Arabidopsis thaliana and recently in crop species, including Maize, Rice and Barley. However, in most crop species, an understanding of the participants in nitrate and ammonium transport across the soil plant continuum remains undefined.In order to grow, plants rely on soil nutrients which can vary both spatially and temporally depending on the environment, the soil type or the microbial activity. An essential nutrient is nitrogen, which is mainly accessible as nitrate and ammonium. Many studies have investigated transport genes for these ions in Zea mays . We also selected genes from published studies . To analyse these genes, the plants were grown in a semi-hydroponic system to carefully control nitrogen delivery and then harvested at both vegetative and reproductive stages. The expression patterns of 26 putative nitrogen transporters were then tested. Six putative genes were found not expressed in our conditions. Transcripts of 20 other genes were detected at both the vegetative and reproductive stages of maize development. We observed the expression of nitrogen transporters in all organs tested: roots, young leaves, old leaves, silks, cobs, tassels and husk leaves. We also followed the gene expression response to nitrogen starvation and resupply and uncovered mainly three expression patterns: (i) genes unresponsiveness to nitrogen supply; (ii) genes showing an increase of expression after nitrogen starvation; (iii) genes showing a decrease of expression after nitrogen starvation.We have mapped a non-exhaustive set of putative nitrate and ammonium transporters in maize. The selected transporters were defined based on previous studies comparing nitrate transport pathways conserved between Arabidopsis and These data allowed the mapping of putative nitrogen transporters in maize at both the vegetative and reproductive stages of development. No growth-dependent expression was seen in our conditions. We found that nitrogen transporter genes were expressed in all the organs tested and in many cases were regulated by the availability of nitrogen supplied to the plant. The gene expression patterns in relation to organ specificity and nitrogen availability denote a speciality of nitrate and ammonium transporter genes and their probable function depending on the plant organ and the environment.The online version of this article (10.1186/s12870-019-1768-0) contains supplementary material, which is available to authorized users. Once absorbed, NO3− and NH4+ undergo a complex process of assimilation, transformation and mobilization within the plant [3− after rainfall or irrigation events [Nitrogen is an essential nutrient required for plant growth. It is a primary constituent of nucleic acids, amino acids and proteins. Although 78% of the atmospheric air is made of Nnt roots . Plants he plant . In agrihe plant –8. The en events . This ca3− and NH4+ involves two physiological mechanisms [NRT2 (Nitrate Transporter 2) and AMT1 (Ammonium Transporter 1) families for NO3− and NH4+, respectively [3− LATS involves the NPF (NRT1/PTR Family) gene family [4+ LATS has been described yet, the recent identification of AMF1 (Ammonium Facilitator 1) proteins in soybean and yeast are promising candidates [The uptake of both NOchanisms , 11. Wheectively . Converse family . Althougndidates .ZmNRT2 genes have been identified in the maize genome: ZmNRT2.1, ZmNRT2.2, ZmNRT2.3 and ZmNRT2.5 [ZmNRT2.1 and ZmNRT2.2 Both genes are closely related, sharing 98% homology in their amino acids sequence [3− inducible genes in seedling roots [ZmNRT2.1 transcripts in the cortex whereas ZmNRT2.2 transcripts could be found in the cortex, the stele and the incipient of root lateral primordia [NRT3.1 gene: ZmNRT3.1A and ZmNRT3.1B [Four ZmNRT2.5 . Only twsequence . Both arng roots –18. In srimordia . The exprimordia , 18. NRTrimordia –21. The mNRT3.1B , neither3− transporter with efflux activity that has been reported as a potential high-affinity chloride transporter [3− specific transport in a pH-dependent manner [ZmNPF6.6 expression was downregulated after 4 days of nitrogen starvation and upregulated specifically after NO3− resupply [Ten genes belonging to the NPF family have been discovered in maize . Little nsporter . The gent manner . Transcrresupply .AMT family contains 8 members divided in four classes, ZmAMT1 to ZmAMT4 [ZmAMT1.1A, ZmAMT1.1B and ZmAMT1.3 belong to the sub-class 1 of the AMT family. ZmAMT1.1B is a low expressed gene in maize. ZmAMT1.1A and ZmAMT1.3 are expressed in the rhizodermis of the apical root zone and act as high affinity NH4+ transporters with Km affinities of 48 and 33 μM, respectively [4+ supply specifically up-regulates the expression of ZmAMT1.1a and ZmAMT1.3 independently of the whole plant nitrogen status [The maize o ZmAMT4 , but onlo ZmAMT4 . ZmAMT1.ectively . NH4+ sun status .4+ was described in rice roots more than 20 years ago [GmbHLHm1, in yeast activated the expression of ScAMF1 which allowed a NH4+ transport-deficient strain of Saccharomyces cerevisiae to grow on an NH4+ enriched-medium. A phylogenetic analysis revealed the presence of AMF1 genes in most plants [ZmAMF1.1 and ZmAMF1.2.Although the low affinity transport of NHears ago , no LATSears ago . The ectt plants . In maizIn recent years, an increasing collection of informative transcriptional gene array data sets have been presented in public sites such as Maize GDB . This daArabidopsis thaliana transport genes [3− transporter genes included in this study, five were found to be not expressed across the tissue samples examined. No transcripts were detected for ZmNPF4.10, ZmNPF6.5, ZmNPF6.7, ZmNPF7.12 and ZmNRT2.3 (data not shown). However, each of the remaining genes were expressed at both growth stages at varying levels within the tissues sampled. Although growth-dependent expression was generally absent, differences in expression levels occurred between the vegetative and reproductive tissue stages for most of the genes examined  genes in reproductive tissues and as leaves transition from young to older leaves.In order to study the expression profiles of maize nitrogen transporter genes, plants were grown using a semi-hydroponic system in the glasshouse that enabled full-plant growth with the ability to access aerial and root samples. Plants were harvested at a vegetative (V7) and reproductive stage R1) of growth from which RNA was extracted from a range of tissues that included roots, old leaves, young leaves, cobs, silks, tassels and husk leaves . Although expressed, ZmNPF6.4, ZmAMT1.3 and ZmAMT3.3 did not respond to the nitrogen starvation (data not shown). The most nitrogen responsive genes were identified as ZmNRT2.1 and ZmNRT2.2, where expression increased more than 10-fold in roots after starvation  have been previously described in A. thaliana. AtNPF7.3/AtNRT1.5 encodes a low-affinity NO3− transporter located in the root pericycle cells near the xylem and is responsible for xylem loading of NO3− [AtNPF7.2/AtNRT1.8 also encodes a low-affinity NO3− transporter [3− from xylem vessels. Together, AtNPF7.3/AtNRT1.5 and AtNPF7.2/AtNRT1.8 are believed to work in concert with each other to load and unload NO3− from the xylem, respectively. The maize NPF7 family contains a total of 12 members [ZmNPF7.12 was not expressed (data not shown) and ZmNPF7.10 was found to be ubiquitously expressed across tissues and developmental stages   Fig. a. Its leAtNRT1.4 , 15. In the leaf . This lo4+ LATS transporter genes were constitutively expressed in our experiments. ZmAMF1.1 and ZmAMF1.2 expression was similar in all the organs independent of the growth stage  and sorghum (SbAMT1.1) [ZmAMT1.1A, its closest homologue, ZmAMT1.1B showed specific expression only in the silks  , 42. Conng stage . A thirdtermined . Our resZmAMT2.1, was found expressed in all organs with some specificity to roots and tassels .Transcripts of the last member of the he roots . A detaiAMT1, AMT2 and AMF1) systems are clearly induced with expected roles in nitrogen redistribution in these important tissues. Activity of nitrate transport systems in flowering tissues (R1) are noticeably less than those of ammonium.To provide a visual summary of indicative gene expression at the vegetative (V7) and reproductive stages (R1), gene expression relative to controls have been presented as colour indicative heat maps Fig. . There iA. thaliana [AtNRT2.1 was demonstrated to be expressed rapidly and strongly after nitrogen starvation [3− HATS gene was found responsive in both roots and shoots. No expression of ZmNRT2.5 could be detected in our control conditions probably because of the younger age of the plants used in the starvation experiment  indicated that AtNAR2.1/AtNRT3.1 expression increased after 24 h of NO3− starvation in a similar fashion to AtNRT2.1 [NRT2.1 and AtNAR2.1/AtNRT3.1 components. A corresponding protein association of ZmNRT2.1 and ZmNRT3.1A was recently demonstrated in maize roots [ots Fig. e, k. HowAtNRT2.1 . The autze roots . Our resZmNPF6.6 decreased after nitrogen starvation (~ 58%) but then returned to base level after nitrogen resupply  after starvation and then reverted to initial levels after nitrogen resupply  seeds were first imbibed 24 h in bubbling reverse osmosis water before sown individually in pots (diameter of 25 cm) filled with diatomaceous earth rocks . The plants were watered via a drip irrigation system and grown in a heated glasshouse. After one week on reverse osmosis water, the plants were supplied with a solution of 7 mM nitrogen containing 0.5 mM MgSO4, 0.5 mM KH2PO4, 0.05 mM KCl, 0.5 mM K2SO4, 2.5 mM KNO3, 0.75 mM CaCl2, 1.25 mM Ca(NO3)2, 1 mM NH4NO3, 0.1 mM Fe-EDTA, 0.1 mM Fe-EDDHA, 25 μM H3BO3, 2 μM MnSO4, 2 μM ZnSO4, 0.5 μM CuSO4 and 0.5 μM Na2MoO4. The nutrient solution was changed weekly to maintain nutrient levels and solution pH around 5.9.Maize (The plants were harvested at a vegetative stage (V7) and a reproductive stage (R1) Fig. . Roots w− 2.s− 1 at canopy level. The plants were supplied with nutrient solution containing 2.5 mM NH4NO3, 0.5 mM MgSO4, 0.5 mM KH2PO4, 1.05 mM KCl, 1.25 mM K2SO4, 0.25 mM CaCl2, 1.75 mM CaSO4, 0.1 mM Fe-EDTA, 0.1 mM Fe-EDDHA, 25 μM H3BO3, 2 μM MnSO4, 2 μM ZnSO4, 0.5 μM CuSO4 and 0.5 μM Na2MoO4. The nutrient solution was changed weekly to maintain nutrient levels and solution pH around 5.9.Seeds were imbibed in bubbling reverse osmotic water for 4 h before sown individually onto a supportive mesh within a PVC seedling tube which contained moist diatomaceous earth rocks . 4 day-old seedlings were transferred to a 700 L ebb-and-flow hydroponic system that allows for continual 15 min fill/drain cycles. The seedling tubes were placed within larger tubes (300 mm × 50 mm), which kept the roots of adjacent plants separate, but allowed for free access of the roots to nutrient solution. The hydroponic system was situated in a controlled environment room with a day/night cycle of 12 h/12 h, temperatures of 28 °C and 21 °C respectively, and a light intensity of 300 μmol.m4NO3. Twenty-six-day-old control, starved and resupplied plants were harvested at midday on the same day , SIN3 component, histone deacetylase complex (ZmSIN3), Cullin (ZmCullin) and Elongation factor 1-alpha (ZmElF1), were chosen as housekeeping genes to normalise gene expression data. Symbol numbers and genome gene ID are described in Additional file Total RNA was isolated using the TRIzol Reagent  according to manufacturer’s protocol. RNA concentrations were estimated using a NanoDrop ND-1000 Spectrophotometer . cDNA was generated using 1 μg of total RNA using SuperScript III Reverse Transcriptase kit . cDNA was mixed with TaqMan OpenArray® Real-Time PCR Master Mix . All samples were run on a QuantStudio 12 K Flex Real-Time PCR (RT-PCR) System  using 48-well plates TaqMan® OpenArray® RT PCR Inventoried Format 112 . Four genes, ubiquitin-conjugating enzyme . Differences in treatment levels were further evaluated for significance with Tukey post-hoc comparisons and a level of Additional file 1:Table S1. List of genes studied. Table S2. One-way ANOVA results of root gene expression between control (C), starved (S) and resupplied (R) plants. Bold text denotes p < 0.05. Table S3. One-way ANOVA results of shoot gene expression between control (C), starved (S) and resupplied (R) plants. Bold text denotes p < 0.05. (DOCX 118 kb)


More importantly, 2D cultures lack cell–cell and cell–extracellular matrix (ECM) interactions, which are critical for regulating cell behavior and functions. There are several three-dimensional (3D) cell culture systems such as Matrigel, hydrogels, micropatterned plates, and hanging drop that overcome these drawbacks but they suffer from technical challenges including long spheroid formation times, difficult handling for high throughput assays, and/or matrix contamination for metabolic studies. Magnetic 3D bioprinting (M3DB) can circumvent these issues by utilizing nanoparticles that enable spheroid formation and growth via magnetizing cells. M3DB spheroids have been shown to emulate tissue and tumor microenvironments while exhibiting higher resistance to toxic agents than their 2D counterparts. It is, however, unclear if and how such 3D systems impact cellular metabolic networks, which may determine altered toxic responses in cells. We employed a Stable Isotope-Resolved Metabolomics (SIRM) approach with 13C6-glucose as tracer to map central metabolic networks both in 2D cells and M3DB spheroids formed from lung (A549) and pancreatic (PANC1) adenocarcinoma cells without or with an anti-cancer agent (sodium selenite). We found that the extent of 13C-label incorporation into metabolites of glycolysis, the Krebs cycle, the pentose phosphate pathway, and purine/pyrimidine nucleotide synthesis was largely comparable between 2D and M3DB culture systems for both cell lines. The exceptions were the reduced capacity for de novo synthesis of pyrimidine and sugar nucleotides in M3DB than 2D cultures of A549 and PANC1 cells as well as the presence of gluconeogenic activity in M3DB spheroids of PANC1 cells but not in the 2D counterpart. More strikingly, selenite induced much less perturbation of these pathways in the spheroids relative to the 2D counterparts in both cell lines, which is consistent with the corresponding lesser effects on morphology and growth. Thus, the increased resistance of cancer cell spheroids to selenite may be linked to the reduced capacity of selenite to perturb these metabolic pathways necessary for growth and survival.Conventional two-dimensional (2D) cell cultures are grown on rigid plastic substrates with unrealistic concentration gradients of O Fu. Fu13C eSimilar to A549 cells, we observed differential selenite inhibition of HBP activity and UTP synthesis, but not of PPP activity and PRPP production in PANC1 2D cell culture I.50 values, respectively >100 µM versus 6.25 µM for A549 and >150 µM versus 10 µM for PANC1  of regulator proteins, including those important for cancer development and survival [O-GlcNAcylation of proteins between spheroids and 2D cells is involved in modulating their differential responses to selenite toxicity. Further investigations will be required to test this hypothesis. It is also foreseeable that enhanced capacity for gluconeogenesis in PANC1 spheroids could improve their survival in a hostile hypoxic tumor microenvironment, where glucose is depleted due to its increased glycolytic conversion to lactate and extracellular release of lactate [Besides differential metabolic responses to selenite for spheroids versus 2D cells, we also noted their intrinsically distinct metabolic activities, i.e., reduced capacity for lactate release, UTP synthesis and HBP in both A549 and PANC1 spheroids , and actsurvival ,57,58,59 lactate ; the latIn summary, both NSCLC and PDAC cancer cell spheroids displayed much higher resistance to anti-cancer selenite toxicity than their 2D cell counterparts in terms of cell growth, which was associated with attenuated response of growth- and survival-requiring central metabolic activities. This resistance can be attributed, at least in part, to the presence of a resistant cell population, presumably as a result of cell–cell and cell–matrix interactions in the spheroids. These interactions also led to reduced intrinsic capacity of UDPGlcNAc synthesis in A549 and PANC1 spheroids and activation of gluconeogenesis in PANC1 spheroids, which could be involved in modulating cancer spheroid metabolism and survival.50 for sodium selenite  in PANC1 cells were initially determined in 96-well plates and further determined in 6-well plates. For 6-well plate (Greiner Bio-one CELLSTAR® 657160), PANC1 cells were grown in DMEM , 2 g/L glucose, 2 mM l-glutamine, 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin) for two days (reaching 60~70% confluency) before titrating with 0, 2, 10, 20, 50, 200 μM sodium selenite for 26 h. At the end of the treatment, cell viability was determined by neutral red assay. In brief, cell media were aspirated and cells were incubated in 2 mL neutral red solution (33 μg/mL in DMEM) at 37 °C and 5% CO2 for 3 h. At the end of the incubation, neutral red solution was aspirated and cells were washed with 2 mL PBS twice. After removing PBS, cells were lysed in 1 mL lysis buffer containing 50% ethanol and 1% acetic acid in water. Lysates were transferred to a 96-well plate  and A540 was measured in a plate reader. Dose response curves were plotted using A540 with background subtraction and normalization to control cells. IC50 was deduced from curve fitting using SigmaPlot.IC2 before treating cells with NanoShuttlesTM-PL  at 1 µL/104 cells overnight according to the vendor’s protocol. NS-loaded cells were then trypsinized, counted, and seeded in a cell-repellent round-bottom 384-well plate at 1000 cells per well. We have separately determined this cell seeding density to be within the linear range of spheroid growth as a function of cell seeding density. The plate was held below a 384-well spheroid drive (n3D) initially for 15 min in the biosafety hood and followed by 45 min at 37 °C/5% CO2 for spheroid formation. The drive was then removed and spheroids were allowed to grow for 3 days before medium change to DMEM containing 0 to 500 µM selenite. Spheroid growth was assayed daily using the PrestoBlue® live cell fluorescent stain reagent  [® reduction was followed for 30 min at 3–5 min intervals for each well in the 384-well plate using a Cytation 3 imaging microplate reader  set at 37 °C with 5% CO2. The slope of the fluorescence versus time was used to plot the selenite dose-response curve  as the Hill equation or the Hill equation modified to two components (sensitive and resistant).min is the plateau value at high concentrations, c, of the inhibitor and n is the Hill coefficient. For biphasic curves, this equation was modified as follows:A549 or PANC1 cells were grown in 6-well plates to 50–70% confluence in DMEM growth medium at 37 °C/5% COMA, USA) , which eurve cf. A, from w2D cell experiments were performed in 10-cm plates with cells grown to 50–70% confluence in DMEM growth medium.5) cells were seeded on 10-cm plates and cultured in DMEM growth medium using dialyzed, exosome-depleted FBS. Exosome-depleted FBS was prepared by ultracentrifugation at 120,000× g for 2 h followed by filtration through a sterile 0.22 μm vacuum filter. Four days after seeding, media were replaced with fresh media containing either 0.2% 12C6-Glc (unlabeled) or 13C6-Glc, without or with 10 μM SeO3 in triplicates. Aliquots of 200 μL media were collected at 0, 12 and 24 h after treatment started and spun at 3500× g for 15 min at 4 °C to remove debris. Aliquots of 100 μL supernatants were subject to protein precipitation by mixing with 400 μL cold acetone, incubating at −80 °C for 30 min before centrifugation at 14,000× g for 10 min at 4 °C. Supernatants were aliquoted and lyophilized for NMR analysis. Such acetone extraction method efficiently precipitated proteins and recovered polar metabolites from the media, similarly as the 10% trichloroacetic acid method [v/v/v) as described previously [PANC1 . The method efficiently quenches the hydrolysis of high-energy metabolites as evidenced by the high energy charge ratios calculated for A549 (ca. 0.9) and PANC1 (0.98) spheroids. The polar extracts were lyophilized overnight and subjected to IC-UHR FT-MS analysis. The cell residues were extracted and analyzed for proteins as described above. Polar metabolites from the media were extracted in 80% acetone as described above.To prepare spheroid cultures for SIRM experiments, 464 µL of NanoShuttlesm/z 200) on MS1 acquisition to capture all 13C isotopologues. The chromatograph was outfitted with a Dionex IonPac AG11-HC-4 µm RFIC&HPIC (2 × 50 mm) guard column upstream of a Dionex IonPac AS11-HC-4 µm RFIC&HPIC (2 × 250 mm) column. Chromatography and mass spectrometric settings were the same as described previously [m/z range of 80 to 700. Metabolites and their isotopologues were identified by chromatographic retention times and their m/z values compared with those of the standards. Peak were integrated and the areas exported to Excel via the TraceFinder 3.3  software package. Peak areas were corrected for natural abundance as previously described [13C incorporation into various metabolites.Ion chromatography-ultra high-resolution Fourier transform-MS (IC-UHR FT-MS) was performed as previously described . Brieflyeviously  with an escribed , after w2O containing 8.81 nmoles of d6-DSS  for 1D 1H-NMR analysis using a 1.7 mm inverse triple-resonance HCN cryoprobe on a Bruker AVANCE III NMR at 16.45 T . A 1H 90° pulse with solvent presaturation was used to acquire 1D 1H spectra with a 8403 Hz spectral width, 2 s acquisition time, 4 s relaxation delay, and 512 transients. The free induction decays were Fourier-transformed with 1 Hz line-broadening and zero-filled to 131,072 points. The “peak picking” routine in the MestReNova software  was used to quantify the 13C satellites of the methyl resonance of lactate, which represented 13C3-lactate based on the splitting patterns. The lactate peak areas were calibrated for nmoles against that of the methyl resonance of d6-DSS and normalized to the cell protein content. The final 13C3-lactate content in the media was expressed as mmole/g protein.Lyophilized medium extracts were redissolved in 35 µL D® rabbit mAb . Slides were mounted in Prolong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific P36935) before imaging. Images were acquired at 40× magnification using a laser scanning confocal microscope Olympus Fluoview™ FV1200.3D Spheroids were suspended in HistoGel (Thermo Scientific HG-4000-012). After solidification, HistoGel pellets were fixed in 10% neutral buffered formalin for 24 h, followed by paraffin embedding, sectioning at 4 μm thickness, and slide mounting. The slides were deparaffinized, rehydrated, subjected to heat-induced epitope retrieval (hier) by microwaving in sodium citrate buffer  for 10 min at a sub-boiling temperature. For immunofluorescence analysis, slides were treated with AlexaFluor™ Tyramide Superboost™ kits  as per manufacturer’s instructions, followed by primary and secondary antibody treatments as described previously . The folm/z 96) following collision with oxygen flowing at 50% with integration time of 1 s. Se concentrations in the digests were calculated from a standard curve of sodium selenite (Na2SeO3) in 5% nitric acid at 5 concentrations from 0.1 to 10 ppm.2D and 3D cells were cultured in 6-well plates, as described in 4.2 and 4.3. A549 cells (passage 15) were treated with 6.25 µM (2D) or 10 µM selenite (3D) for 24 h with 200 µL media sampled at 0 and 24 h after selenite treatment. Both 2D and 3D PANC1 (passage 20) cells were treated with 10 µM selenite for 24 h and media sampled as described above. Then 10 µL aliquots of media were digested in triplicate in 0.5 mL concentrated nitric acid  at 150 °C for 10 min with maximal pressure of 200 psi and power of 150 W using Discover SP microwave digestor . At the end of 24 h treatment, cells were washed in cold PBS, quenched in cold 70% methanol, and extracted for polar metabolites as described above. The protein pellets were then extracted using SDS/Tris buffer and protein concentrations were measured by microBCA assay as above. Both polar and protein extracts were aliquoted in duplicates and lyophilized. The residue pellets were dried in a Vacufuge (Eppendorf) and pre-digested in 100 µL nitric acid overnight. All lyophilized extracts and predigested residue pellets were digested in 250 µL concentrated nitric acid as above and the final digests were diluted to 5% nitric acid with nanopure water. ICP-MS was preformed using an 8800 Triple Quadrupole ICP-MS  with a micromist nebulizer. Selenium was monitored in MS/MS mode at the second quadrupole as the oxide (SeO,


The specific reaction of aldehyde and amino groups was used to attach lysozyme aptamers with specific sequences to NH2-Fe3O4 NPs to produce Apt-NH2-Fe3O4 NPs. The synthesized materials were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), hysteresis loop analysis, and thermogravimetric analysis (TGA). The optimal experimental conditions for adsorption of lysozyme were investigated. The effects of initial lysozyme concentration, adsorption time, pH, reaction temperature, and ionic strength were determined. The maximum adsorption capacity and relevant activity of Apt-NH2-Fe3O4 NPs was 460 mg·g−1 and 16,412 ± 55 U·mg−1 in an aqueous lysozyme solution. In addition, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis analysis, lysozyme could be separated from crude fresh egg white using Apt-NH2-Fe3O4 NPs with an amount up to 113 ± 4.2 mg·g−1 and an activity up to 16,370 ± 46 U·mg−1.Lysozyme is in high demand due to its many favorable characteristics such as being naturally occurring, non-toxic, and easy to digest and absorb. Recently, superparamagnetic nanoparticles with strong magnetic responsiveness have attracted significant interest for enzyme purification. The aptamer of the enzyme can be chemically synthesized rapidly at a large scale using simple and low-cost preparation methods. Therefore, Fe N-acetyl glucosamine in mucopolysaccharides [Lysozyme is an alkaline enzyme mainly present in animals, plants, and microbes. It is usually derived from egg whites and it can damage bacterial cell walls by catalyzing the hydrolysis of 1,4-b-linkages between muramic acid and charides ,3. Therecharides ,6,7. TheRecently, magnetic nanoparticles have attracted significant interest in a wide range of applications, such as cell separation, enzyme immobilization, and targeted drug administration ,9,10. MaAptamers are single-stranded DNA or RNA sequences, typically 15–40 bases in length, that are selected from a library of synthetic nucleotide combinations by SELEX  techniques . The apt3O4 NPs were prepared by a chemical co-precipitation method and were then functionalized with amino groups using 3-aminopropyltriethoxysilane (APTES) to produce NH2-Fe3O4 NPs. The lysozyme aptamer was then attached to the NH2-Fe3O4 NPs using a specific reaction of the aldehyde and amino groups to produce Apt-NH2-Fe3O4 NPs. Different adsorption conditions were tested to determine the optimal conditions for the adsorption of lysozyme in water to the prepared nanomaterials. The variables tested included initial lysozyme concentration, adsorption time, pH, reaction temperature, and ionic strength. The Apt-NH2-Fe3O4 NPs were then used for the separation of lysozyme from crude fresh egg whites.Herein, we report a new method for purification of lysozyme that exploits a selective adsorption process by magnetic nanoparticles with a lysozyme sequence aptamer . Magneti2·4H2O), ferric chloride hexahydrate (FeCl3·6H2O), ammonia (25%), sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), toluene, and anhydrous ethanol were purchased from Beijing Chemical Factory Reagent Company . A phosphate buffer solution (PBS) was prepared using NaH2PO4 and Na2HPO4. The lysozyme-specific aptamer sequences with aldehyde modification  were purchased from the Lianxing Biotechnology Company . Ultra-pure water with 18.2 MΩ·cm resistivity was used in all experiments. All chemicals were used as received without any further purification.Lysozyme was purchased from Sigma . 3-aminopropyltriethoxysilane (APTES), ferrous chloride tetrahydrate (FeCl3O4 NPs:Synthesis of magnetic Fe FeCl2·4H2O (3 g) and FeCl3·6H2O (9.1 g) were mixed in 200 mL of distilled water that had previously been purged with nitrogen and the solution was stirred for 10 min. After adding 85 mL of ammonia (25%), the solution was stirred at 25 °C for 30 min and then heated to 70 °C with continual stirring for 30 min with nitrogen purge. The reaction products were collected with a magnet after the reaction system had cooled to room temperature and were repeatedly washed with distilled water to remove unreacted material. Finally, the products were freeze-dried and magnetic Fe3O4 NPs were obtained [obtained .2-Fe3O4 NPs:Synthesis of magnetic NH The surfaces of Fe3O4 NPs were coated with APTES using a silanization reaction. After dissolving 4 g of magnetic Fe3O4 in 50 mL of anhydrous toluene, the solution was homogenized using ultrasonic waves for 15 min and then 22 mmol of APTES was added via syringe. Nitrogen gas was then introduced into the reaction system, which was stirred for 5 h at 25 °C. After the reaction was completed, the reaction products were collected with a magnet, washed repeatedly with ethanol, and dichloromethane and freeze dried. The NH2-Fe3O4 NPs were then obtained.2-Fe3O4 NPs with lysozyme aptamers (Apt-NH2-Fe3O4 NPs):Synthesis of magnetic NH NH2-Fe3O4 NPs (1 g) were dissolved in PBS solution  containing 10 nM lysozyme aptamer and the mixture was shaken at 200 rpm for 6 h at 35 °C. The reaction products were separated using a magnet, washed repeatedly with distilled water, and freeze dried. The magnetic Apt-NH2-Fe3O4 NPs were then obtained.3O4 NPs were investigated by transmission electron microscopy . Samples were prepared for TEM observation by ultrasonic dispersion. Take some powder sample into a centrifuge tube with ethanol and ultrasonically dispersed into a suspension. Then, a few drops of the suspended droplets were taken and dropped on a copper mesh (3 mm in diameter) using a capillary and dried to obtain a powder sample observed by an electron microscope. X-ray diffraction  analysis was performed using the Bruker High-Resolution D8 Advance XRD unit with a Ni-filtered CuK (λ = 1.54 Å). Magnetization performance of the magnetic Apt-NH2-Fe3O4 NPs was characterized by measuring the hysteresis loop . The material was demagnetized before measuring the hysteresis loop and then the sample was magnetized to saturation, the magnetizing current was evenly reduced, and the coordinates of the hysteresis loop corresponding to different magnetization currents were recorded. The magnetic content of the Apt-NH2-Fe3O4 NPs was measured using thermogravimetric analysis (TGA) with the following test conditions: rate of nitrogen purge, 100 mL·min−1; rate of temperature rise, 20 °C·min−1 and temperature range, 30–900 °C. The functional surface groups of Apt-NH2-Fe3O4 NPs were characterized by Fourier transforms infrared spectroscopy . Samples were prepared by the KBr tablet method. The KBr was dried at 140 °C for 5 h before the measurement and the ratio of the powder sample to the KBr powder was 1:100. The mixing sample was fully ground and compressed, which was used for infrared measurement and the spectrum range was 400 to 4000 cm−1. The corresponding potential values of Apt-NH2-Fe3O4 NPs were calculated by measuring the potential values at different pH conditions using a zeta potential meter . The magnetic Apt-NH2-Fe3O4 NPs were dispersed in distilled water by ultrasonic treatment and the pH values of the samples were adjusted with HCl and NaOH solutions. The morphology and particle size of the magnetic Fe2-Fe3O4 NPs, 10 mg of material and 4 mL of a specific lysozyme buffer solution were mixed in a 5 mL centrifuge tube. Experiments were repeated at different lysozyme concentrations (0.25–5.0 mg·mL−1) and at various pH values (4.0–11.0). Adsorption experiments were performed at a stirring speed of 180 rpm for different times. The magnetic nanoparticles were separated from the supernatant using a magnet after the adsorption equilibrium was reached and were then washed with deionized water to remove any lysozyme that was not completely adsorbed onto the materials. The supernatant was analyzed by measuring its absorbance at 280 nm , and the amount of lysozyme adsorbed was calculated by determining the lysozyme concentration in the supernatant, according to the following equation:q is the adsorption capacity of Apt-NH2-Fe3O4 NPs to lysozyme (mg·g−1), C0 is the initial lysozyme concentration in the solution (mg·mL−1), eC is the lysozyme concentration at equilibrium (mg·mL−1), m is the amount of Apt-NH2-Fe3O4 NPs (g), and V is the volume of the lysozyme solution (mL).To measure the adsorption equilibrium of magnetic Apt-NH2-Fe3O4 NPs was added to 1 mL of the lysozyme separation solution and the mixture was stirred at 180 rpm for 60 min at 37 °C. The magnetic nanoparticles were then separated by a magnet and washed several times with deionized water to remove unabsorbed substances. The collected magnetic nanoparticle adsorbed with lysozyme was stored at −20 °C for the next elution test and further assayed for the activity of the isolated lysozyme. The supernatant was analyzed by measuring its absorbance at 280 nm and the amount of lysozyme adsorbed was calculated by determining the lysozyme concentration in the supernatant.Egg white liquid was initially prepared according to a previously published method . The egg2-Fe3O4 NPs were subjected to a desorption test. The nanoparticles were added to a glycine solution  and the eluate was collected and analyzed for concentration, purity, and enzyme activity. After separating lysozyme from egg white, the Apt-NHThe purity of the eluate was measured using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 12% polyacrylamide separation gel and an 8% polyacrylamide concentration gel.450) was adjusted to 1.3 by diluting the bacterial solution in PBS . After dissolving 10 mg of lysozyme in 1 mL of PBS , it was then diluted to 100 U·mL−1 and 0.5 mL of this standard enzyme solution was added to 2.5 mL of the aforementioned bacterial solution. The decrease in absorbance per minute was measured using a microplate reader. Lysozyme activity was calculated using the equation below:The freeze-dried bacterial strains were first resuscitated and activated. Then, their optical density at 450 nm , (311), (400), (422), (511), (440) that were present at 30.49, 35.66, 43.25, 53.65, 57.32, 63.02, respectively, which indicated that the Fe3O4 NPs were synthesized successfully [3O4 NPs did not change after amino functionalization and grafting of lysozyme aptamers. It was proved that the good crystal structure and good magnetic properties of the nanoparticles were maintained, thus ensuring the magnetic separation of the adsorbent in the experiment. As shown in −1. The characteristic absorption peak of Fe–O at 599 cm−1 was seen, indicating that Fe3O4 NPs were successfully synthesized. The stretching vibration peaks of the Si–O bond were observed at 1048 cm−1 and 1095 cm−1, indicating that a Si–O bond existed on the surface of the NH2-Fe3O4 and Apt-NH2-Fe3O4 NPs. The stretching vibration peaks of the C–N bond were observed at 1428 cm−1 and a vibration peak associated to imine formation between aldehyde of aptamer and NH2 is visible at 1625 cm−1, indicating that aptamer functionalized on the Apt-NH2-Fe3O4 NPs. The vibration peaks observed at 3440 cm−1 on Fe3O4 NPs may be attributed to –OH. The significant vibration peaks observed at 3420 cm−1 and 2980 cm−1 represented –NH and –CH2, indicating that Fe3O4 NPs were successfully functionalized with amino groups by APTES, and which demonstrated that silica was successfully encapsulated on the surface of Fe3O4 NPs. This can prevent agglomeration of the magnetic nanoparticles.As shown in 3O4, NH2-Fe3O4, and Apt-NH2-Fe3O4 NPs, respectively. The process of weight loss of Fe3O4 NPs could be divided into two relatively independent processes. The first weight loss process took place at 50–200 °C and the weight loss of 0.8%, mainly due to the evaporation of water. The second process occurred at 200–650 °C with a weight loss of 1.9%, mainly due to the decomposition of the surface groups of the Fe3O4 NPs. The TGA curve of NH2-Fe3O4 NPs could be divided into two relatively independent processes. The first weight loss process took place at 50–210 °C and the weight loss was 1.4%, which was mainly due to the decomposition of the surface groups on the magnetic nanoparticles. The second process took place at 210–650 °C with a weight loss of 6.5%, mainly due to the decomposition of the surface groups of the NH2-Fe3O4 NPs. The TGA curve of the Apt-NH2-Fe3O4 NPs was shown in 3O4 content of the magnetic core was about 89.4%. These magnetic nanoparticles contained higher Fe3O4 content and stronger magnetic force.As shown in 2-Fe3O4 NPs, from which it was observed that the magnetic nanoparticle had a magnetic saturation of 70.9 emu·g−1 and the magnetic nanoparticle particles did not exhibit coercivity with an applied magnetic field. As shown in 2-Fe3O4 NPs is shown in 2-Fe3O4 NPs, the initial lysozyme concentration, absorption time, pH value, temperature, and ionic strength were evaluated. The effect of initial lysozyme concentration was shown in −1. This concentration maximum could be attributed to the saturation of binding sites on the Apt-NH2-Fe3O4 NPs. The effect of adsorption time on the lysozyme immobilization was studied at a lysozyme concentration of 4.0 mg·mL−1 and the temperature was 37 °C. The experimental results showed that the adsorption time reached the maximum at 60 min  of the two models.Adsorption isotherms: The adsorption behavior of a lysozyme solution is usually described using the Langmuir and Freundlich isotherm models. The most suitable isotherm model for the data in this study was determined by comparing the correlation coefficients 1qe=1eC is the concentration of lysozyme in the solution (mg·mL−1), eq is the amount adsorbed at the concentration equilibrium state (mg·g−1), maxq is the maximum adsorption capacity at equilibrium (mg·g−1), and b is the adsorption constant.The Freundlich isotherm model assumes heterogeneity of adsorption surfaces and is expressed by the following equation :(4)lnqe=2-Fe3O4 NPs. The parameters of Langmuir and Freundlich are shown in the r2 obtained from the Langmuir model was slightly higher than that obtained from the Freundlich model, indicating that lysozyme adsorption by Apt-NH2-Fe3O4 NPs may be explained using the Langmuir model.2-Fe3O4 NPs, this experiment was investigated. According to the experimental conditions optimized in the above experiments, we weighed 10 mg of magnetic Fe3O4 nanoparticles and 4 mL of 4 mg·mL−1 lysozyme solution in a 5 mL centrifuge tube at 37 °C and shook it for 120 min at 180 rpm. The kinetic models can be used in this case assuming that the measured concentrations are equal to the adsorbent surface concentrations [tq is the adsorption amount at different times (mg·g−1), q1 is the equilibrium adsorption amount obtained by fitting (mg·g−1), and k1 is the first-order reaction rate constant (min−1).Adsorption kinetics modeling: In order to examine the controlling mechanism of the lysozyme adsorption process based on Apt-NHtrations . The quatq is the adsorption amount at different times (mg·g−1), q2 is the equilibrium adsorption amount obtained by fitting (mg·g−1), k2 is the second-order reaction rate constant (g·mg−1·min−1), and k2q22 is the initial adsorption rate (mg·g−1·min−1).The quasi-second-order kinetic model is also commonly used to describe and analyze the adsorption of lysozyme. From this model, it is possible to deduce whether the surface reaction is a rate control step. It is represented by the following equation :(6)tqt=1r2 value of the quasi-secondary model (0.9921) was compared with the quasi-first-order model (0.8545) was larger and closer to 1, and the experimental values (460 ± 3.4 mg·g−1) of the equilibrium adsorption amount and the calculated values q2 of the quasi-secondary model agreed well, indicating that the quasi-secondary model is better to predict the adsorption kinetics of lysozyme on Apt-NH2-Fe3O4 NPs.The fitting curves of the quasi-first-order and quasi-second-class models are shown in 2-Fe3O4 NPs: As seen in 2-Fe3O4 NPs. This indicated that the lysozyme concentration in egg white was high and that the magnetic Apt-NH2-Fe3O4 NPs could separate lysozyme from the egg white (−1, and the activity of the separated lysozyme was 16,370 ± 46 U·mg−1. The conventional separation/purification methods for lysozyme are ultra-filtration, precipitation, and chromatography. We present a simple one-step purification of lysozyme from whole chicken egg white samples. The resulting Apt-NH2-Fe3O4 NPs possessed an excellent adsorption and purification of lysozyme. It is easy to recycle and can be reused many times.Separation of lysozyme from egg white using Apt-NHgg white d. In add−1 according to the difference in absorbance before and after the reaction. The activity of the separated lysozyme from egg crude was 16,370 ± 46 U·mg−1.The absorbance value at 450 nm was measured using a microplate reader. Standard enzyme activity was calculated to be 18,400 ± 53 U·mg3O4 NPs were successfully synthesized and were functionalized with amino and lysozyme aptamers to finally obtain Apt-NH2-Fe3O4 NPs for the separation of lysozyme. The resulting Apt-NH2-Fe3O4 NPs possessed an excellent adsorption of lysozyme in water, with a maximum adsorption capacity of 460 ± 3.4 mg·g−1 and activity of the immobilized lysozyme was 16,412 ± 55 U·mg−1 under the optimal conditions. The optimum initial lysozyme concentration, adsorption time, pH, and reaction temperature were 4 mg·mL−1, 60 min, 9.0, and 37 °C, respectively. Furthermore, the lysozyme adsorption process using Apt-NH2-Fe3O4 NPs could be explained using the Langmuir model and a quasi-first-order kinetic model. In addition, Apt-NH2-Fe3O4 NPs could be successfully used to separate lysozyme from egg white with an amount up to 113 ± 4.2 mg·g−1, and the activity of the separated lysozyme was 16,370 ± 46 U·mg−1. The isolated lysozyme showed high activity and purity. Its purity was demonstrated using SDS-PAGE electrophoresis analysis. The method described here for adsorption and separating enzymes is simple, specific, mild, uses magnetic separation, and has potential in the food field.To summarize, magnetic Fe


The obtained CC-CNT@NiO cathode exhibits a high capacity of 527 mAh·g−1 at 2 A·g−1 and an excellent rate capability with a capacitance retention of 78% even at 40 A·g−1. The all-solid-state asymmetric supercapacitor fabricated with these two electrodes delivers a high energy density of 63.3 Wh·kg−1 at 1.6 kW·kg−1 and retains 83% of its initial capacitance after 5000 cycles. These results demonstrate that our simple aqueous reduction method to combine CNT and metal oxides reveals an exciting future in constructing high-performance supercapacitors.We have successfully prepared iron oxide and nickel oxide on carbon nanotubes on carbon cloth for the use in supercapacitors via a simple aqueous reduction method. The obtained carbon cloth–carbon nanotube@metal oxide (CC-CNT@MO) three-dimensional structures combine the high specific capacitance and rich redox sites of metal oxides with the large specific area and high electrical conductivity of carbon nanotubes. The prepared CC-CNT@Fe Transition metal oxides such as RuO2, MnO2, NiO, and Fe2O3 [2O3 have attracted considerable attention. Fe2O3 is attractive for its low cost, abundance, nontoxicity, and eco-friendliness [2O3 composites have been achieved. For example, Guan et al. deposited iron oxide on CNTs by atomic layer deposition (ALD) and the obtained CNTs@Fe2O3 presented a specific capacitance of 580.6 F·g−1 at 5 A·g−1 [2O3@C structures, and the composite exhibited a specific capacitance of 787.5 F·g−1 at 5 mV·s−1 [Supercapacitors offer long cycling life, superior charge–recharge ability, high power density, and wide operating temperature –3. Howevnd Fe2O3 –15 have nd Fe2O3 . To addrnd Fe2O3 . Among tndliness –20. Some 5 A·g−1 . Zhang e5 mV·s−1 . These m2O3 on a carbon cloth–carbon nanotubes (CC-CNT) substrate with NaBH4 as reductant and ferric chloride as the reactant. NaBH4 is an active reducing agent with which most metal chlorides can be reduced to metal [Considering all the above issues, we proposed a simple solution-processing method to synthesize Feto metal –24 and tto metal .2O3-coated CNT on carbon cloth (CC-CNT@Fe2O3) as anode and NiO-coated CNT on carbon cloth (CC-CNT@NiO) as cathode were prepared. CNTs significantly improved the conductivity and enhanced the capacity of Fe2O3 up to 226 mAh·g−1 at 2 A·g−1, and capacity of NiO to 527 mAh·g−1 at 2 A·g−1. Furthermore, by assembling the two electrodes, an asymmetric supercapacitor (ASC) with a high energy density of 63.3 Wh·kg−1 at 1.6 kW·kg−1 was obtained. Besides, the ASC exhibits excellent cycling stability with capacitance retention of 83% after 5000 cycles. All these results demonstrate that our method is promising for constructing CNT-based metal oxide composites for high-performance supercapacitors.Through aqueous reduction, Fe2 is grey, it becomes black after CNT growth and finally yellow after Fe2O3 growth. The mass loading of Fe2O3 can be adjusted through the concentration of FeCl3. As shown in 2O3. The morphology of the as-obtained CC-CNT@Fe2O3 is shown in 2O3 coats all carbon fibers well. The space between CNTs provides enormous room for Fe2O3 growth, and the obtained CNT@Fe2O3 are entangled with each other and sticky to the CC  pore size distribution of CC-CNT@Fe2O3 are shown in 2·g−1, much larger than that of pure carbon cloth (0.2 m2·g−1). The pore size distribution . The EDX spectrum confirms the existence of C, O, and Fe elements . As shown in o the CC . N2 adsoEM image  indicate2O3 (JCPDS Card No. 25-1402). The samples show the same XRD features at different mass loadings  test yields the same result  at current densities of 2, 5, 10, 20, 30, and 40 A·g−1, respectively  was carried out to explore the resistance of the electrode . Therefore, the EIS results confirm the good conductivity of CC-CNT@Fe2O3 3D structures, which contributes much to the superior electrochemical performance of the CC-CNT@Fe2O3 anode.the slope can be determined to be about 0.58, indicating a battery-like behaviour of Fe2O3 . We alsoectively , indicatlectrode . The mea2O3. As shown in 2 adsorption–desorption isotherm and BJH pore size distribution of CC-CNT@NiO are shown in 2·g−1, much larger than that of pure carbon cloth (0.2 m2·g−1). Pore size distribution  at current densities of 2, 5, 10, 20, 30, and 40 A·g−1, respectively  as the electrolyte, and a cellulose membrane (140 μm thick) as the separator. To assemble the device with the best performance, charge balance between the positive and negative electrodes were considered by adjusting the mass loading of the active materials and verified by the areal ratio of CV at the same scan rate of 20 mV·s−1 (2O3 vs 0.23 mg NiO), CV curves of the ASC at different voltage windows were measured to find out the optimal operating voltage window. As shown in 2O3 and CC-CNT@NiO electrodes. When the scan rate increases from 2 to 100 mV·s−1, the CV shapes are still maintained, suggesting the ultrafast charge–discharge ability and excellent reversibility of the device. The GCD curves of the device measured at 2, 5, 10 and 20 A·g−1 are shown in Figure S10b , respectively. Cycling stability of the device was conducted at 10 A·g−1 for 5000 cycles. As shown in −1 at 1.6 kW·kg−1 and retains 25.2 Wh·kg−1 at a high power density of 16.2 kW·kg−1. These values are superior to the previously reported NiO-based or Fe2O3-based solid-state ASCs [2O3 electrodes. Therefore, the rationally designed CNT@metal oxide 3D structures made by the aqueous reduction method are promising for constructing high-performance supercapacitors.An all-solid-state asymmetric supercapacitor (ASC) was constructed with CC-CNT@Feure S10b . An obvi20 A·g−1 . The speate ASCs –45. Thes2O3 and NiO were individually synthesized on carbon nanotubes on carbon cloth by a simple aqueous reduction method. The prepared negative CC-CNT@Fe2O3 and positive CC-CNT@NiO electrodes both show high capacity, excellent rate capability, and high conductivity. Additionally, an all-solid-state asymmetric supercapacitor with CC-CNT@NiO as the positive electrode, CC-CNT@Fe2O3 as the negative electrode, and PVA/KOH gel as the electrolyte delivered a high energy density of 63.3 Wh·kg−1 at 1.6 kW·kg−1. Moreover, the device exhibits superior cycling stability with 83% retention of initial capacitance after 5000 cycles. The outstanding electrochemical performance of the device stems from the rationally designed CNT@metal oxide 3D structures. More importantly, our simple method provides a new strategy for constructing CNT@metal oxide composites for high-performance supercapacitors.FeAll the reagents were of analytical grade and used without further purification. The preparation of the positive and negative electrodes and the device structures are illustrated in 2, 11 mg) was treated with a mixture of H2SO4/HNO3 (3:1 volume ratio) at 70 °C for 2 h and then cleaned by sonication in deionized water and finally kept in a drying oven for 12 h. Afterward, the CC was immersed in catalyst, which consists of 10 mM Ni(NO3)2 and 1 mM Al(NO3)3 in alcoholic solution before CVD treatment. Flow rates of C2H2, H2, and Ar were set to 10, 20, and 50 sccm. The growth time was 30 min at 700 °C, and the mass loading of CNTs was 1.30 to 1.70 mg·cm−2. The as-prepared CNTs on CC (CC-CNT) was subsequently treated by oxygen plasma to make it hydrophilic and then immersed in diluted hydrochloric acid to remove residual catalyst and finally rinsed by deionized water and dried in an oven.Carbon nanotubes (CNTs) were grown on carbon cloth (CC)  by CVD with a pretreatment of the CC. Typically, the CC  aqueous solution for 12 h, and then dried at 70 °C in drying oven for 2 h, and transferred into NaBH4 (0.1 M) aqueous solution for 6 h at room temperature afterwards. Finally, the obtained samples were washed by deionized water and dried in a drying oven for 12 h. CC-CNT@NiO: The NiO was coated on the CC-CNT by the same process with NiCl2 (0.1 M) aqueous solution instead of FeCl3 (0.1 M).The morphologies and microstructures of the as-prepared samples were characterized by using a field-emission scanning electron microscope  and a transmission electron microscope  coupled with an energy dispersive X-ray spectrometer. Crystal structures were tested by X-ray diffraction . The vibrational information of chemical bonds of samples was characterized by micro-Raman spectroscopy . The element composition and chemical bonding of samples were examined by X-ray photoelectron spectroscopy . The pore size distribution was measured by the Barrett–Joyner–Halenda method (ASAP 2020).2O3 or CC-CNT@NiO as the binder-free working electrode. Electrochemical impedance spectroscopy (EIS) curves were measured in frequencies from 100 kHz to 0.01 Hz at open-circuit voltage with an AC voltage perturbation amplitude of 5 mV. The mass of the active material was weighed by a microbalance  with a tolerance of less than 0.01 mg.The half-cell electrochemical properties were tested in a standard three-electrode system at room temperature on an electrochemical work station . 2 M KOH aqueous solution was used as the electrolyte, a Pt plate as the counter electrode, a saturated calomel electrode (SCE) as the reference electrode, and the CC-CNT@FeFile 1Specific capacitance calculation and additional figures.File 2Video showing the ASC powering a blue LED for about 27 seconds.


Geotrichum candidum AS 2.361 was previously reported by our group as an active strain for the enantioselective reduction of ketones. Although some other Geotrichum strains were also found from the terrestrial sources, information on their stability and reusability is scarce. Herein, the stabilities—in terms of pH tolerance, thermostability, and storage stability, and reusability—of G. candidum AS 2.361 were described for the asymmetric reduction of a series of aromatic ketones. Two differently immobilized cells  as well as free cells were prepared. For three substrates (1-(3-bromophenyl) ethan-1-one (1b), 1-(2-chlorophenyl) ethan-1-one (1d), and acetophenone (1g)) immobilized cells on agar showed a great improvement in the bioreduction activities compared to the free cells, increasing yields up to 97% with ee values of 99%. Cells immobilized on agar/calcium alginate could maintain more than 90% of the original activities within the assayed pH ranges of 3.5–11, while free cells were highly sensitive to alkaline and acidic conditions. Concerning thermostability, immobilized cells on agar kept 99% of their original activities after incubation at 60 °C for 1 h, while almost no activity was detected for the free cells under the same condition. Immobilized cells were stable at 4 °C for 80 days without any activity loss, while free cells started to decrease the activity after storage at 4 °C for six days. The immobilized cells retained almost 99% activity after four reuse cycles, while free cells lost almost all the activities at on the third cycle.Marine-derived fungus  Although in both cases the enantioselectivity of 2i was not unsatisfactory, the inversion of the configuration of the (S)-alcohol [(S)-2i] (produced by the free cells) to the (R)-alcohol [(R)-2i] (produced by the immobilized cells) showed strong influence of the immobilization depending on the type of substrates and/or support materials used. The immobilization process may have brought about some changes in the physical properties, which favoured the action of (R)-selective carbonyl reductases rather than (S)-selective enzymes. Another possibility may be that the immobilization has activated another dehydrogenase which is different from the enzymes catalyzing the reduction in free cells [G. candidum AS 2.361 cells, with beneficial influence on the enzyme activity. In order to prove the usefulness and importance of applied procedures, the reaction was up-scaled to 1000 mL using 1a as represented substrate and immobilized cells on agar as represented biocatalysts under the same reaction conditions . Gratifyingly, in the preparative-scale reaction immobilization cells with agar catalyzed quantitatively substrate 1a to the desired product (S)-2a. The obtained results highlighted the potential industrial use of the applied procedure.The data presented in ee cells . HoweverG. candidum AS 2.361, we then turned our attention to its stabilities. Generally, buffer pH can affect the stability, and/or the activity/selectivity of an enzymatic reaction, that is, pH tolerance is a key factor for the stability of a biocatalyst. Fundamentally, the immobilization process could strongly enhance the stability of an enzyme. Thus, we are interested in the pH effect on the bioreduction activity of G. candidum AS 2.361 cells in the forms of both free cells and immobilized cells. Here, reactions were all performed in 50-mL screw-capped plastic vials to prevent evaporation of ketones/alcohols. The reaction mixture contained 3 g of free resting cells or 4.5 g of immobilized cells, 0.5 g of glucose in 10 mL given buffer (100 mM) with given given pH values between 3.5–11.0 , with the substrates of the final concentration of 10 mM. The solution was shaken at 25 °C for 24 h. As shown in 1a) to alcohol (S)-2a (yield >99%) within the assayed pH ranges of 3.5–9.0. When the buffer pH increased from 9.0 to 11.0, both immobilized cells catalyzed slightly less substrates 1a into the desired alcohols (S)-2a (yield 90%), corresponding to approximately 90% of the original activity at pH 3.5–9.0. By contrast, the free cells were highly sensitive to alkaline and acidic conditions. As illustrated in 1a, with the product yield of 99%. When the pH values continuously increased from 7.0 to 11.0, the catalytic activity of free cells dramatically decreased, and almost no activities were detected at pH 11.0. Remarkably, the reaction product ee remained above 99% in the measured pH ranges , with no significant changes of biocatalytic activities.Encouraged by the acceptable biocatalytic activities obtained from both free cells and immobilized cells of G. candidum AS 2.361 is one of the most important application criteria for different applications. Thus, the thermostability of G. candidum AS 2.361 cells was evaluated as well. Generally, the immobilized cells are superior to the free cells with respect to thermostability [2HPO4-KH2PO4 buffer , containing 0.5 g glucose and 10 mM substrate. The reaction mixture was shaken at 25 °C for 24 h. As shown in G. candidum 2.361 on both agar and calcium alginate were stable at 30 °C or 40 °C for 12 h, without any activities loss . Notably, the immobilized cells on calcium alginate kept >99% of their original activity after incubation at 50 °C for 1 h. When the temperature increased to 60 °C, a slight decrease in the activity  was detected for the immobilized cells on calcium alginate. Remarkably, the immobilized cells on agar still showed a retention of >99% of their original activity after incubation at 60 °C for 1 h. After 3 h of incubation at 60 °C, the immobilized cells on agar lost 49% of the initial activity, and the immobilized cells on calcium alginate lost 57% of the initial activity, but both lost their activity almost completely after incubation at 70 °C for 1 h. On the other hand, the results showed that the free cells were stable at 30 °C for 1 h, without any activity loss, but started to lose the activity significantly when the temperature increased to 40, 50, and 60 °C. After incubation at 60 °C for 1 h, almost no activity was detected for the free cells. From the above results obtained, it is clear that the activity and stability of the immobilized cells are more resistant against heat than that of free cells. In the case of free cells and immobilized cells of strain G. candidum, AS 2.361 derivatives such effect of temperature on changes in activity and stability can be related to the protein unfolding and thus to the enzyme denaturation. The results also indicated that the profile curves for both immobilizations on agar and calcium alginate shifted towards higher temperatures suggesting that between enzyme and the tested supports there is existing a strong interaction, enhancing the conformation stability of the free cells.The thermal stability of cells of tability . Thus, tG. candidum AS 2.361, both immobilized cells and free cells were stored in physiological saline  at 4 °C and tested for the activity in bioreduction of ketone 1a. Samples (3 g free cells and 4.5 g immobilized cells) were withdrawn at various time intervals  and added to 10 mL Na2HPO4-KH2PO4 buffer , containing 0.5 g glucose and 10 mM substrate to initiate the reaction. The reaction of the mixture was then shaken at 25 °C for 24 h. As shown in G. candidum 2.361 exhibited excellent storage properties, which is essential for its industrial application potential. It needs to emphasize that in the current study of storage stability; all the cells were stored in physiological saline  at 4 °C and were withdrawn at regular intervals for activity test. Effect of other factors such as storage solvent and storage temperature might cause the loss of activity and stability of cells of G. candidum AS 2.361, which should be considered for future studies.Storage stability is an essential factor for the practical application of (bio)catalysts . To inveG. candidum AS 2.361, reactions were performed to examine the recyclability of both the immobilized cells and free cells. Every reaction was performed in 50-mL screw-capped plastic vials to prevent evaporation of ketones/alcohols. The reaction mixture contained 3 g of free resting cells or 4.5 g of immobilized cells, 0.5 g of glucose in 10 mL Na2HPO4-KH2PO4 buffer , with the substrate 1a of the final concentration of 10 mM. The solution was shaken at 25 °C for 24 h. After the completion of the reaction, the cells were separated by centrifugation , washed twice with the same buffer ) and then reused in the next cycle under the same reaction conditions. As shown in 1a. The immobilized cells on agar showed high activity and complete conversion of substrate 1a for four cycles. Loss of 70% of initial activity was detected at the fifth cycle and almost complete loss of the activity was detected on the seventh cycle. The immobilized cells on calcium alginate showed high activity and enantioselectivity towards substrate 1a, without any loss of activity or enantioselectivity for three cycles. Losses of 64% and 85% and of the initial activity were observed for cycles four and five, whereas almost no activity (3.7% yield of desired product) was retained inthe seventh cycle. The free cells could maintain approximately 92% of their original activity during the second cycle but were almost deactivated in the third cycle. Notably, the recycling of the immobilized or free cells showed no significant impact on the enantioselectivities of the biocatalysts, as supported by the observation that the product ee remained consistently higher than 99.0% during each cycle of the reaction (data not shown). The results demonstrate the high repeated use by the immobilized cells, suggesting that the immobilized G. candidum AS 2.361 on agar or calcium alginate would be promising for application in actual industrial production. Biocatalysts (cells of G. candidum AS 2.361) inactivation in this specific case of changes in activity and stability becomes one of the greater problems when they are used as industrial catalysts. Inactivation of a cell starts by some reversible conformational changes, and finally the cell may also suffer some chemical modifications, aggregation, etc. That way, most strategies to stabilize cells are directed to the slowdown of these initial conformational changes. The conformational changes start by some weak point of the cell conformation and then get more generalized along the whole cell’s conformation until reaching full cell inactivation. However, it is not difficult to imagine that the weakest point of a cells conformation, or at least the way that the cells structure follows during inactivation, may be different under different inactivating conditions [Reusability is one of the most important characteristics of a (bio)catalyst for practical application . In the nditions .G. candidum AS 2.361 culture media components were purchased from Huankai Microbial .Chemicals used in the current study were all purchased from Sigma-Aldrich  and were used directly without any purification. 1H 500 and 13C 125 MHz, respectively,  and internally referenced to residual solvent signals. Data for 1H NMR are recorded in terms of chemical shift (d ppm), multiplicity , integration, coupling constant (Hz), and assignment. Data for 13C NMR are recorded as chemical shift. Optical rotations were measured at 20 °C on an MCP 300  (sodium D line). Column chromatography was performed with a silica gel , using petroleum ether (PE) and ethyl acetate (EtOAc) as eluents. Thin-layer chromatography (TLC) was performed on 0.20 mm silica gel 60-F plates. Organic solvents were removed using a rotary evaporator. Free cells of G. candidum AS 2.361 were obtained as described in a previous report by our group [Geotrichum candidum AS 2.361 used in this study was deposited and commercially available in Guangdong Culture Collection Center (http://www.gimcc.net/index.asp), which belongs to the World Federation for Culture Collections (Budapest Notification no. 309).NMR data were obtained with a Bruker Advance 500 instrument (ur group . Geotricw/v). After stirring thoroughly for 5 min, the mixture was transferred into a clean plate. The solution was allowed to solidify, forming a solid agar layer. The agar layer was cut into small pieces (3 × 3 × 3 mm3), and then stored in a phosphate buffer  at 4 °C after washing with distilled water.5 g of free resting cells were resuspended to 4 mL distilled water (1.25 g/mL). The resuspension was added to a 50-mL screw-capped glass vial containing 20 mL sterilized agar (5% 2 (4% v/v). The formed stable bead layers were kept in a 20–22 °C water bath for 2 h. The collected spherical particles was stored in CaCl2 (4%) solution at 4 °C after washing thoroughly with distilled water.5 g of free resting cells were resuspended to 10 mL distilled water (0.2 g/mL). The resuspension was added to a 50-mL screw-capped glass vial containing 5 mL sterilized sodium alginate (40 g/L) solution. After stirring for 5 min, the mixture was added dropwise into a sterilized solution of CaClAfter washing with water to remove the non-adhering support matrix, the immobilized cells were successively suspended in ethanol of different concentrations  for 15 min. The samples were air dried at room temperature in between. Using a Baltec MCS 010 model sputter , the washed immobilized cells were coated with 8–10 nm of gold by argon ion sputtering, before used for scanning electron microscopy on a Joel JMS 6480 LV computer (TECHCOMP(CHINA)LTD., Guangzhou, China).2HPO4-KH2PO4 buffer , with the substrates of the final concentration of 10 mM. The solution was shaken at 25 °C for 24 h. After separating from the cells by centrifugation, 2 mL of the obtained supernatant was saturated with NaCl, and extracted with n-hexane/i-PrOH . The obtained organic layer was dried over Na2SO4 and injected into HPLC for yield and ee measurement. The yield and enantioselectivity excess (ee) were recored on a chiral HPLC using a Shimadzu LC-10AT VP series and a Shimadzu SPD-M10Avp photo diode array detector (190–370 nm) with a Chiralcel AD-H column , flow rate: 0.5 mL/min, column temperature 25 °C). The retention time of substrates and desired products are summarized as below: 11.85 min for 1a, 12.71 min for (R)-2a, 13.23 min for (S)-2a; 10.66 min for 1b, 16.45 min for (R)-2b, 17.39 min for (S)-2b; 11.21 min for 1c, 16.83 min for (R)-2c, 17.99 min for (S)-2c; 10.71 min for 1d, 13.32 min for (R)-2d, 13.98 min for (S)-2d; 10.49 min for 1e, 14.60 min for (R)-2e, 16.35 min for (S)-2e; 10.78 min for 1f, 15.09 min for (R)-2f, 16.09 min for (S)-2f; 11.46 min for 1g, 15.93 min for (R)-2g, 17.93 min for (S)-2g; 10.94 min for 1h, 14.74 min for (R)-2h, 16.49 min for (S)-2h; 16.75 min for 1i, 20.98 min for (R)-2k, 24.71 min for (S)-2i; 16.42 min for 1j, 22.33 min for (R)-2j, 27.39 min for (S)-2j.Reactions were all performed in 50-mL screw-capped plastic vials to prevent evaporation of ketones/alcohols. The reaction mixture contained 3 g of free resting cells (0.3 g/mL) or 4.5 g of immobilized cells (0.45 g/mL), 0.5 g of glucose in 10 mL NaS)-2a, [αc 1.00, CHCl3), {lit.[S)-1-(2-bromophenyl)ethanol [αc 1.00, CHCl3)}; (S)-2b, [αc 1.00, CHCl3), {lit.[S)-1-(3-bromophenyl)ethanol [αc 1.00, CHCl3)}; (S)-2c, [αc 1.00, MeOH), {lit.[S)-1-(4-bromophenyl)ethanol [αc 1.07, MeOH)}; (S)-2d, [αc 1.00 in MeOH), {lit.[S)-1-(2-chlorophenyl)ethanone [αc 0.85 CHCl3)}; (S)-2e, [αc 1.00 in MeOH), {lit.[R)-1-(3-chlorophenyl)ethanone [αc 0.71, CHCl3)}; (S)-2f, [αc 1.00 in MeOH), {lit.[R)-1-(4-chlorophenyl)ethanone [αc 0.53 in Et2O)}; (S)-2h, [αc 1.00 in MeOH), {lit.[c 0.85, CHCl3)}; (S)-2i, [αc 0.05 in MeOH), {lit.[R)-2-chloro-1-phenylethanone [αc 1.00, CHCl3)}; (R)-2j, [αc 0.35 in MeOH), {lit.[R)-2-bromo-1-phenylethanone [αc 1.00 in CHCl3)}.The absolute configuration of products was assigned by measuring and comparing the optical rotation of isolated alcohols and those reported in previous studies ,34,35,363), {lit. (S)-1-(23), {lit. (S)-1-(3H), {lit. (R)-1- was dissolved in 1000 mL of Na2HPO4-KH2PO4 buffer  containing 50 g glucose and 450 g immobilzed cells of G. candidum AS 2.361 on agar. The reaction mixture was shaken at 25 °C with the speed of 220 rpm for 24 h. The cells were separated by centrifugation and the obtained supernatant (about 1000 mL) was saturated with NaCl and then extracted with ethyl acetate (2 × 1000 mL). The combined organic layers were dried with Na2SO4 and evaporated under reduced pressure. The crude product mixture was purified by flash chromatography on silica gel  to yield 1.90 g (95%) of a colourless oil (S-2a). The NMR data of isolated (S)-2a was in accordance with literature [1-(2-bromophenyl) ethanone . The obtained organic layer was dried over Na2SO4 and injected into HPLC for yield and ee measurement, as described above in Reactions were all performed in 50 mL screw-capped plastic vials to prevent evaporation of ketones/alcohols. The reaction mixture contained 3 g of free resting cells (0.3 g/mL) or 4.5 g of immobilized cells (0.45 g/mL), 0.5 g of glucose in 10 mL given buffer (100 mM) with given given pH values between 3.5–11 , with the substrates of the final concentration of 10 mM. The solution was shaken at 25 °C for 24 h. After separating from the cells by centrifugation, 2 mL of the obtained supernatant was saturated with NaCl, and extracted with 2HPO4-KH2PO4 buffer , containing 0.5 g glucose and 10 mM substrate. The reaction mixture was shaken at 25 °C for 24 h. The yields and enantioselectivity excess (ee) were obtained by a chiral HPLC, as described above in All tested cells including immobilized cells and free cells were first incubated at various temperatures (30–70 °C) for 1, 3, 6, 9, and 12 h, respectively, prior to the addition to the reaction mixture. Then, the cells (3 g free cells or 4.5 g immobilized cells) were resuspended to 10 mL Naw/v) at 4 °C and tested for the activity in bioreduction of ketone 1a. Samples (3 g free cells (0.3 g/mL) and 4.5 g immobilized cells (0.45 g/mL)) were withdrawn at various time intervals  and added to 10 mL Na2HPO4-KH2PO4 buffer , containing 0.5 g glucose and 10 mM substrate to initiate the reaction. The reaction of the mixture was then shaken at 25 °C for 24 h. The yields and enantioselectivity excess (ee) were obtained using a chiral HPLC, as described above in Both immobilized cells and free cells were stored in physiological saline , with the substrate 1a of the final concentration of 10 mM. The solution was shaken at 25 °C for 24 h. After the completion of the reaction, the cells were separated by centrifugation , washed twice with the same buffer ) and then reused in the next cycle under the same reaction conditions. 2 mL of the obtained supernatant was saturated with NaCl, and extracted with n-hexane/i-PrOH . The obtained organic layer was dried over Na2SO4 and injected into HPLC for yield and ee measurement, as described above in Every reaction was performed in 50 mL screw-capped plastic vials to prevent evaporation of ketones/alcohols. The reaction mixture contained 3 g of free resting cells (0.3 g/mL) or 4.5 g of immobilized cells (0.45 g/mL), 0.5 g of glucose in 10 mL NaG. candidum AS 2.361. To achieve this goal, two differently immobilized cells  and free cells were prepared for the asymmetric reduction of 14 aromatic ketones. The immobilization was characterized by FTIR and SEM, revealing that the cells were successfully entrapped on agar and calcium alginate. pH tolerance studies revealed that the immobilized cells gave 99% yield of the product (ee > 99%) at pH values in the range of 3–9, whereas the free cells exhibited an optimal activity at pH 7.0, yielding 99% of the desired product with >99% ee but were found to be very sensitive to alkaline and acidic conditions. Thermostabilities studies showed that immobilized cells on agar maintained 99% of the original activity during 1 h of incubation at 60 °C or 12 h of incubation at 40 °C. The immobilized cells maintained 99% of the original activity and enantioselectivity after storage at 4 °C for 80 days. Batch experiments showed that the immobilized cells on agar could be reused for four cycles without significant loss of activity and enantioselectivity. In summary, immobilized G. candidum AS 2.361 on agar or calcium alginate showed better acid–alkaline resistance, higher thermostability, better storage stability, and reusability as well as lower production costs compared to free cells. These results also indicated that immobilized G. candidum AS 2.361 can provide a solution for practical application and is a useful choice for the preparation of chiral alcohols.In this study, we reported the activity, stability  and reusability of a marine-derived fungus


Toxoplasma gondii can both inhibit and induce host cell apoptosis; however, the parasitic factors involved remain unclear. The T. gondii virulence factor ROP18 (TgROP18) has been reported to regulate host cell apoptosis; nevertheless, results for this regulation have been rarely reported or have provided contradictory findings. Human purinergic receptor 1 (P2X1) is an ATP-gated ion channel that responds to ATP stimulation and functions in cell apoptosis mediation. The precise roles of TgROP18 in T. gondii pathogenesis, and the relationship between TgROP18 and host P2X1 in host cell apoptosis are yet to be revealed.Apoptosis plays a critical role in the embryonic development, homeostasis of immune system and host defense against intracellular microbial pathogens. Infection by the obligate intracellular pathogen TgROP18 and the host P2X1 was measured by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation (co-IP) assay. Calcium influx and mitochondrial membrane depolarization were determined by FCM after JC-1 staining. The translocation of cytochrome C (Cyt C), Bax and Bcl2 proteins, expression of the apoptotic proteins PARP and caspase activation were detected by western blotting.Apoptosis rates were determined by flow cytometry (FCM) and TUNEL assay. The interaction between T. gondii strain  were significantly inhibited compared with their uninfected controls. TgROP18 inhibited ATP-induced apoptosis of SF268 with P2X1 expression, but had no effect on RAW264.7 or THP-1 cells without detectable P2X1 expression. It was further identified that TgROP18 interacted with P2X1, and overexpression of ROP18 in COS7 cells significantly inhibited cell apoptosis mediated by P2X1. Moreover, TgROP18 also inhibited P2X1-mediated Ca2+ influx, translocation of cytochrome C from the mitochondria to the cytosol, and ATP-triggered caspase activation.The apoptosis rates of glial or immune cells  infected by any Toxoplasma gondii infection inhibits ATP-induced host cell apoptosis, regardless of strain virulence and host cell lines. TgROP18 targets the purinergic receptor P2X1 of the SF268 human neural cells and inhibits ATP-induced apoptosis through the mitochondrial pathway, suggesting a sensor role for the host proapoptotic protein P2X1 in this process.The online version of this article (10.1186/s13071-019-3529-1) contains supplementary material, which is available to authorized users. Toxoplasma gondii, an obligate intracellular protozoan, can infect humans and nearly all warm-blooded animals [T. gondii [Toxoplasma gondii infection shows no or mild symptoms in immune competent hosts; however, the symptoms may be severe in immunocompromised patients and after congenital infections [T. gondii strains are categorized into the highly virulent type Ӏ (RH) strain with a lethal dose (LD) of one parasite, and non-virulent type II  and type III (CEP) strains with an LD50 of more than 1000 parasites [ animals . It is e. gondii . Toxoplafections . Based oarasites , 4.Toxoplasma gondii infection can both inhibit and induce host cell apoptosis. These opposing effects might involve complicated factors that modulate the finely balanced interaction between the parasite and the pro- and anti-apoptotic signals of the host, such as the host cell type, the virulence of T. gondii and others [Toxoplasma gondii ROP18 is a Ser/Thr kinase secreted by the rhoptries into the PV and host cytosol during invasion [TgROP18 [TgROP18 can degrade host cell proteins activating transcription factor 6 beta (ATF6β) or transcription factor P65 and help the parasite resist the host innate immune response [TgROP18 and its significance to T. gondii pathogenesis are yet to be established.d others . For exad others , while id others , 8. Apopd others . Toxoplainvasion . Among t[TgROP18 . TgROP18response , 13. Morresponse  and inhiresponse . HoweverT. gondii have evolved many ways to interfere with the highly regulated host cell apoptotic pathways [Apoptosis can be triggered by either a physiologic or a pathologic stimulus, exhibiting cytoplasmic shrinkage, chromatin condensation, nuclear fragmentation, plasma membrane blebbing, and finally formation of apoptotic bodies which are efficiently absorbed by neighboring cells with phagocytic activity and degraded within lysosomes . It playpathways , 21.Both immune response and apoptosis are triggered and regulated by signaling molecules. One of the signaling molecules is adenosine triphosphate (ATP). Inside a cell, ATP serves as a major energy source, whereas outside of the cell, ATP functions as a potent autocrine and paracrine signaling molecule for immune response –25. For + and Ca2+ influx and K+ efflux, which leads to depolarization of the plasma membrane [T. gondii is not known.ATP signaling is mediated by cell-surface P2 purinergic receptors, which consist of two families of receptors, P2X and P2Y receptors , 25. Themembrane . The celmembrane . WhetherT. gondii infection inhibited the ATP-induced apoptosis of SF268, THP-1 and RAW264.7 cells, regardless of strain virulence and host cell lines tested. Furthermore, we showed that TgROP18 targeted the purinergic receptor P2X1 of the human neural cells and inhibited ATP-induced apoptosis through the mitochondrial pathway.In this study, we found that in situ cell death detection kit and JC-1 mitochondria membrane potential detection kit were purchased from KeyGen ; protein A-agarose immunoprecipitation reagent and normal rabbit IgG from Santa Cruz Biotechnology ; HA-tag rabbit mAb, HA-tag mouse mAb, β-actin rabbit mAb, caspase-3 rabbit mAb, caspase-7 rabbit mAb, caspase-9 rabbit mAb and PARP rabbit mAb from Cell Signaling Technology ; Bax rabbit mAb, Bcl2 rabbit mAb, Cyt C rabbit mAb, P2X1 rabbit pAb and cytochrome C oxidase IV (COXIV) rabbit mAb from Abcam ; and DDDDK-Tag Mouse mAb from Abclonal .The apoptosis inducer ATP was purchased from Sigma-Aldrich , the P2X1 antagonist NF449 from Tocris Bioscience , Fluo-4 AM from Dojindo Laboratories , cell mitochondria isolation kit from Beyotime Biotechnology  and Lipofectamine® 3000 transfection kit from Thermo Fisher Scientific . An isothiocyanate (FITC)-Annexin V/propidium iodide (PI) apoptosis detection kit, TUNEL 2. The mouse monocyte/macrophage cell line RAW264.7 (ATCC), the Cercopithecus aethiops kidney fibroblast cell line COS7 (ATCC), and the human foreskin fibroblast cell line HFF (ATCC) were cultured in Dulbecco’s modified Eagle medium  supplemented with 10% (v/v) FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37 °C and 5% CO2.The human monocyte/macrophage cell line THP-1  and human glioblastoma cell line SF268 (ATCC) were propagated in Roswell Park Memorial Institute 1640 medium  supplemented with 10% (v/v) fetal bovine serum , 100 U/ml penicillin and 100 µg/ml streptomycin  at 37 °C and 5% COrop18, RH-ROP18-GFP-Flag, type II strain ME49 and type III strain VEG were cultured in HFFs in DMEM supplemented with 1% (v/v) FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37 °C and 5% CO2. Three to five days later, when most of the cells were ready to be ruptured by the tachyzoites, the cells were scraped and harvested by passing through a syringe multiple time. The tachyzoites were then purified by passing them through a 3-µm filter , pelleting at 2500× rpm for 10 min and resuspending in DMEM. The tachyzoites were counted with a hemocytometer.Type Ӏ strain RH, RH-Δ2. Before induction of apoptosis, cells were washed with phosphate buffered saline (PBS) to remove non-adherent parasites, and then the THP-1 cells, SF268 cells and RAW264.7 cells were all treated with 1 mg/ml ATP for 4 or 6 h, and the apoptotic level of the cells was analyzed by the AnnexinV-FITC/PI and TUNEL assay.Before infection, THP-1 were cultured in the presence of 100 ng/ml phorbol 12-myristate 13-acetate  for 24 h for differentiation into macrophages as described previously . THP-1, rop18 tachyzoites were separated from the host cells by centrifugation, then the SF268, THP-1 and RAW264.7 cells were infected with the RH-wild type and RH-Δrop18 tachyzoites at MOI = 13. Cells were washed with PBS at 12 h post-infection and then treated with 1 mg/ml ATP for an additional 12 h to induce apoptosis. The apoptotic levels of the cells were analyzed by AnnexinV-FITC/PI assay.RH-wild type and RH-Δ+/PI−, annexinV-FITC+/PI+ and annexin V-FITC-/PI+ quadrants were counted as apoptotic cells.The apoptotic levels of RAW264.7, THP-1 and SF268 cells were determined with annexin V- FITC/PI kit. Cells were briefly digested with 0.25% trypsin (Thermo Fisher Scientific) and washed twice with cold PBS. Then, they were resuspended in 500 μl of binding buffer, 5 μl of annexin V- FITC and 5 μl of PI and incubated at room temperature for 15 min in the dark. Annexin V-FITC binds to phosphatidylserine on the outer membrane of apoptotic cells, while PI enters and stains cells with compromised membrane integrity and then binds to and labels DNA. A flow cytometer  was used for data collection, and FlowJo software for data analysis. The FSC-H/FSC-A dot plot was used to exclude clumped cells to select single cells. The cells in the annexin V-FITCIn situ labeling of fragmented DNA was performed using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method following the manufacturer’s protocol. RAW264.7, THP-1 and SF268 cells were fixed with 4% formaldehyde and then permeabilized with 0.1% Triton-X-100 in 10 mM PBS. After washing 3 times with PBS, the cells were incubated in biotin-labeled dUTP solution for 60 min at 37 °C. After washing 3 times with PBS, the cells were incubated in streptavidin-fluorescein solution for 30 min at 37 °C. Then, the stained cells and total cells were calculated under a fluorescence microscope , and the mean percentage of the stained cells among the total cells was calculated from 10 representative fields per coverslip. Finally, the mean percentage was calculated from the quadruplets in each treatment group. Four repetitive experiments were performed for statistical analysis.COS7 cells were cultured in plates the day before transfection. pECFP-N1-ROP18 and pEYFP-C1-P2X1 were co-transfected into COS7 cells for the experimental group. pECFP-N1 and pEYFP-C1 were co-transfected into COS7 cells as a negative control and pEYFP-CFP was transfected into COS7 cells as a positive control . Forty-eg for 10 min at 4 °C. The supernatants were harvested and incubated with the primary antibody anti-HA antibody , anti-FLAG antibody  or rabbit normal IgG for 2 h at 4 °C with gentle rotation. Then, protein A-agarose (Santa Cruz Biotechnology) was added to the mix and incubated overnight at 4 °C with gentle rotation. The beads were collected by centrifugation at 500×g for 5 min at 4 °C and then resuspended in SDS-PAGE sample buffer . The samples were boiled, loaded onto the gels for SDS-PAGE and then analyzed by Western blotting (WB) [SF268 and COS7 cells were grown in T75 flasks to 100% confluence. SF268 cells were infected with or without the RH-ROP18-GFP-Flag strain for 36 h. COS7 cells were transfected with pcDNA3.1-P2X1-HA (or pcDNA3.1-P2X1Δ339-399-HA) individually or together with pcDNA3.1-ROP18-3×Flag for 48 h. The cells were washed 3 times with PBS and lysed using cell lysis buffer (Beyotime) with 1 mM phenylmethanesulfonyl fluoride . Cell lysates were centrifuged at 14,000×antibody 24S, CST,COS7 cells were transfected with pcDNA3.1-ROP18-3×Flag individually or together with pcDNA3.1-P2X1-HA. At 48 h post-transfection, the COS7 cells were treated with 60 µg/ml ATP for 24 h to induce apoptosis. SF268 cells were treated with 4 µM NF449 (P2X1 receptor antagonist) for 2 h and then subjected to 1 mg/ml ATP treatment for 12 h. The normal control cells were treated with neither NF449 nor ATP. The apoptosis rate of the cells was analyzed by FCM after annexin V-FITC/PI staining.2+ can bind to Fluo-4 intracellularly to emit green fluorescence. Time series reads were collected every 2 s for 100 s, and the mean value was calculated as the baseline read F0 (referred to as background). Following 60 µg/ml ATP stimulation, the green fluorescence intensity, reflecting the level of the free intracellular calcium ions in each group, was recorded for 500 s by FCM. The intracellular calcium level was quantified by the ratio of the strongest fluorescence signal to the basal signal (F/F0).COS7 cells were grown in 24-well plates and transfected with pcDNA3.1-ROP18-Flag individually or together with pcDNA3.1-P2X1-HA plasmids for 72 h. The cells were then harvested and washed twice with Hank’s balanced salt solution . Two hundred and fifty microliters of 5 μM Fluo-4 AM  was added to the cells and incubated for 30 min at 37 °C. After being washed twice with HBSS, the cells were analyzed by FCM. Fluo-4 AM is virtually non-fluorescent, and acetoxymethyl (AM) ester moiety allows this dye to cross the cell membrane, whereupon endogenous esterases cleave the AM group to liberate the active dye and Carop18 tachyzoites (MOI = 13). Twelve hours later, 1 mg/ml ATP was added to the cells to induce apoptosis for 12 h. The cells were then digested with 0.25% trypsin and rinsed twice with PBS. Five hundred microliters of incubation buffer containing 1 μl of JC-1 was added to the cells and incubated for 15 min at 37 °C in 5% CO2. The cells were then visualized under a fluorescence microscope (Nikon) and subjected to FCM (BD Biosciences). Twenty views were obtained under GFP and RFP fluorescence filters, and the fluorescence intensities were determined using ImageJ software. This experiment was repeated four times for statistical analysis. For FCM, JC-1 was excited with a 488 nm argon laser, and JC-1 green and red fluorescence were recorded in the FL1 and FL2 channels. A minimum of 10,000 cells within the gated region were analyzed. The value was calculated by the relative intensities of green to red fluorescence. Staining with the lipophilic, cationic dye JC-1 can discriminate polarized and depolarized mitochondria, because in polarized mitochondria, the normal green fluorescent JC-1 dye forms red fluorescent aggregates in response to their higher membrane potential, while in depolarized mitochondria, the red fluorescent aggregates are converted to the monomeric form and exhibit green fluorescence [SF268 cells were infected with RH or RH-Δrescence .g for 10 min at 4 °C to remove nuclei and unbroken cells. Then, the supernatant was collected and centrifuged again at 12,000×g for 10 min at 4 °C to obtain the cytosolic (supernatant) and mitochondrial (pellet) fractions. Cyt C, Bcl2 and Bax proteins in the cytosol and mitochondria were detected by western blotting. The intensity of Cyt C, Bcl2 and Bax bands with unsaturated exposure from four independent experiments were analyzed using ImageJ software, and the proportions of Cyt C, Bcl2 and Bax to the loading control Actin or COXIV were calculated.Mitochondrial and cytosolic proteins were separated using a cell mitochondria isolation kit. SF268 cells were harvested and washed twice with cold PBS, and then incubated in 100 μl of ice-cold mitochondrial lysis buffer with gentle rotation at 4 °C for 15 min. The cell suspension was then transferred into a glass homogenizer and homogenized on ice for 30 strokes using a tight pestle. The homogenate was subjected to centrifugation at 600×Protein samples were diluted in 6× SDS PAGE loading buffer and then boiled for 10 min. The boiled samples were loaded onto 15% SDS-polyacrylamide gels for electrophoresis and then transferred to a polyvinylidene fluoride (PVDF) membrane. After transfer, the PVDF membrane was blocked in PBS/5% bovine serum albumin (BSA)/0.05% Tween-20 at 37 °C for 2 h with gentle shaking, followed by probing with the primary antibody at 4 °C overnight. Then, the membrane was incubated with the secondary antibody at 37 °C for 2 h. The PVDF membranes were visualized by enhanced chemiluminescence (ECL) detection and photographed as recommended by the manufacturer.P < 0.05 and **P < 0.01 were considered to indicate a significant difference. The statistical comparisons presented in the figures are provided in Additional file All experiments were repeated four times. The data were analyzed using GraphPad Prism 5 . Statistical significance was determined using the Kruskal–Wallis H-test with Bonferroni correction, and *T. gondii RH (type I), ME49 (type II) and VEG (type III), individually, for 2 or 22 h, and then were treated with ATP for 4 or 6 h to induce apoptosis. Our FCM results obtained with annexin V-FITC/PI staining indicated that there were significantly fewer apoptotic SF268 cells in any of the infected groups than that in the uninfected control, at both 6 and 28 h post-infection   Fig. a, b. HowTgROP18 inhibits cell apoptosis and identify its targets in host cells, we performed a genome-wide screening of human targets for TgROP18 of the RH strain with the bimolecular fluorescence complementation (BiFC) technique. The technique is based on two non-fluorescent fragments (YFP N-terminus and C-terminus) of the yellow fluorescent protein (YFP) that can reconstitute the fluorophore only when the two fragments are brought together by interactions between proteins that are covalently linked to each fragment [TgROP18 [TgROP18 with P2X1 in the cytosol of COS7 cells  and co-transfected with pcDNA3.1-ROP18-3×Flag into COS7 cells. The results showed that Flag-tagged TgROP18 could not be immunoprecipitated by P2X1 carrying the C-terminal deletion when compared with the full-length P2X1, which was used as a positive control , indicating that P2X1 was indeed involved in ATP-induced apoptosis , although both of their apoptosis indexes were significantly lower than the ATP-treated group  for 2 hTgROP18 inhibited host cell apoptosis through binding to the proapoptotic protein P2X1 of the host cell, untransfected COS7 cells and COS7 cells transfected with pcDNA3.1-TgROP18 and pcDNA3.1-P2X1, individually or together for 48 h, were stimulated with ATP for an additional 24 h. Normal cells  served as the control. The expression of these two proteins was verified by western blotting   Fig. d.TgROP18 and P2X1 were overexpressed in COS7 cells for 72 h, and the intracellular Ca2+ concentration was measured for 600 s following the addition of 60 µg/ml ATP into the culture medium. We found that P2X1 increased Ca2+ influx after ATP stimulation, and this process could be inhibited by co-expression of TgROP18 in the COS7 cells  , indicating that infection of the RH strain inhibited mitochondrial membrane depolarization induced by ATP. Moreover, the intensity ratio of green to red fluorescence also decreased in the RH + ATP treatment group compared with the RH-Δrop18 + ATP treatment group , implying that depolarization of the mitochondrial membrane was inhibited by TgROP18. Therefore, an increase in green/red fluorescence ratio means depolarization of the mitochondrial membrane and this phenomenon can be changed by the expression of TgROP18.Previous studies have shown that RH regulates apoptosis of host cells mainly through the mitochondrial pathway . To idenTgROP18, we next measured the release of Cyt C from mitochondria to the cytosol and the translocation of Bax (a pro-apoptotic Bcl2 family protein) and Bcl2 (an anti-apoptotic Bcl2 family protein) from the cytosol to mitochondria in each group. The western blot results revealed a relatively higher level of Cyt C in the mitochondrial fraction, but a lower level of Cyt C was detected in the cytosolic fraction in the RH + ATP treatment group compared with the RH-Δrop18 + ATP treatment group   Fig. a, c1, c2df = 3, P = 0.005) and decreased level of proapoptotic protein Bax  relative to COXIV was found in the mitochondrial fraction of the RH + ATP treatment group compared with the ATP-treated group     between the ATP-treated and RH + ATP groups  in immune cells or neural cells  [T. gondii virulence factors also play a role in modulating ATP-induced host cell apoptosis needs to be further investigated.Although H strain ; however pathway . Several kinase) , type 2C kinase)  and dens kinase) . WhetherT. gondii infection inhibits ATP-induced host cell apoptosis, regardless of strain virulence and host cell lines. Toxoplasma gondii virulence factor TgROP18, which targeted the C-terminus of P2X1, inhibited apoptosis mediated by P2X1. When apoptosis was induced by ATP, the P2X1 mediated Ca2+ influx was inhibited by the presence of TgROP18 in COS7 cells overexpressing both P2X1 and TgROP18. Furthermore, TgROP18 inhibited depolarization of the mitochondrial membrane, Cyt C translocation from the mitochondria to the cytoplasm, and ATP-triggered caspase 9, 7 and 3 activation in SF268 cells. Taken together, these findings support the conclusion that TgROP18 inhibits ATP-induced apoptosis by interfering with P2X1 function and the mitochondrial apoptotic pathway.The present findings showed that Additional file 1: Table S1. Summarized information of statistical comparisons presented in the figures.Additional file 2: Figure S1. TUNEL assay of ATP-induced apoptosis of infected SF268 cells. SF268 cells were infected with RH, ME49 or VEG strain and treated with ATP in the same way as described in Fig. a1, a2 Analysis of apoptosis by fluorescence microscopy following TUNEL assay. TUNEL-positive cells were visualized as indicated by green fluorescence staining and DAPI (blue) staining to discern all cell nuclei. The percentage (%) of TUNEL-positive cells was determined . b1, b2 Quantification of the data in panels a1 and a2. The experiments were repeated four times. The values were analyzed using the Kruskal–Wallis H-test and Bonferroni correction (**P < 0.01).Additional file 3: Figure S2. TUNEL assay of ATP-induced apoptosis of infected RAW264.7 cells. RAW264.7 cells were infected with the RH, ME49 or VEG strain and treated with ATP in the same way as described in Fig. P < 0.01).Additional file 4: Figure S3. TUNEL assay of ATP-induced apoptosis of infected THP-1 cells. THP-1 cells were infected with the RH, ME49 or VEG strain and treated with ATP in the same way as described in Fig. P < 0.01).Additional file 5: Figure S4. Effect of T. gondii virulence factor ROP18 on ATP-induced apoptosis of RAW264.7 and THP-1 cells. RAW264.7 and THP-1 cells were infected with RH or RH-Δrop18 tachyzoites (MOI = 13) or left uninfected to serve as the normal control (N) or positive control (ATP treatment). At 12 h post-infection, 1 mg/ml ATP was added to the cells for an additional 12 h, except in the normal control group. a Representative flow cytometry data. b Quantification of the flow cytometry data. The percentages of apoptotic cells were separately determined for each group of cells. The experiments were repeated four times for Kruskal–Wallis H-test statistical analysis .Additional file 6: Table S2. Summarized information for Toxoplasma gondii modulation of immune and neural cell apoptosis.Additional file 7: Figure S5. Western blot analysis of P2X1 in SF268, RAW264.7, HFF and THP-1 cells. RAW264.7, HFF, THP-1 and SF268 cells were grown in a T25 flask to 100% confluence and then harvested and lysed. Total proteins for each sample were subjected to SDS-PAGE and western blotting analysis with P2X1 antibody.