide derivatives, some of them in different isomeric form. With this method, some proanthocyanidins with novel structures were determined for the first time. Tandem Mass Spectrometry (MSn) was demonstrated to be a useful analytical tool of choice for characterizing PAs, since more information about the structural details of the different molecules can be elucidated from the fragmentation of the precursor ions. The occurrence of proanthocyanidins is in accordance with chemical constituents of this genus, since tannins were previously identified in P. clappertoniana [Effective, rapid and sensitive HPLC/ESI-IT-MS and FIA-ESI-IT-MSrtoniana .P. biglobosa can be attributed to the proanthocyanidin derivatives present in this plant.The antiradical activity of the extracts and the fractions was evaluated using the DPPH assay, with gallic acid and quercetin as antiradical reference compounds. The study suggests that the high antiradical activity of extracts and fractions from


They also exhibited high specific capacitance of 140 F/g at 0.2 A/g and good electrochemical stability with 94% capacitance retention after consecutive 2500 cycles. The graphitization degree of the HGCS contributed to 60% of their specific capacitance, and their specific capacitance per unit surface area was as high as 0.2 F/m2, which was much higher than in the most cases of porous amorphous carbon materials reported before. In addition, the HGCS showed a high adsorption capacity of 182.8 mg/g for methylene blue (MB), which was 12 times as high as that in the case of carbon spheres before graphitization.Hierarchical micro-/mesoporous graphitic carbon spheres (HGCS) with a uniform diameter of ~0.35 μm were synthesized by Fe-catalyzed graphitization of amorphous carbon spheres resultant from hydrothermal carbonization. The HGCS resultant from 3 h at 900 °C with 1.0 wt % Fe catalyst had a high graphitization degree and surface area as high as 564 m BET > 1000 m2/g) which can improve the accumulation of ions at the electrode/electrolyte interface via forming electrical double layers [2/g, which showed a superior specific capacitance as high as 165 F/g at 1 A/g [2/g and exhibited a maximum specific capacitance of 226 F/g at 1 A/g [As an important electrochemical energy storage device, supercapacitors are attracting more and more attention ,2. Theire layers . Generalat 1 A/g . DespiteConsequently, an alternative strategy via enhancing graphitization degree of porous carbon materials was proposed, and several types of hierarchical graphitic carbons with well-controlled crystalline structure, large surface area, high porosity, high electric conductivity, and good chemical and electrochemical stability were developed ,12,13,143)3·9H2O as the carbon source and catalytic precursor, respectively. Their multifunctional application potentials were demonstrated by examining their electrochemical performance and adsorption capacity for toxic dye.To address the issue, in this work, hierarchical graphitic carbon spheres (HGCS) were prepared via combined hydrothermal carbonization and catalytic graphitization using glucose and Fe, commercial ferric nitrate (Fe(NO3)3·9H2O, 99.0%, Lia Chemical Co., Ltd., Wuhan, China), sodium polyacrylate n, AR; Damao chem. Co., Ltd., Tianjin, China), methylene blue trihydrate , and potassium hydroxide .The main starting materials used included glucose (C2/g (3)3·9H2O (0.5–2.0 wt % Fe with respect to carbon spheres) were dissolved in 50 mL alcohol under 25 °C, forming a homogeneous solution to which the presynthesized carbon spheres were carefully added to form a uniform gel. The gel was dried at 80 °C/12 h, and then fired at a heating rate of 5 °C/min to a given temperature (800–1100 °C) and held for 1–5 h in an alumina tube furnace protected by flowing Ar (99.999 vol % pure). The resultant HGCS were subject to characterization directly without any further purification.The preparation process of HGCS typically involved the following two steps: (1) low temperature hydrothermal carbonization to prepare carbon spheres and (2) high temperature catalytic graphitization of presynthesized carbon nanospheres to obtain HGCS. In the first step, an aqueous solution of glucose (0.7 mol/L) was combined with PAANa (1.0 wt % with respect to carbon) forming a transparent solution. Since PAA anions can reduce the surface energy of carbon spheres , PAANa w2/g . To prepThe crystalline phases in samples were identified by powder X-ray diffraction (XRD) analysis using a Philips X’Pert PRO diffractometer . The morphologies of samples were observed by means of a field-emission scanning electron microscope  and transmission electron microscope  equipped with an energy dispersive spectrometer ). Raman spectra were recorded using a Horiba Jobin-Yvon Labram-HR800 Raman spectrometer ; a 532 nm diode laser was used with an integration time of 30 s, a spectral resolution of 1 nm, and an approximate power level of 2 mW. To calculate the mean diameter and determine the size distribution, at least 200 carbonaceous spheres were examined in each case. Nitrogen adsorption–desorption isotherms were determined  to calculate the specific surface area and pore size distribution. The specific surface area was calculated according to the Brunauer Emmett Teller (BET) model and the pore size distribution was determined using the Barret–Joner–Halenda (BJH) method. The MB adsorption was evaluated using an ultraviolet-visible spectrophotometer .Electrochemical behavior of as-prepared HGCS was investigated in a three electrode configuration at 25 °C using Hg/HgO, Pt foil, and 6M KOH aqueous solution respectively as the reference electrode, the counter electrode, and the electrolyte. The working electrode was prepared by coating carbon spheres and HGCS with conducting nickel foam, and then used directly in the circuit. Cyclic voltammetry (CV) and galvanostatic charge/discharge (GCD) curves were used to evaluate electrochemical performances of samples. The former was determined between −1.0 and 0 V potential window at scan rates of 5, 10, 20, 50, 100, and 200 mV/s, and the latter determined between −1.0 and 0 V at current densities of 0.1, 0.2, 0.5, 1, 2, 5, and 10 A/g.C (F/g) is the specific capacitance, I (A) the instant current shown in the CV curve, m (g) the sample mass in the working electrode, v (V/s) the scan rate, Δt (s) the discharge time, and V (V) the voltage upon discharging.Based on the CV and GCD curves, the specific capacitance values were calculated by the following equations:The cycling stability was evaluated based on the capacitance retention after 2500 consecutive galvanostatical charge and discharge cycles at 5 A/g.The adsorption tests were carried out using MB as an adsorbate. Typically, 20 mg of as-prepared samples were added into an MB solution with different concentrations (0–50 mg/L), and stirred at 25 °C for 30 min. After centrifugation, the concentration of supernatant solution was measured using a UV-visible spectrophotometer. The adsorption kinetics was also investigated and adsorption isotherms were determined following the procedures reported in our earlier publication [3O4 was detected in the CS fired at 800 °C, but it was replaced by Fe at a higher heating temperature (>800 °C) (3)3·9H2O had initially decomposed at a low temperature (800 °C) to Fe3O4 which was further reduced at a higher temperature to Fe catalyst [The carbon spheres presynthesized via hydrothermal carbonization  were the>800 °C) b, suggescatalyst ,23.−1, respectively  to the latter (ID) (IG/ID) indicated the graphitization degree of HGCS [G/ID values of HGCS resultant from 3 h firing at 800, 900, 1000, and 1100 °C were determined as 0.93, 0.97, 0.96, and 0.97, respectively, indicating that the graphitization degree of the samples increased initially with increasing the temperature from 800 to 900 °C, but almost did not change upon further increasing the temperature to above 900 °C, which, along with the XRD results  was reached upon using 1.0 wt % Fe catalyst. These results  and mesopores (the adsorption in the relative pressure range of 0.1–0.8) were present in the HGCS [2/g. The SEM image presented in the HGCS ,27. GiveIn order to better understand the morphology and structure of the HGCS sample, TEM analysis was carried out. As shown in 2/g), which could be explained as follows. (1) After catalytic graphitization, the average size of the HGCS was decreased (from 0.48 to 0.35 μm) (3)3 9H2O precursor not only acted as a catalyst, but also as an oxidizing agent during the graphitization process. It reacted with carbon spheres, releasing some gaseous phases such as NOx, CO, and CO2, which was beneficial to the physical activation of micro/meso pores formation [As stated above, TEM  reveals 0.35 μm) , and corormation ; and (3)−1 in the potential window from −1.0 to 0 V and galvanostatic charge-discharge (GCD) measurements at various current densities from 0.1 to 10 A/g. The distorted quasi-rectangular CV and asymmetric triangular GCD curves in the case of presynthesized carbon spheres  the presynthesized carbon spheres were solid and their specific surface area was low (The capacitive properties of the starting carbon spheres (180 °C/12 h) and as-prepared HGCS  were respectively studied by cyclic voltammetry (CV) at various scan rates from 5 to 200 mV·s spheres a,b were  spheres ), it can was low , which a2/g) [2/g) [On the other hand, in the case of the HGCS, the quasi-rectangular and symmetric CV curves a,b witho2/g) , althougg) [2/g) , even thBET = 666 m2/g [BET = 564 m2/g for the latter). The former showed a low specific capacitance of 59 F/g at 0.2 A/g which was similar to that of the presynthesized carbon spheres (prepared at 180 °C for 12 h). Since the specific capacitance of the HGCS at 0.2 A/g was 140 F/g, it can be reasonably estimated that the graphitization degree contributed to 60% of their specific capacitance. Moreover, the surface area normalized specific capacitance  was calculated according to the equation of Cs = C/SBET, where C and SBET are the specific capacitance and the BET surface area, respectively. As demonstrated in 2, which was much higher than in the cases of most documented porous amorphous carbon materials, such as activated porous carbon (0.13 F/m2) [2) [To evaluate the effect of graphitization degree of HGCS on their electrochemical performance, the specific capacitance values of amorphous mesoporous carbon spheres and as-prepared HGCS with similar specific surface area were compared (S666 m2/g  for the 13 F/m2)  and hierThe cycling stability was evaluated based on the capacitance retention after 2500 consecutive charge-discharge cycles between −1.0 to 0 V at a current density of 5 A/g e. The HGeq (mg/g) is the equilibrium adsorption amount, Q0 (mg/g) the maximum adsorption amount, b (L/mg) the constant term related to the energy of adsorption, ec (mg/L) the equilibrium concentration of dye solution, and Fk and n are the Freundlich constants representing the adsorption capacity of the adsorbent and favorable extent of the adsorption process, respectively  [Adsorption behaviors of presynthesized carbon spheres and HGCS prepared at 900 °C/3 h with 1.0% Fe catalyst were also examined and compared using MB as an adsorbate. The adsorption isotherms  of the sctively) .2) (Q0) of the as-prepared HGCS was calculated to be 182.8 mg/g, which is about 12 times as high as that (15.4 mg/g) in the case of the presynthesized carbon spheres. This can be mainly attributed to the much larger specific surface area and hierarchically porous structure of the former.The calculated adsorption parameters and correlation coefficients (R2)  suggest tq (mg/g) is the adsorption amount at time t, k1, and k2 (g·mg–1·min–1) are the pseudo-first-order and pseudo-second-order rate constants, respectively. Both the pseudo-first-order and the pseudo-second-order models describe the relationship between adsorption rate and MB concentration. As indicated in 2) above 0.99, suggesting that the adsorption kinetics of the presynthesized carbon spheres and as-prepared HGCS both followed this model and the adsorption rate was irrelevant to the MB concentration.The following pseudo-first-order and pseudo-second-order kinetic models were also used to investigate the adsorption mechanism of the presynthesized carbon spheres and as-prepared HGCS b;(5)Pse2/g were synthesized by a facile combined hydrothermal carbonization and catalytic graphitization method using glucose as the carbon source and ferric nitrate as the catalyst precursor. The optimal weight ratio of Fe catalyst for graphitization of presynthesized amorphous carbon spheres was 1.0 wt %, and the optimal temperature and dwelling time were 900 °C and 3 h, respectively. The as-prepared HGCS exhibited a high specific capacitance of 140 F/g at 0.2 A/g, and good electrochemical stability with 94% capacitance retention after consecutive 2500 cycles. Moreover, the degree of graphitization contributed to 60% of the specific capacitance of the HGCS, this fundamental understanding provided an important clue for the development of high performance supercapacitor electrode materials. Moreover, the HGCS showed a good adsorption capacity of 182.8 mg/g for MB, which was 12 times as high as that of the presynthesized carbon spheres before graphitization.Hierarchical micro-/mesoporous graphitic carbon spheres with an average size of 0.35 μm and specific surface area of 564 m


Candidatus Liberibacter asiaticus” strain YNJS7C, isolated from a navel orange tree in Yunnan Province, China, is presented here. The YNJS7C strain has a genome size of 1,258,986 bp, with a G+C content of 36.6%, 1,174 predicted open reading frames, and 53 RNA genes.The draft genome sequence of “ Candidatus Liberibacter asiaticus” strain YNJS7C, isolated from a navel orange tree in Yunnan Province, China, is presented here. The YNJS7C strain has a genome size of 1,258,986 bp, with a G+C content of 36.6%, 1,174 predicted open reading frames, and 53 RNA genes.The draft genome sequence of “ Candidatus Liberibacter asiaticus” is associated with citrus huanglongbing , one of the most devastating diseases in citrus production worldwide  was reported previously  tree showing typical HLB symptoms in Jianshui City  of Yunnan Province, China. Total plant DNA was extracted from infected citrus leaf midribs using the plant DNA extraction kit . Library preparation was performed using the Illumina TruSeq version 2 paired-end library preparation kit (300-bp insert size), and sequencing was carried out on the Illumina HiSeq platform . Quality control filtering and trimming were conducted using CLC Genomics Workbench 9.5 . The HiSeq sequencing generated a total of 7.87 × 107 reads with an average size of 150 bp. Using the whole-genome sequence of “Ca. Liberibacter asiaticus” strain A4 (GenBank accession number CP010804)    . De novodr.org/) . The strQXDO00000000. The version described in this paper is version QXDO01000000. The sequence data have been deposited at the NCBI Sequence Read Archive (SRA) under BioProject number PRJNA488522.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number


The randomized trial PRODIGE 7 failed to show the benefit of oxaliplatin hyperthermic intraperitoneal chemotherapy (HIPEC) in colorectal peritoneal metastasis treatment (CR PM). This systematic review focuses on the association of cisplatin (CDDP) with mitomycin C (MMC) in HIPEC in CR PM.2/L) plus MMC (3.3 mg/m2/L) at a temperature of 41.5–42.5 °C for 60–90 min. Major complications occurred in less than 30% of patients with limited hematological toxicity (less than 15%). In addition, in a phase 2 trial, an adjuvant HIPEC benefit was demonstrated in colorectal cancer patients with high risk for peritoneal failure . After a recurrence, an iterative procedure permitted similar recurrence-free disease (13 vs. 13.7 months) with an acceptable morbidity (18.7% of severe complications).Experimental studies demonstrated that hyperthermia, in addition to CDDP ± MMC treatment, gradually improved the cytotoxic effect by increasing early apoptosis, eATP interaction, intracellular CDDP concentration (by 20%) and p73 expression. Recent studies with highly selected patients reported unusual prolonged survival with a median overall survival (OS) of approximately 60 months, with a HIPEC combination of CDDP (25 mg/mThe combination of CDDP and MMC seems to be an interesting protocol as an alternative to high-dose and short-term oxaliplatin. Recently, the effectiveness of CDDP has been validated in ovarian peritoneal metastasis in a phase III trial offering a HIPEC solution [Colorectal peritoneal metastasis (CR PM), with an occurrence rate of 40% , 2, is tsolution . Is it psolution , 26, 27.This systematic review agrees with the guidelines outlined in the preferred reporting items for systematic reviews and meta-analyses statement (PRISMA). On PubMed, combinations of the following terms were used: “cisplatin,” “peritoneal metastasis,” “peritoneal carcinomatosis,” “colorectal cancer,” “experimental,” “hyperthermia,” “HIPEC” and “colon.” We first identified 105 articles with the combination of “peritoneal carcinomatosis” AND “colorectal cancer” AND “cisplatin” and 132 articles with the combination of “experimental” AND “hyperthermia” AND “cisplatin” words. To expand this search, we analyzed 63 articles involving “colorectal cancer” AND “hyperthermia” AND “cisplatin,” 50 articles involving “colon” AND “hyperthermia” AND “cisplatin”, and 37 articles with the combination of “colon” AND “HIPEC” AND “cisplatin”. An initial screening of the title and abstracts was performed.Only studies including a surgical treatment of CR PM with HIPEC including cisplatin with/without MMC were selected. We first deleted duplicate articles and many articles about pressurized intraperitoneal aerosol chemotherapy – cancer (and no HIPEC). We separated experimental and clinical studies. The following studies were excluded: case-control studies, editorials, no full-text available, and studies in languages other than English. Finally, we analyzed 22 articles: 13 clinical and 9 preclinical studies .For clinical studies, we extracted the study design, number of patients, peritoneal cancer index, follow-up, postoperative mortality and morbidity and survival.Some experimental studies demonstrated that hyperthermia can affect cell membranes, the cytoskeleton, and the synthesis of macromolecules and can increase drug-induced DNA damage and inhibit the repair of drug-induced DNA damage . CDDP apin vitro results, Bhagwandin [in vitro drug sensitivity testing in 27 patients with peritoneal surface malignancies  undergoing CRS plus HIPEC . Seventeen tumors (63%) displayed in vitro sensitivity to the agents used. However, there was no significant difference in survival for patients whose tumors displayed in vitro drug sensitivity vs. those whose tumors did not . There was no correlation between in vitro drug sensitivity and the histopathology of the primary neoplasm (p=0.309). Today, it is not recommended to use these assays during the decision-making process in such patients because of the lack of clinical validation.In view of these interesting agwandin  performeThe toxicity and effectiveness of the HIPEC procedure with MMC or CDDP were evaluated in preclinical studies.Makrin showed that this procedure had a detrimental effect on the strength of colonic anastomosis, especially during the early postoperative period (until day 10) . The burThe effectiveness of these HIPEC procedures was evaluated in two experimental studies.increased life span) at 37 °C=29.88, 199.32 and 108.52, ILS% at 43 °C=62.69, 260.50 and 178.05, respectively). The most pronounced effect on survival was achieved by a combination of IL-2, CDDP and hyperthermia at 43 °C .Bevanda suggested the synergistic effect of hyperthermia, chemotherapy and immunotherapy and that interleukin-2 (IL-2) significantly increased antitumor activity and the survival rate of mice with CR PM . He compMore recently, Yun proposed the development of a novel hydrogel drug delivery system through the combination of 5-fluorouracil (5-FU)-loaded polymeric micelles and CDDP in biodegradable thermosensitive chitosan (CS) hydrogels . The resTaken together, all experimental results suggest that CDDP and MMC are efficient drugs to control CR MP and that CDDP is more active in a hyperthermic situation.2/L) plus MMC (3.3 mg/m2/L) at a temperature of 41.5–42.5 °C for 90 min. The same year, he published a second study with the same protocol [2/L of perfusate or CDDP 25 mg/m2/L plus MMC 3.3 mg/m2/L, and perfusion lasted 60–90 min at 41.5–43 °C. Baratti published three studies [2 plus MMC 16 mg/m2 at a temperature of 41.5 °C for 60 min.The HIPEC protocol with CDDP and MMC is not standardized. These two chemotherapeutic agents were mainly described by Italian teams. They were the first to publish this HIPEC protocol with oncological results. Cavaliere, in 2000 , publishprotocol . In 2011 studies , 24, 25  studies  proposedTwo Japanese teams described HIPEC with these two chemotherapeutic agents. Yonemura  used theIn 2016, Lin  publishePostoperative mortality after CRS/HIPEC CR PM treatment with CDDP ± MMC was described between 0%  and 3% , equivalThe median survival rates described for patients treated with HIPEC were very different, from 16  to 60.1 The peritoneal carcinomatosis index (PCI) could explain these survival differences. The majority of patients (53%) treated in Huang’s study  had an iIn addition, Baratti demonstrated that major complications independently affect long-term disease-specific survival (DSS) . Five-yeExtraperitoneal metastasis appeared as another pejorative survival criterion. In 2018, Baratti  comparedTwo authors, namely, Virzi and Baratti, evaluated the HIPEC protocol with CDDP and MMC at the time of primary curative surgery in patients with colorectal cancer at high risk for metachronous peritoneal metastases , 46.and positive peritoneal washing cytological examination). The protocol was well tolerated and safe. Major morbidity occurred in 17% of cases and operative death in none. The 5-year progression-free and peritoneal progression-free survival rates were 74.1% and 90.9%, respectively. The 5-year overall survival was 83.3%, although one patient died shortly after 5 years.Virzi published  a prospeBaratti  publishe2 CDDP and 16 mg/m2 MMC at the first intervention were perfused at the second with 35 mg/m2 MMC. The median interval between the initial and second CRS with HIPEC was 19 months (range 12–111 months). The mean PCI was 8 vs. 16 for the first procedure. No patient died postoperatively, and the overall morbidity was 43.7%, with 18.7% severe complications. After a median follow-up of 20 months, RFS following repeated CRS and HIPEC was comparable (13 vs. 13.7 months) to that registered after the first procedure.In some cases, after CR PM treatment by CRS/HIPEC, recurrences may be confined to the peritoneal cavity and are completely resectable. Vaira  evaluate2/L) plus MMC (3.3 mg/m2/L) at a temperature of 41.5–42.5 °C for 60–90 min. The survival results described for patients treated with HIPEC combined with CDDP and MMC were very different. We first explain these differences by the peritoneal carcinomatosis index (PCI), which is a very different function of cohorts. However, PCI has been demonstrated by many multivariate analyses to be the most important adverse prognostic factor after CRS-HIPEC [2/L) plus MMC (3.3 mg/m2/L) at a temperature of 41.5–42.5 °C for 60–90 min could be the rational base to support the design of future randomized trials vs. cytoreductive surgery alone in the treatment of colorectal PM. This protocol represents a potential alternative for now. We expect, in the next 10 years, new drug delivery systems, immunotherapy combined with chemotherapeutic, and development of nanovectorization, a new therapeutic area, to improve actual survival and decrease morbidity [Residual microscopic metastases after cytoreductive surgery remain a therapeutic challenge. The adjuvant application of intraperitoneal chemotherapy has become the standard of care. However, the chemotherapeutic drug protocol is not standardized. In 2018, two randomized phase III studies evaluated combined therapy (CRS+HIPEC) vs. CRS alone for peritoneal metastases. PRODIGE 7 failed to show a difference in overall survival between patients treated with oxaliplatin HIPEC adjunction for CR PM. Overall survival was high in both groups but did not differ significantly  . In paraRS-HIPEC , 43, 49.RS-HIPEC , 19, 50.RS-HIPEC  did not RS-HIPEC . No clinorbidity .Today, the CDDP+MMC-based HIPEC is the only protocol to demonstrate an adjuvant HIPEC benefit in colorectal cancer patients at high risk for peritoneal failure  . During The combination of CDDP and MMC might be a valid HIPEC protocol in CR PM. Recent studies evaluating this protocol demonstrated prolonged survival  with limited toxicity. Major complications occurred in less than 30% of cases with few hematological toxicities (less than 15%). This protocol is the only one to demonstrate an adjuvant HIPEC benefit for CRC patients at high risk for peritoneal failure . After a recurrence, an iterative procedure is possible with similar recurrence-free disease (13 vs. 13.7 months) and acceptable morbidity (18.7 % severe complications). Randomized trials are now needed to confirm this hypothesis.


Arabidopsis thaliana plants grown under exclusive ammonium or nitrate supply. We identified and quantified 68 proteins with differential abundance between both conditions. These proteins revealed new potential important players on root response to ammonium nutrition, such as H+-consuming metabolic pathways to regulate pH homeostasis and specific secondary metabolic pathways like brassinosteroid and glucosinolate biosynthetic pathways.Nitrogen is an essential element for plant nutrition. Nitrate and ammonium are the two major inorganic nitrogen forms available for plant growth. Plant preference for one or the other form depends on the interplay between plant genetic background and environmental variables. Ammonium-based fertilization has been shown less environmentally harmful compared to nitrate fertilization, because of reducing, among others, nitrate leaching and nitrous oxide emissions. However, ammonium nutrition may become a stressful situation for a wide range of plant species when the ion is present at high concentrations. Although studied for long time, there is still an important lack of knowledge to explain plant tolerance or sensitivity towards ammonium nutrition. In this context, we performed a comparative proteomic study in roots of  The first one is an anion  and the second one, a cation , thus, both N sources differ extremely in their chemical properties [3− as fertilizer, as well as the high nitrification rates commonly observed in agricultural soils when urea or NH4+ are applied, have made that crop species are mostly adapted to nitrate nutrition. However, the increasing use of ammonium-based fertilizers formulated with nitrification inhibitors, which have been proven useful to mitigate the effect of agriculture on the environment [4+ concentration and pH [Nitrogen (N), despite being one of the essential macronutrients for plant development, is often a limiting element in agricultural soils. The two major inorganic N sources for plants in soils are nitrate  or cation channel [3− and NH4+ assimilation is also different in terms of H+ balance, reductants consumption and redox balance [3− assimilation produces OH- whereas NH4+ assimilation produces two H+ [+ production and consumption must be accurately controlled according to the N form absorbed to maintain the cytoplasmic pH and a favorable electrochemical gradient across cell membrane [42− and in the action of H+-pumps . The “biochemical pH-stat” is based on the activation of H+-consuming metabolic pathways [NO channel ,13. This channel ,15. More balance . For thes two H+ ,18. Thusmembrane . This copathways .4+ as main N source, may entail benefits for plants. For instance, since ammonium nutrition is known to stimulate N assimilation machinery, an increase in protein yield has been reported in grains of wheat grown with NH4+ as N source [2 concentrations, some authors have argued that C3 plants growing under ammonium nutrition responded more positively to elevated CO2 than such plants growing under nitrate nutrition [Although ammonium nutrition at high concentration  is mostly reported as an unwanted situation that may affect crops yield, the metabolic adaptation to the presence of NHN source . SimilarN source ,22. BesiN source ,24,25,26utrition ,27. Howeutrition ,29.+ balance.Although plant response upon ammonium nutrition has been extensively studied, the molecular mechanisms governing the responses that lead plants to adapt their metabolism to tolerate this situation remain largely unknown. In this context, to find new actors, mechanisms and processes associated with plants ammonium response, we have performed a quantitative proteomic study in the root of Arabidopsis plants grown under a non-toxic ammonium condition, using nitrate nutrition as control. This approach provided some new clues for future research related to metabolic pathways and signaling processes involved in root adaptation to ammonium nutrition, such as the induction of secondary metabolism and the putative association between the gamma-aminobutyric acid (GABA) shunt, malate, and enzymes participating in biochemical pH-stat to regulate H4+ nutrition is an important step to design practices for mineral N nutrition management in plants. In general, it is considered that the deleterious effect of ammonium nutrition at high concentrations in plants is a consequence of the excessive NH4+ uptake and accumulation in tissues, and plants with an enhanced synthesis of N-reduced compounds are more tolerant to ammonium nutrition [4+ content, free amino acid and total soluble protein contents in roots. In this work, roots of ammonium-fed plants showed an increase of amino acid and soluble protein contents, whereas only a slight increase of internal NH4+ content compared with those grown with NO3−. These results indicate that plants were not facing a stressful situation  in both N conditions  and a V-type proton ATPase subunit E3 (P0CAN7)  in tomato, and the H+-transporting plasma membrane ATPases  in Arabidopsis [+-ATPase gene expression and activity [+ pumps and specifically ATPases on pH regulation associated with ammonium nutrition.A quantitative proteomic analysis, with isobaric tags for relative and absolute quantitation (iTRAQ), was used to analyze relative abundance of proteins in four independent pools of Arabidopsis roots per treatment . Peptides of six or more amino acids in length, and with a maximum of two missed cleavages were exclusively considered for the analyses. For protein quantification, only proteins identified in at least three out of four samples per treatment and with two or more unique peptides identified were considered. Following these criteria , we identified 4469 proteins and quantified 799, out of them 68 proteins were differentially abundant (nditions . Among tnditions . Importanditions . Differeutrition . Despitechanisms . These m pH-stat . Curious(P0CAN7) . ConsistN source ,35. Furtbidopsis ,37. In aactivity . It has activity . Future Gene ontology (GO) enrichment analysis for cellular component  and biolAs stated, the GO enrichment analysis highlighted the regulation of the glucosinolate (GLS) biosynthetic process by the N source provided b. GLS arRegulation of secondary metabolism has been reported in several species exposed to ammonium stress such as in tomato  or in Ar+-sink unit of the revised biochemical pH-stat mechanism under aerobic conditions [The C/N balance in plants is regulated by the availability of C skeletons, energy, and reductants for the N assimilatory pathways . One of nditions ,61. Thisnditions ,30,62.As previously mentioned, this proteomic study also revealed a number of proteins associated with secondary metabolism  and seve+ such as phosphoenolpyruvate carboxykinase,  and glutamate decarboxylase  generated is diverted into amino acids synthesis . Thus, t2+ (via Ca2+/CaM) or H+ concentrations and thus, it has also been related to cell pH regulation [4+ uptake, transport and storage appears essential in order to maintain cell pH, reductant, and electrochemical homeostasis upon plants growth under ammonium nutrition.The increased 2OG production by isocitrate dehydrogenase (ICDH)  may indugulation ,68. GABAgulation . Additiogulation ,71. Thus+ balance and plasma membrane electrical potential deserve special attention in future research.Finally, the present study provides new hints of metabolic pathways and signaling processes that can be involved in root adaptation to ammonium nutrition, a process that although thoroughly studied continues being still poorly understood. Among the novel points that arise from this study, the connection between ammonium nutrition and secondary metabolism and the putative association between GABA shunt and TCA cycle associated enzymes to regulate HArabidopsis thaliana ecotype Col-0 were sterilized and cultured with a modified Murashige and Skoog solution  supplemented with 0.5% sucrose and 20.5 mM MES (pH 6.5) [4)2SO4 for ammonium-based nutrition or 1 mM Ca(NO3)2 for nitrate nutrition. To equilibrate the Ca2+ supplied together with the NO3−, NH4+-fed plants were supplemented with 1 mM CaSO4. The experimental design of this study, in terms of N concentration, pH, volume, and renew frequency of nutrient solution, was selected according to results obtained in previous nutritional studies with Arabidopsis plants [Plants used in this study were cultured as described in . Briefly(pH 6.5) . Nitroges plants ,15.−2·s−1 light intensity, 60% relative humidity, and 22 °C (day conditions); 10 h, 70% relative humidity, and 18 °C (night conditions).Plants were stratified at 4 °C for four days in the dark and then moved into a growth chamber under the following controlled conditions: 14 h, 200 µmol·mFirst, plants were germinated and cultured during 9 days in 0.6% agar Petri dishes with the nutrient solution described above. After this time, seedlings were transferred to sterile 24-well plates containing 1 mL of the same nutrient solution used for seed germination without agar (one plant per well). Then, plates were kept under continuous shaking (120 rpm) for 12 additional days and the liquid nutrient solution was renewed on days 5 and 9. Four independent experiments were carried out, each one with 10, 24-well plates. Each plate contained 12 plants per treatment. When harvesting, shoots and roots of plants within each plate and treatment were pooled separately, dried with paper towels and the biomass was recorded. For proteomic and metabolic analysis, all the roots within each experiment and treatment (120 plants) were pooled together, immediately frozen in liquid nitrogen and stored at −80 °C.g and 4 °C for 20 min and then, supernatants were recovered.Root extracts for ammonium and total free amino acids quantification were obtained by adding 20 µL of ultrapure water per milligram of tissue. The homogenates were incubated at 80 °C during 5 min and centrifuged at 16,000Ammonium content was determined following the phenol hypochlorite method . For tot2, 1 mM EDTA, 1 mM EGTA, 10 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM ε-aminocaproic acid and 10 µM leupeptin. Homogenates were then centrifuged at 16,000g for 20 min at 4 °C and the supernatants recovered. Soluble protein content was determined by a dye binding protein assay (Bio-Rad Bradford Protein assay) using BSA as a standard for the calibration curve. Phosphoenolpyruvate carboxylase (PEPC), NAD-dependent malic (NAD-ME), NADP-dependent isocitrate dehydrogenase (ICDH), and malate dehydrogenase (MDH) were assayed as described in [2, 5 mM NaF, 0.25 mM NADH, 6.4 U of malate dehydrogenase mL−1, 2 mM NaHCO3 and 3 mM phosphoenolpyruvate); for NAD-ME activity assay (50 mM HEPES-KOH (pH 8), 0.2 mM EDTA-Na2, 5 mM DTT, 2 mM NAD, 5 mM malate, 25 μM NADH, 0.1 mM Acetyl CoA and 4 mM MnCl2); for NADP-dependent malic enzyme (NADP-ME) activity assay (100 mM Tris-HCl (pH 7), 10 mM MgCl2, 0.5 mM NADP and 10 mM malate); for ICDH assay (100 mM Tricine-KOH (pH 8), 0.25 mM NADP, 5 mM MgCl2 and 5 mM isocitrate); for MDH assay (100 mM HEPES-KOH (pH 7.5), 5 mM MgSO4, 0.2 mM NADH and 2 mM oxaloacetate). Phosphoenolpyruvate carboxykinase (PEPCK) was assayed using a reaction buffer composed by 100 mM HEPES-KOH (pH 6.8), 25 mM DTT, 100 mM KCl, 90 mM KHCO3−, 1 mM ADP, 6 mM MnCl2, 0.2 mM NADH, 7 U of malate dehydrogenase mL−1 and 6 mM phosphoenolpyruvate, as described in [For soluble protein and enzyme activities determination, frozen root powder was homogenized with extraction buffer (10 µL per milligram of tissue). Extraction buffer was composed by 0.1% Triton X-100, 10% glycerol, 0.5% polyvinylpolypyrrolidone, 50 mM HEPES pH 7.5, 10 mM MgClribed in . The folribed in . Enzyme ribed in . All reag. Glucosinolates were determined from supernatants by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS) analyses using an Acquity UPLC from Waters  interfaced to a Synapt G2 QTOF from Waters with electrospray ionization as described previously [For glucosinolate determination around 25 mg of frozen root powder were extracted by adding 1 mL of MeOH:water (70:30). The mixtures were homogenized in a Tissue Lyser  and incubated for 15 min at 80 °C to inactivate myrosinase. Then, homogenates were centrifuged for 20 min at 16,000eviously . Glucosig and 4 °C and total protein precipitated from 200 µL of supernatant with methanol and chloroform . Mixtures were spun (in a vortex) and centrifuged for 1 min at 14,000g. The aqueous phase was then removed, an additional 450 µL of methanol added, and the centrifugation step was repeated. After discarding the methanol phase, protein pellets were dried in a vacuum centrifuge and resuspended into 7 M urea, 2 M thiourea, and 4% CHAPS. Global experiments were carried out with four independent biological samples in each experimental condition. Each sample corresponded to a pool of 120 plants. Protein extracts (150 μg) were precipitated with methanol/choloroform, and pellets dissolved in 7 M urea, 2 M thiourea, 4% (v/v) CHAPS. Protein was quantified with the Bradford assay kit . A shotgun comparative proteomic analysis of total root extracts using an iTRAQ 8-plex experiment was performed [w/w, was used for protein enzymatic cleavage at 37 °C for 16 h. Each root tryptic digest was labeled by incubation (1 h) according to the manufacturer’s instructions with one isobaric amine-reactive tags, as follows: Tag113, ammonium media-1; Tag114, ammonium media-2; Tag115, ammonium media-3; Tag116, ammonium media-4; Tag117, nitrate media-1; Tag118, nitrate media-2; Tag119, nitrate media-3; Tag121, nitrate media-4. Then, every set of labeled samples was independently pooled and evaporated until <40 μL by vacuum centrifugation. Unless otherwise stated all reagents were purchased from Sigma-Aldrich .Proteins were extracted from 50 mg of root fresh weight (FW) homogenized in 0.5 mL of an extraction buffer composed by 7 M urea, 2 M thiourea, 4% CHAPS, 2% Triton X-100, 50 mM DTT, and 0.5% plant protease inhibitor and phosphatase inhibitors cocktails . Then, homogenates were centrifuged for 15 min at 10,000erformed . iTRAQ lThe peptide pool was injected to an Ettan LC system with a X-Terra RP18 pre-column (2.1 × 20 mm) and a high pH stable X-Terra RP18 column   at a flow rate of 40 μL/min, increasing in this way the proteome coverage. Elution of peptides was made with a mobile phase B of 5–65% linear gradient over 35 min . Eleven fractions were collected, evaporated under vacuum and reconstituted into 20 μL of 2% acetonitrile, 0.1% formic acid, 98% MilliQ water previous to mass spectrometric analysis.m/z up to 1250 m/z; scan time: 250 ms). Top 35 peaks were selected for fragmentation. Minimum accumulation time for MS/MS was set to 100 ms . Product ions were scanned in a mass range from 100 m/z up to 1700 m/z and excluded for further fragmentation during 15 s.The split of peptides was made by reverse phase chromatography using an Eksigent nanoLC ultra 2D pump fitted with a 75 μm ID column (Eksigent 0.075 mm × 25 cm). Samples were desalted and concentrated with a 0.5 cm length 300 μm ID pre-column, which was packed with the same chemistry as the separating column. Mobile phases were 100% water 0.1% formic acid (buffer A) and 100% Acetonitrile, 0.1% formic acid (buffer B). The column gradient (237 min) used was a two-step gradient, first from 5% B to 25% B in 180 min and second, from 25% B to 40% B in 30 min. Column was equilibrated in 95% B for 10 min and 5% B for 15 min. Along the entire process, the pre-column was in line with column, and flow during the gradient was maintained at 300 nL/min. The separated peptides eluted from the column were analyzed using an AB Sciex 5600 TripleTOF™ system . Information data was acquired upon a survey scan , and Andromeda was used as a search engine [p value < 0.05.Analyses of raw data  were performed with MaxQuant software . Peak lih engine . The seahttp://mapman.gabipd.org/es/mapman, version 3.6.0) [A. thaliana Columbia tair10 genome as background population [p ≤ 0.05.Functional classification of the differentially abundant proteins was carried out according to MapMan software (n 3.6.0) . Gene onpulation . Over-reProteomics data statistical analysis is described in the above section.t-test with a p value < 0.05.For biomass and metabolic data, statistical analysis was carried out using IBM SPSS 22.0 software . The significance of the results was assessed using independent samples Student


Vaccinium spp.) are rich in bioactive compounds. However, the biological activity of polysaccharides from blueberry has not been reported so far. This study evaluated the anti-tumor and immunological activities of a polysaccharide (BBP3-1) from blueberry in S180-bearing mice. The experimental results indicated that BBP3-1 (100 mg·kg−1·d−1) inhibited the tumor growth rate by 73.4%. Moreover, this group, compared with the model control, had shown an effect of increasing both the spleen and thymus indices (p < 0.05), increasing phagocytosis by macrophages (p < 0.05), boosting the proliferation and transformation of lymphocytes (p < 0.01), promoting the secretion of TNF-α, IFN-γ, and IL-2 (p < 0.05) and improving NK cell activity (p < 0.01). From this study, we could easily conclude that BBP3-1 has the ability to inhibit tumor progression and could act as a good immunomodulator.Blueberries ( Ericaceae Vaccinium group of deciduous shrub plants and is popular because of its high health care value, as it is recommended by the FAO as one of the five major healthy fruits  and a maximum release well , centrifuged at 152 g for 2 min, then incubated in a 5% CONK cells .The serum samples and medium supernatants from spleen lymphocytes cultured for 48 h were applied to 3 wells each. The content of IFN-γ, TNF-α, and interleukin IL-2 were examined by ELISA kit. Summary procedures: Preparing reagents, samples and standard → Add prepared sample and standard, incubated 30 min at 37 °C → Plate washed five times, adding HRP-Conjugate Reagent incubated 30 min at 37 °C → Plate washed five times, adding Chromogen Solution A and B incubated 30 min at 37 °C → Add stop solution → Measure within 15 min → Calculation.t-test were used. Data are reported as the mean ± SD (standard deviation) and average. Statistical calculations were performed using SPSS software .All experiments were repeated at least three times. Analysis of variance and This study, to our knowledge, is the first report on anti-cancer and immune adjustment of a polysaccharide (BBP3-1) isolated from blueberry. It is found that the BBP3-1 has the strongly inhibited the growth of S180 tumor and the best dose is 100 mg/kg body mass in this experiment. In addition, the BBP3-1 can up-regulate the immunity of S180 tumor-bearing mice by increasing the thymus index and spleen index, enhancing lymphocyte conversion and multiplication capacity, promoting macrophage phagocytosis, promoting immune factor secretion , and enhancing NK cell activity. However, elucidation of the detailed mechanism requires further experiments.


To enhance risk stratification for Wilms tumour (WT) in a pre-operative chemotherapy setting, we explored the prognostic significance and optimal age cutoffs in patients treated according to International Society of Paediatric Oncology Renal Tumour Study Group (SIOP-RTSG) protocols.Patients(6 months-18 years) with unilateral WT were selected from prospective SIOP 93–01 and 2001 studies(1993–2016). Martingale residual analysis was used to explore optimal age cutoffs. Outcome according to age was analyzed by uni- and multivariable analysis, adjusted for sex, biopsy(yes/no), stage, histology and tumour volume at surgery.P<0.001). Using previously described age categories, EFS was lower for patients aged 2-4, 4-10 and 10–18 years as compared to patients aged 6 months-2 years. OS was lower for patients 4–10 years and 10–18 years, but not for 2–4 years. Higher stage, histological risk group and tumour volume were independent adverse prognostic factors.5631 patients were included; median age was 3.4 years(IQR: 2–5.1). Estimated 5-year event-free survival (EFS) and overall survival (OS) were 85%(95%CI 83.5–85.5) and 93%(95%CI 92.0–93.4). Martingale residual plots detected no optimal age cutoffs. Multivariable analysis showed lower EFS with increasing age and OS (4 years) in children with WT treated with pre-operative chemotherapy. These findings encourage the consideration of age in the design of future SIOP-RTSG protocols. As treatment for Wilms tumour (WT) is evolving towards further risk adaptation, there is an increasing interest in additional factors that can help to stratify treatment intensity based on the patient’s individual risk. One of these factors appears to be a patient’s age at diagnosis. Older age has been suggested to be an adverse prognostic factor for recurrence and mortality–4 while S1 Table).Treatment stratification of WT has been primarily based on pathological stage and histology. More recently, potential molecular prognostic markers such as copy number changes and loss of heterozygosity (LOH) of specific chromosomal regions are emerging.–15 CurreAge as a prognostic factor was first described in 1976 when D’Angio et al.–8Whether the age of 2 years is the optimal cutoff for risk stratification was debated in a later study by the UK Children’s Cancer Study Group, suggesting that the age of 4 years may be a more relevant cutoff in the setting of minimal adjuvant chemotherapy. The AssoIn the current study, we aimed to assess the prognostic significance of age in a large, prospectively registered cohort of paediatric patients with WT treated with pre-operative chemotherapy according to recent SIOP protocols. Moreover, we aimed to identify relevant age cutoffs for future stratification purposes.Patients with histologically proven stage I-IV WT, aged 6 months-18 years, treated according to SIOP 93–01 and SIOPPre-operative chemotherapy consisted of 4 weeks of vincristine and actinomycin-D in case of localized disease, and 6 weeks of vincristine, actinomycin-D and doxorubicin in case of metastatic disease. Biopsy before start of treatment was not recommended as a standard procedure, but was allowed without upstaging if performed by a percutaneous fine needle or trucut procedure. This was a routine procedure in the Children’s Cancer and Leukemia Group (CCLG), including the UK and Republic of Ireland, that participated in SIOP 2001 but not SIOP 93–01. Post-operative treatment stratification depended on SIOP stage and histological risk group , 25, andIn the current analysis, high-risk tumours included diffuse anaplastic and/or blastemal-type WT after pre-operative chemotherapy. Intermediate risk tumours were either stromal, epithelial, mixed or regressive type or focal anaplasia, while low risk was defined as completely necrotic tumours after pre-operative chemotherapy. Central pathology review was performed for 83.4% of patients in SIOP 2001, and for 94.4% of patients in SIOP 93–01. Tumour volume was not a factor for treatment stratification, with the exception of German Paediatric Oncology Haematology (GPOH) centers, where patients with non-stromal, non-epithelial intermediate risk WT and a tumour volume >500mL at surgery, received ‘high-risk’ post-operative chemotherapy (four drugs).P<0.10) and/or were known confounders based on previous literature, were entered into the multivariable model, stratified by national/regional group and study protocol (SIOP 2001 and SIOP 93–01). Patient characteristics were compared using Pearson’s Chi-Squared test for categorical variables, and Mann Whitney’s U-test/Kruskall-Wallis for continuous variables.To search for potential relevant prognostic age cutoffs in relation to event-free survival (EFS) and overall survival (OS), martingale residual plots were evaluated. Cox regrTumour volume at surgery was missing in 18·3% of cases, and was imputed using multiple imputation techniques  on 100 generated datasets, assuming it was missing at random. Missing volume was associated with center/consortium and not with patient characteristics, and there were no indications that it might be related to unobserved characteristics or the missing volume itself. Resulting model estimates were combined using SAS PROC MIANALYZE software (version 9.4).S1 Fig). Baseline characteristics are summarized in Table 1. Median follow-up at time of data capture was 6·3 years . Median age at diagnosis was 3·4 years (IQR: 2–5·1) with only 189 patients (3·4%) aged 10 years or older. Median age was 3·6 (IQR 2–5·2) for females and 3·2 (IQR 1·9–4·9) for males (P<0.001). The age distribution of females showed two peaks, at around 1 year and 4 years. For males there was an early peak in the age distribution but bimodality was less apparent than for females . Forty-five percent (N = 2554) presented with overall stage I disease, 23% (N = 1271) with stage II, 17% (N = 949) with stage III and 15% (N = 857) with stage IV. WT’s were histologically classified as low-risk in 5·6% (N = 315), intermediate risk in 82% (N = 4566), high-risk blastemal type in 8·3% (N = 466) and high-risk diffuse anaplastic in 4·9% (N = 278). Biopsies were performed in 208 cases (10·5%) in SIOP 93–01 and 1159 cases (31·7%) in SIOP 2001. Tumour volume at surgery was available for 4599 patients (81.7%), of whom 14·1% (N = 649) had a tumour volume of >500ml at surgery.Out of 7262 registered patients with histologically proven WT, 5631 met the inclusion criteria (78%) (Table 2).The distribution of stage, histological risk and tumour volume differed between age groups, with the frequency of metastatic disease, high-risk histology types (most markedly for diffuse anaplastic WT) and high-volume tumours increasing with age  suggested that an increase in age was linearly associated with the risk of an event. However, since no specific change point (knot) could be clearly observed, hence, no optimal cutoffs for categorizing age could be identified. Therefore, in further analyses, age was included as a linear factor (per year), as well as categorized according to previous studies at the ages of 2, 4 and 10 years.Martingale residual plots (N = 784) were relapses. In univariable analysis, significant differences in EFS and OS were found between age categories 6 months– 2 years, 2–4, 4–10 and ≥10 years . The 5-year EFS was 91·2% (95% CI 89·7–92·8) for ages 6 months-2 years, 86·3% (95% CI 84·7–87·9) for 2–4 years, 79·3% (95% CI 77·5–81·1) for 4–10 years and 73·5% (95% CI 66·8–80·9) for 10–18 years (log rank P<0·0001). OS was 96·8% (95% CI 95·9–97·8) for ages 6 months-2 years, 94·1% (95% CI 92·9–95·2) for 2–4 years, 89·5% 95% CI 88·1–90·9) for 4–10 years and 84·6% (95% CI 79·0–90·7) for 10–18 years (P<0·0001).5585 patients were included in the survival analysis, after censoring 46 patients without available follow-up data. Estimated 5-year EFS and OS of the total cohort was 85% (95% CI 83·5–85·5) and 93% (95% CI 92·0–93·4) respectively. A total of 836 events occurred, of which 93.8% , after stratifying for national/regional study group and study protocol, and including sex, overall stage, histological risk group, biopsy and tumour volume at surgery. Other independent prognostic factors for EFS were overall stage III and IV, histological subtype  and tumour volume at surgery (Table 3).When replacing the age categories by age per year in the multivariable model, the linear trend observed in the Martingale residual plot was confirmed for EFS . The same conclusions were obtained when imputing missing volume (S2 Table), and when limiting the analysis to SIOP 2001 patients only .Age categorized as 6 months– 2 years, 2–4 years, 4–10 years and 10–18 years, was a significant prognostic factor for EFS in multivariable analysis  did not retain significance in multivariable analysis. However, patients aged 4–10  and 10–18 years  revealed lower OS compared to patients aged 6 months– 2 years. Other factors that were significantly associated with OS included overall stage III and IV, histological classification and tumour volume at surgery (Table 4). When including age as a continuous variable in the multivariable model, this did not reach statistical significance . These conclusions were maintained when imputing missing volume (S4 Table). When limiting the analysis to SIOP 2001 patients, age (categorized or continuous) did not retain significance .For OS, the age category 2–4 years .This study, which included 5631 patients with unilateral WT registered over 23 years in the recent SIOP trials, demonstrated that age is an independent prognostic factor for EFS in patients treated with pre-operative chemotherapy. Although optimal age cutoffs for risk stratification could not be identified, the prognostic significance of previously described cutoffs was confirmed for EFS (2 and 4 years) and OS (4 years). Despite the observation that older patients more frequently have a higher stage at diagnosis, high-risk histology types and large-volume tumours, age retained independent prognostic significance. Interestingly, the strong prognostic value of tumour volume ≥500mL confirmed previous findings from the posthoc analysis of the SIOP 2001 randomized trial. While prS6 Table). A large study on prognostic molecular markers (N = 1114) showed that 1p/16q loss/LOH was not independently associated with EFS when correcting for 1q gain, but suggested prognostic value in the group of patients lacking 1q gain.[We observed that the presence of diffuse anaplasia increases with age, and is a strong adverse prognostic factor. We could not include molecular markers in the analysis, since copy number status was only available for a subset of the SIOP 2001 cohort (N = 586), as previously described. Gain of kers N = 14 showed 1q gain., 28, 29  1q gain., 30Furthermore, Wilms tumour predisposition syndromes may be a relevant factor to consider in relation to age at diagnosis and survival, but could not be assessed in this study due to incomplete data. Wilms tumour predisposition syndromes have been associated with a younger age at diagnosis and depending on the genetic aberration, a more favorable tumour biology. On the other hand, these syndromes carry a higher risk of bilateral/second tumours and subsequent renal failure. Wilms tumour predisposition was not always evaluated or recognized in the past, and incompletely registered, as this was beyond the objectives of SIOP 93–01 and 2001. Therefore, we were unable to reliably distinguish between patients with and without a Wilms tumour predisposition syndrome in the current study. As genomic sequencing becomes more widely implemented in paediatric oncology, more data will become available to unravel these associations in the SIOP-RTSG UMBRELLA protocol.Other limitations of this study included the long period of time during which treatment evolved based on the results of two successive clinical trials, and missing data requiring imputation. When limiting the analysis to SIOP 2001 only, a more uniform but slightly smaller cohort, age retained significance in relation to EFS but not OS.The two most recent COG protocols have provided some insight into the outcomes after reduced treatment for young patients, 7, but Overall, these results encourage the consideration of age in the design of future SIOP-RTSG protocols, albeit after validation of 1q gain, other molecular markers and age as independent prognostic factors in the UMBRELLA SIOP-RTSG protocol.S1 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


Water soluble tetrazolium salt assay showed that fludioxonil decreased Jurkat T cell and Ramos B cell viability. Jurkat T cell viability decreased at 24 and 48 h, but Ramos B cell viability decreased only at 48 h. JC-1 dye revealed decreased mitochondrial membrane potential in fludioxonil-treated Jurkat T cells and Ramos B cells. To evaluate apoptosis, annexin-V conjugated FITC, AF488, and propidium iodide (PI) were used and to evaluate cell cycle arrest PI was used. Apoptosis and cell cycle arrest were induced by fludioxonil (10−7–10−5 M) in the Jurkat T cells at 24 and 48 h and Ramos B cells at 48 h. Moreover, the protein levels of pro-apoptotic proteins, such as p53, BAX, and cleaved caspase 3, were increased and anti-apoptotic protein Bcl-2 was decreased by fludioxonil. Expression of the Fas receptor related to the extrinsic apoptosis pathway was increased by fludioxonil. Additionally, cyclin D1 and cyclin E1 were decreased by fludioxonil. In the present study, fludioxonil induced immunotoxicity in human T cells and B cells through apoptosis and cell cycle arrest. Therefore, the present study suggests that fludioxonil induces the cellular toxicity in immune cells.Fludioxonil is fungicide used in agriculture, which is present in fruits and vegetables. In this study, the effects of fludioxonil on human immune cell viability, apoptosis, cell cycle arrest, and mitochondrial membrane potential were examined in human immune cells, such as Jurkat T cells and Ramos B cells. To examine the cell viability, Jurkat T cells and Ramos B cells were treated with fludioxonil (10 Pesticides are chemical substances that kill living fungi, weeds, insects, and rodents. Though pesticides benefit human life via increasing agricultural productivity through protecting crops from fungi, weeds, and insects, etc., pesticides are toxic to humans and theenvironment. Many pesticides have been used in agriculture, such as organochlorines (OCs), organophosphorus (OPs), carbamate (CBs), pyrethroids, chlorophenoxys, triazines amid, and phthalimides. These pesticides remain in the air, water, and soil ,2. AccorFludioxonil is a phenylpyroll fungicide and is an antifungal antibiotic, produced in 1993 by Sygenta . FludioxThe immune system plays the role of the defense system from a variety of agents, such as pathogens, viruses, bacteria, toxins, and foreign materials. The immune system is classified into innate and adaptive immune systems, and the adaptive immune system is classified into humoral immunity and cellular immunity . The innCell cycle is a mechanism that replicates itself and induces cell proliferation. This mechanism is proceeded by various proteins, such as cyclin and cyclin-dependent kinases (Cdks). Therefore, deregulation of cell cycle-related protein induces cell cycle arrest, causing apoptosis ,17Apoptosis was originally introduced by Kerr in 1972 as a form of programmed cell death . BiochemIn this study, the cytotoxicity of fludioxonil in human immune cells, such as T cells and B cells, was investigated. To investigate the cytotoxicity of fludioxonil, cell proliferation, cell cycle arrest, apoptosis, mitochondrial membrane potential, and apoptosis- and cell cycle-related gene expression were evaluated.Fludioxonil was purchased from Sigma-Aldrich Corp. . Fludioxonil was dissolved in 100% dimethyl sulfoxide . The final concentration of DMSO in the cell culture media was 0.1%.The human Jurkat T cell line, human Ramos B cell line, and human lung diploid fibroblast cell line, WI-26, were obtained from the Korean Cell Line Bank (KCLB) . Jurkat T cells and Ramos B cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS), 10 U/mL penicillin, 100 µg/mL streptomycin, and 10 mM HEPES. WI-26 cells were maintained in Dulbecco’s Modified Eagle’s medium  added to 10% FBS, 10 U/mL penicillin, 100 µg/mL streptomycin, and 10 mM HEPES.5 cells/mL in the 24-well plates  and 107 cells/mL in 90 mm cell culture dish (SPL Life Science). The seeded cells in 24-well plate were used to analyze apoptosis, cell cycle arrest, and mitochondrial membrane potential, and seeded cells in 90 mm dish were used for the extraction of proteins for Western blot analysis. Then, T cells and B cells were treated by fludioxonil at 10−7–10−5 M for 24 or 48 h and incubated at 37 °C in 5% CO2.The human Jurkat T cells and Ramos B cells were seeded at a density of 6 × 104 cells/well in 200 µL in 96 well plates (SPL Life Science), and incubated in a humidified 5% CO2 atmosphere at 37 °C for 24 h. Incubated cells were treated with various concentrations of fludioxonil  for 24 or 48 h. After 24 and 48 h, the cell viability was evaluated using EZ-Cytox . EZ-Cytox 20 µL was added to each well and incubated at 37 °C for 1 h. The absorbance was measured at 450 nm using a microplate reader . The human Jurkat T cells, Ramos B cells, and WI-26 cells were seeded at a density of 5 × 10−7 M–10−5 M) for 24 and 48 h were collected to 15 mL tubes, and collected cells were centrifuged at 400× g for 10 min and removed the supernatant. The cells were washed by phosphate-buffered saline (PBS) and resuspended by PBS, then the Jurkat T cells and Ramos B cells were fixed by 70% ethanol and the cells were incubated in 4 °C for 1 h. After cells were fixed, these cells were washed by PBS and stained with propidium iodide (PI) premix, which combined PI (Sigma-Aldrich Corp.) and RNase A . Then, cells, treated with PI, incubated at 4 °C for overnight. These cells, stained with PI and fixed with ethanol, were analyzed by flow cytometry  to evaluate cell cycle arrest. A total of 30,000 cells were analyzed by flow cytometry.In the 24-well plate, Jurkat T cells and Ramos B cells treated with fludioxonil (10−7 M to 10−5 M) for 24 and 48 h. The treated Jurkat T cells and Ramos B cells were collected to 15 mL tubes, and stained with FITC, Alexa Fluor 488-annexin V/PI  for 30 min. After staining, Jurkat T cells and Ramos B cells were washed by PBS. Then the stained Jurkat T cells and Ramos B cells were counted and 30,000 cells were analyzed by flow cytometry. Early apoptotic cells were defined as positive annexin V/negative PI and late apoptotic cells were defined as positive annexin V/positive PI.In the 24-well plate, Jurkat T cells and Ramos B cells were treated with fludioxonil (10–7 M–10–5 M) for 24 and 48 h and collected each 15 mL tubes, centrifuged at 400× g for 10 min, and the supernatant was removed. Remaining cells in the 15 mL tubes were stained by JC-1 dye  10 µg/mL at 37 °C in 5% CO2 incubator for 15–30 min. The stained cells were washed by PBS twice and resuspended in media. The stained cells were seeded in 0.2% gelatin-coated 24-well plates, and stained cells were incubated at 37 °C in 5% CO2 overnight. The stained cells were observed with an Olympus CKX 41 microscope  under green fluorescence and red fluorescence. The red fluorescence indicates healthy mitochondria and the green fluorescence indicate depolarized membrane potentials of mitochondria.In the 24-well plate, the immune cells were treated with fludioxonil  buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, and 0.1% SDS). The protein concentrations were determined by a solution of bicinchoninic acid  which was mixed with copper II sulfate solution (Sigma-Aldrich Corp.) at a ratio of 50:1. The total cell proteins (30 µg) were separated in a 15% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane . The membrane was immersed in primary antibodies in tris-buffered saline (TBS) containing 5% bovine serum albumin , as shown in After treatment with fludioxonil in 10Fas gene expression, the human glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as an internal control as shown Total RNA was isolated from human Jurkat T cells and Ramos B cells using TRIzol Reagent . After isolating RNA, cDNA was synthesized using moloney murine leukemia virus reverse transcriptase (M-MLV RT) (iNtRON Biotechnology) from the isolated mRNA. Fas gene was amplified from cDNAs of Jurkat T cells and Ramos B cells to confirm gene expression levels. In this analysis, to compare −5 M). Additionally, in Jurkat T cells treated with fludioxonil for 48 h, cell viability was decreased by dose dependent in 10−8–10−5 M (−7–10−5 M in a dose-dependent manner (−9–10−5 M) for 24 and 48 h, cell viability did not change  for 24 or 48 h. To confirm the effects on the cell cycle by fludioxonil, Jurkat T cells and Ramos B cells were stained by PI. Separation of cells in G0/G1, S phase, and G2/M was based on the linear fluorescence intensity after PI. In Jurkat T cells treated with fludioxonil (10−7–10−5 M) for 24 or 48 h, the G0/G1 phase was increased by fludioxonil in a concentration-dependent manner. However, the S phase was decreased by fludioxonil (Flow cytometry was used to confirm the changes in cell cycle induced in Jurkat T cells and Ramos B cells that were control or exposed to fludioxonil (10dioxonil A. At Ramdioxonil B. −7–10−5 M) for 24 or 48 h. In Jurkat T cells, protein expression of cyclin D1 and cyclin E1 was decreased by exposure to fludioxonil (10−7–10−5 M) at 24 and 48 h compared to control, but there was significance at the highest concentration of fludioxonil (−5 M) for 48 h, protein expression of cyclin D1 was decreased, and cyclin E1 was decreased with exposure to fludioxonil (10−7–10−5 M) at 24 and 48 h  compared to control, but significance was present only at a high concentration of fludioxonil (10−5 M) (−7–10−5 M), but significance was presented at only at a high concentration of fludioxonil (10−5 M)  A. In the(10−5 M) B. Apopto−6 M, and at treatment of fludioxonil for 48 h in Jurkat T cells, green fluorescence presented at fludioxonil 10−7 M. Additionally, the green fluorescence was increased by fludioxonil in a concentration-dependent manner , Bcl-2, and cleaved caspase-3, in Jurkat T cells and Ramos B cells or not . The pro and BAX B.Fas by the treatment of fludioxonil in Jurkat T cells and Ramos B cells  were employed for cell viability assay. In human immune cells, fludioxonil decreased the Jurkat T cells’ and Ramos B cells’ viability. In the Jurkat T cells, cell viability was decreased in 24 and 48 h. On the other hand, Ramos B cells’ viability was decreased in only 48 h. The cell viabilities in Jurkat T cells and Ramos B cells were decreased at 48 h by fludioxonil (10−7–10−5 M). Thus, in the other experiments, these concentrations of fludioxonil (10−7–10−5 M) were used to decrease the cell viability of Jurkat T cells and Ramos B cells. Decreases in cell viability mean that fludioxonil has an effect on cell proliferation. Decreased immune cell proliferation showed that immune cell counts diminished, thus, decreases in immune cell counts may induce immunodeficiency. T cells and B cells are known to play a role in adaptive immunity, are major types of immune cells [First, the effects of fludioxonil on human immune cells, such as Jurkat T cells and Ramos B cells were identified and investigated. To investigate the cell viability, fludioxonil concentrations , late apoptosis (Annexin V+/PI+), and green fluorescence related with mitochondrial membrane potential loss were increased by treatment with fludioxonil for 24 and 48 h. In Ramos B cells, early apoptosis, late apoptosis, and green fluorescence were increased by treatment with fludioxonil for 48 h. Annexin V is phospholipid-binding protein that binds to phosphatidylserine (PS). PS is exposed at the cell surface which underwent apoptosis and recognized the apoptotic cells [Fas gene expression was confirmed. In Jurkat T cells, Fas was increased by fludioxonil treatment for 24 and 48 h. However, in Ramos B cells treated with fludioxonil for 24 and 48 h, fludioxonil did not change Fas. As a result, fludioxonil induces apoptosis via the increase in pro-apoptotic-related proteins, Fas gene, a decrease in anti-apoptotic-related protein, and induction of dysfunction of mitochondrial membrane potential. Thus, these results present that fludioxonil may induce intrinsic and extrinsic apoptotic pathways and dysfunction of mitochondrial membrane potential in human immune cells, Jurkat T cells, and Ramos B cells.The apoptosis by fludioxonil in Jurkat T cells and Ramos B cells were confirmed by decreasing mitochondrial membrane potential, analysis of annexin V/PI staining, and altered protein expression of apoptosis-related genes and increased gene expression of ic cells . The dysic cells ,42. Protic cells ,44. Thesic cells . These pic cells . The othic cells . Fas genIn conclusion, it was found that fludioxonil may induce apoptosis by inhibiting cell proliferation through cell cycle arrest. Apoptosis was induced by the intrinsic apoptotic pathway mediated in mitochondria and the extrinsic apoptotic pathway and dysfunction of mitochondrial membrane potential. In addition, Jurkat T cells appeared to be more sensitive than Ramos B cells in this study. Therefore, as shown in