However, the molecular mechanisms of drug actions remain poorly understood. Here, we report the cryo–electron microscopy structures of the human α2AAR-GoA complex bound to norepinephrine and three imidazoline derivatives . Together with mutagenesis and functional data, these structures provide important insights into the molecular basis of ligand recognition, activation, and signaling at the α2AAR. Further structural analyses uncover different molecular determinants between α2AAR and βARs for recognition of norepinephrine and key regions that determine the G protein coupling selectivity. Overall, our studies provide a framework for understanding the signal transduction of the adrenergic system at the atomic level, which will facilitate rational structure-based discovery of safer and more effective medications for α2AAR.The α 2A adrenergic receptor structures revealed distinct agonist activation and facilitate design of therapeutics for α2AAR.Active α While the molecular mechanisms of drug action (including norepinephrine and epinephrine) on β adrenoceptors (β1AR and β2AR) have been extensively investigated  and α2CAR and the active α2BAR–G protein complex structures  using a chimeric Gqi protein and Norepi as a reference agent (Emax of Norepi), which is in contrast to the full agonist properties for the other three drugs, suggesting that OXY is a potential Gi/o-biased agonist at the α2AAR.In this study, we determined the structure of the αce agent . Of inte2AAR and the heterotrimeric GoA in the presence of the four different agonists. We kept the long intracellular loop 3 (ICL3) and used the wild-type receptor for our structural studies (fig. S1A). The complexes were prepared as described in Materials and Methods, and the agonist-bound α2AAR formed a stable nucleotide-free complex with GoA in the presence of an antibody fragment scFv16 (fig. S1B) , 3.0 Å (BRI), 3.6 Å (DEX), and 3.4 Å (OXY)  via a salt bridge and a hydrogen bond .The phenyl ring of DEX lacks the two hydroxyls of catecholamines; instead, only two methyl groups and no polar substituents are present. As a result, DEX interacts with TM5 and TM6 mainly through hydrophobic interactions , which iRI . Al5.43 in β adrenoceptors, which is a cysteine in α2AAR, participates in the polar network through hydrogen bonding with N6.55 (6.55) and TM7 (F7.39) of α2AAR are also replaced with two asparagine residues (N6.55 and N7.39) in β adrenoceptors, both of which are involved in the polar interaction networks with Norepi  in α2AAR, together with F6.51, F6.52, Y7.43, and W6.48, form an aromatic cage, which may play key roles in high-affinity Norepi binding . Rec2AAR from inactive to active states. The outward movement of TM6 (12.6 Å) and inward movement of TM7, which are hallmarks of GPCR activation  upon agonist binding , and most of the interactions with the receptor are identical for agonists and antagonists . These structural features suggest a conserved mechanism for the allosteric coupling between the orthosteric pocket and G protein coupling domain for adrenoceptors.In spite of these differences in conformational changes of the orthosteric pocket and in ligand recognition, the α2 and β2 adrenoceptors have adverse physiological functions, with α2 inhibiting the activity of adenylyl cyclase via Gi/o and β2AR stimulating it via Gs, they have long served as model systems for studying G protein coupling selectivity. 2AR (α2AAR and α2BAR) and Gs with β2AR. Here, we used the Norepi-bound α2AR-GoA for comparison with other adrenoceptors, as the interfaces with GoA for different agonist-bound α2AR are almost identical (fig. S6A).Given that α2AAR-GoA and α2BAR-GoA are almost identical  of the Gαo cluster with surrounding hydrophobic residues from TM3, TM5, TM6, and ICL2. However, several differences in polar interactions with the GoA heterotrimer were observed between α2AAR and α2BAR. For example, the last amino acid Y354G.H5.26 of Gαo adopts a distinct side-chain conformation and forms a hydrogen bond with R6.29 in α2BAR. In contrast, this interaction is not observed in α2AAR . These distinct interactions between α2AAR-GoA and α2BAR-GoA suggest the versatility of Go coupling with GPCRs.The overall interaction interfaces for αdentical . The majin α2AAR . A hydro2ARs-GoA and β2AR-Gs complex structures shows that the relative positions for Gαo and Gαs are different, with the extreme C terminus of Gαo helix α5 shifting 2.5 Å toward TM7 and a large displacement of the αN helix. This is associated with the smaller outward displacement of TM6 and inward movement of TM7 in α2ARs, resulting in a smaller cavity formed by TM3/5/6/7 for GoA than for Gs (2AR (fig. S6D).The comparison of αn for Gs . This st2ARs and GoA, hydrophilic residues R380G.H5.12, D381G.H5.13, and Q384 G.H5.16 on one side of the α5 helix of the Gαs protein form a cluster of hydrogen-bonding interactions with hydrophilic residues from TM3 and TM5 of β2AR . Although α2AAR- β2-TM5 showed decreased Gi coupling activity (~3-fold), the Gs activity also decreased substantially (~10-fold) compared with that of the wild-type α2AAR . By contrast, β2AR-α2A-TM5 showed markedly increased activity for both Gi and Gs coupling (over 100-fold) compared with that of the wild-type β2AR . These data suggest that the TM5 intracellular domain plays important roles in G protein coupling and that the amino acid sequence of α2AAR has stronger ability to couple with both G proteins than that of β2AR, despite the weaker interactions with G protein  and were infected with recombinant baculovirus containing the α2AAR gene at a density of 4 × 106 cells ml−1 in the presence of 5 μM rauwolscine. After 48 hours of infection at 27°C, the cells were spun down and cell pellets were stored at −80°C until use.The human wild-type α−1), and benzamidine (160 μg ml−1)]. Cell membranes were then spun down and solubilized with a buffer consisting of 20 mM Hepes (pH 7.5), 500 mM NaCl, 1% n-Dodecyl-b-D-Maltopyranoside (DDM), 0.2% sodium cholate, 0.03% Cholesteryl Hemisuccinate (CHS), 5 μM rauwolscine, leupeptin (2.5 μg ml−1), and benzamidine (160 μg ml−1). Nickel-NTA sepharose was added to the solubilized receptor and rotated for 2 hours at 4°C. The resin was spun down and washed in batch for three times with a buffer composed of 20 mM Hepes (pH 7.5), 500 mM NaCl, 0.1% DDM, 0.02% sodium cholate, 0.03% CHS, 5 μM rauwolscine, leupeptin (2.5 μg ml−1), and benzamidine (160 μg ml−1). The washed resin was poured into a glass column, and the receptor was eluted in the wash buffer supplemented with 250 mM imidazole.Thawed cell pellets were resuspended in hypotonic lysis buffer  and stirred at room temperature for 15 min. Cell membranes were spun down and resuspended with solubilization buffer  using a Dounce homogenizer. The samples were stirred at 4°C for 40 min and then centrifuged for 30 min to remove insoluble debris.Thawed cell pellets were resuspended in lysis buffer . The protein was then eluted with E2 buffer supplemented with 250 mM imidazole.Ni-NTA resin preequilibrated in solubilization buffer was added to the supernatant and shaken for 2 hours at 4°C. After incubation, the Ni-NTA resin was spun down and poured into a glass column and washed with 50 ml of solubilization buffer. The heterotrimeric GoA was gradually exchanged into E2 buffer  by repeated concentration and dilution using a 50-kDa molecular weight cutoff Millipore concentrator. The concentrated heterotrimeric GoA was aliquoted, flash frozen in liquid nitrogen, and stored at −80°C before use.The sample was then dephosphorylated by treating with 5 μl lambda phosphatase . The protein was purified with Ni-NTA column followed by size exclusion chromatography using a Superdex 200 Increase 10/300GL column . The monomeric peak fractions were collected, concentrated, and stored at −80°C until use.2AAR with heterotrimeric GoA was formed in a buffer composed of 20 mM Hepes (pH 7.5), 100 mM NaCl, 0.1% DDM, 1 mM MgCl2, 10 μM GDP, and 100 μM agonist. The α2AAR-GoA complex was then treated with 50 U of apyrase (NEB) on ice overnight and exchanged on an anti-Flag M1 column into a buffer containing 20 mM Hepes (pH 7.5), 100 mM NaCl, 0.0075% lauryl maltose neopentyl glycol , 0.0025% GDN , 0.001% CHS, 100 μM agonist, and 2 mM CaCl2 in a stepwise manner. After elution by adding 5 mM EDTA and Flag peptide (0.2 mg ml−1), the complex was concentrated and incubated with excess scFv16 for 1 hour on ice and then loaded onto Superdex 200 Increase 10/300GL column  with a running buffer of 20 mM Hepes (pH 7.5), 100 mM NaCl, 0.00075% MNG, 0.00025% GDN, 0.0001% CHS, and 100 μM agonist. The monomeric peak fraction of the α2AAR-GoA complex was collected and concentrated to ~10 mg/ml for cryo-EM.The complex of αThe gold film  in a ratio of 1:2 and transferred into 384-well microplates. On the day of the experiment, incubation started by adding the agonists for 90 min  or 150 min , or 120 min (for the direct comparison of α2AAR and β2AR with the α2AAR-β2-TM5 and β2AR-α2A-TM5 chimeras), respectively. Accumulation of second messenger was stopped by addition of the detection reagents (IP1-d2 conjugate and anti-IP1cryptate TB conjugate). After a further 60 min, time-resolved fluorescence resonance energy transfer (FRET) was measured using the Clariostar plate reader . FRET signals were calculated as the ratio of emissions at 665 and 620 nm and ligand-induced changes in FRET (deltaFRET) normalized to the maximum effect of norepinephrine (100%) and vehicle (0%). For the mutant α2AAR-D1283.32A, the maximum effect of dexmedetomidine was used as 100%. Normalized concentration-response curves from 4 to 11 experiments each done in duplicates were analyzed using the algorithms for four-parameter nonlinear regression implemented in PRISM 8.0  to derive EC50 and Emax values.The determination of receptor-mediated G protein signaling by wild-type and mutant receptors was performed applying an IP accumulation assay  according to the manufacturer’s protocol and in analogy to previously described protocols  or 150 min (α2AAR-D1283.32A), respectively. Chemoluminescence was monitored with a Clariostar plate reader and analyzed by normalizing the raw data relative to basal activity (0%) and the maximum effect of norepinephrine (100%). For the mutant α2AAR-D1283.32A, normalization was done by analyzing the raw data as fold change relative to basal (=100%). Three to nine individual experiments each done in duplicate were analyzed by nonlinear regression, applying the algorithms in Prism 8.0  to get dose-response curves representing average EC50 and Emax values.Determination of receptor stimulated β-arrestin-2 recruitment was performed applying the PathHunter assay  measuring fragment complementation of β-galactosidase as described  at a 3:1 PEI/DNA ratio. Per 1.2 ml of cell suspension, 200 ng of α2AAR and 800 ng of single-stranded salmon sperm DNA (Sigma-Aldrich) were used. Fifty thousand cells per well were transferred to a 48-well plate coated with poly-d-lysine and incubated at 37°C and 5% CO2 for 48 hours. Cells were fixated using 4% paraformaldehyde , washed once , and blocked for 60 min [skim milk powder (30 g liter−1) in wash buffer], before anti-FLAG mouse immunoglobulin G  was added. After 60 min, wells were washed twice (300 μl) and blocked for a further 60 min, before 200 μl of anti-mouse rabbit IgG-HPR  was added. After 60 min, cells were washed thrice, before the addition of substrate buffer . Reactions were kept in the dark for 5 to 10 min and stopped by addition of 1 M H2SO4 (200 μl). The resulting solution (150 μl) was transferred to clear, flat bottom 96-well plates, and absorption at 492 nm was measured with the Clariostar microplate reader. Data were normalized to the expression level of wild-type α2AAR (100%) and mock transfected HEK293T cells (0%), respectively. N = 5 independent experiments were performed, with each condition in triplicate.Receptor surface expression was analyzed using an enzyme-linked immunosorbent assay directed against the N-terminal FLAG-tag as previously described (


Organic solvents such as hexane, heptane, toluene, and xylene are used during the wet milling process. The phase evolution, powder morphology, and electrochemical properties of the wet-milled Li6PS5Cl powders and electrolytes are studied. Compared to dry milling, the processing time is significantly reduced via wet milling. The nature of the solvent does not affect the ionic conductivity significantly; however, the electronic conductivity changes noticeably. The study indicates that xylene and toluene can be used for the wet milling to synthesize Li6PS5Cl electrolyte powder with low electronic and comparable ionic conductivities. The all-solid-state cell with the xylene-processed Li6PS5Cl electrolyte exhibits the highest discharge capacity of 192.4 mAh·g−1 and a Coulombic efficiency of 81.3% for the first discharge cycle.Lithium argyrodite Li Inorganmproving .6PS5Cl) are typically prepared by mechanical milling and subsequent annealing processes. The precursor materials  are dry-milled in a high-speed planetary ball mill, and the obtained powder mixture is annealed in a vacuum furnace or in an Ar-filled ampule to obtain a crystalline argyrodite phase. The lithium argyrodite electrolytes prepared via the aforementioned process exhibit a room temperature (25°C) ionic conductivity exceeding 10–3 S·cm−1 , P2S5 , and LiCl  powders were mixed in a planetary ball mill  using organic solvents such as hexane , heptane , toluene , and xylene . Typically, the milling was performed using 2 g of the powder mixture with zirconia balls (d = 5 mm) in a zirconia jar (80 ml) at a rotational speed of 500 rpm for a duration of 2 h. The slurry was vacuum dried at 150°C for 6 h followed by annealing at 550°C in a tube furnace for 4 h under an argon atmosphere. Argon flow rate was 50 sccm. For comparison, Li6PS5Cl powder was also synthesized via a dry ball milling process, details of which have been reported elsewhere . The microstructures were examined via field-emission scanning electron microscopy (FE-SEM) .The phase analysis of the synthesized powder samples was performed using X-ray diffraction (XRD)  with Ni-filtered Cu-Kα radiation. Surface chemical analysis was conducted via the X-ray photoelectron spectroscopy (XPS)  using a Mg Kα  excitation source. The carbon and hydrogen contents of the Li6PS5Cl powder (55 mg) was placed in a zirconia mold (10 mm) equipped with two stainless-steel rods (d = 10 mm) and cold-pressed at 300 MPa to prepare a disk-shaped pellet sample . The relative densities of the pellet samples were estimated to be approximately 92%, calculated from the bulk density of the electrolyte pellet sample and the theoretical density of Li6PS5Cl (1.64 g·cm−3) (The conductivities (ionic and electronic) were measured via electrochemical impedance spectroscopy (EIS)  over a frequency range of 0.01 Hz–1 MHz at 30, 40, 60, 80, 100, and 120°C. The Li g·cm−3) .ionic+electronic, was calculated using the equation σionic = t/RA, where R is the total resistance, t is the thickness, and A is the area of the pellet sample, respectively. The ionic conductivities were determined by subtracting the electronic conductivities by DC polarization technique from the total conductivities.The alternating current (AC) impedance spectra were obtained from the electrolyte pellet samples with two stainless-steel (SS) rods acting as current collectors under open-circuit conditions with an excitation potential of 20 mV. The total conductivity, σ6PS5Cl/Ni symmetrical cells were prepared in a similar way as previously mentioned. Three voltages  were applied to the Ni/Li6PS5Cl/Ni symmetrical cells for 2 h, and the resulting current signal was recorded. The resistances were determined from the V-I curves, and the electronic conductivity, σelectronic, was calculated from the thickness and area of the Li6PS5Cl electrolyte pellet sample.The electronic conductivity was measured via direct current (DC) polarization technique . The Ni/0.8Co0.1Mn0.1O2 (NCM)/Li6PS5Cl/super P|Li6PS5Cl|Li-In were assembled by cold pressing at 300 MPa using a zirconia mold (d = 10 mm) equipped with stainless-steel rods. The ratio of NCM:Li6PS5Cl:super P was 70:29:1 by weight. The cathode loading of NCM was 26.8 mg·cm−2. Wet-milled and dry-milled Li6PS5Cl powders were used as the solid electrolyte and the composite cathode, respectively to exclude the solvent effect on reactions at the cathode/solid electrolyte interface. The charge–discharge behavior of the ASSCs was studied at 80°C using a battery test system  with a cutoff voltage of 1.9–3.6 V (vs Li–In). The current density was set to 0.535 mA cm−2. Charging and discharging were carried out in constant current (CC)–constant voltage and CC modes, respectively.All-solid-state cells (ASSCs) of LiNi2S, P2S5, and LiCl powder mixtures obtained after milling (dry and wet) and annealing. For the as-milled powder mixture samples  time in the case of wet-milled and dry-milled samples was 6 and 16 h, respectively. Considering the highly reduced synthesis time to achieve the same composition, it was concluded that wet ball milling is an efficient method to synthesize Li6PS5Cl powder.In the XRD patterns of the post-annealed powder samples , the pea6PS5Cl/SS symmetrical cells measured at 25°C. In all the spectra, only the spike (straight line) of the blocking electrodes was observed. The ionic conductivity at room temperature can be derived from the total resistance, which is determined from the intersection of the spike with the real axis  at the lower frequency side. The ionic conductivities of the Li6PS5Cl electrolyte samples are listed in 6PS5Cl electrolyte pellet samples were estimated to be in the range of 1.0–1.9 mS·cm−1. This indicates that the organic solvent does not affect the lithium-ion conduction in the wet-milled Li6PS5Cl. By contrast, the ionic conductivity of the dry-milled Li6PS5Cl electrolyte pellet sample was found to be 2.39 mS·cm−1, which is slightly higher than that of the wet-milled samples. The observed conductivity reduction in the wet-milled Li6PS5Cl electrolyte samples could be associated with the particle size (6PS5Br) electrolyte with large crystallites  were due to the grain boundary resistance.cle size . Yu et atallites . As can σ) was found and the activation energy can be determined using the equation of σ = Aexp(−Ea/kBT), where σ is the total conductivity, A is the pre-exponential factor, kB is the Boltzmann constant, and T is the absolute temperature. The activation energies are 0.202, 0.240, and 0.245 eV for the hexane, heptane, and xylene-processed Li6PS5Cl electrolytes, respectively. These values are almost same to the dry-milled Li6PS5Cl electrolyte (0.221 eV), indicating that there are no unwanted secondary phases in the wet-milled Li6PS5Cl powder samples, as can be seen in The Arrhenius plots of the ASSCs is shown in 6PS5Cl/Ni symmetrical cells in which the Li6PS5Cl powder was prepared using different solvents. When the voltage was applied, the current signal sharply decreased with time and saturated on the order of 10–5 to 10–7 A, depending on the solvent employed during the wet ball milling process. For all cells, the steady state was achieved within 2 h. The currents of the hexane-, toluene-, and xylene-processed Li6PS5Cl samples saturated to ∼10–6 A, whereas for the heptane-processed Li6PS5Cl sample, the current saturated to ∼10–5 A.6PS5Cl/Ni symmetrical cells exhibit a linear relationship between voltage and steady-state current values  of the dry- and wet-milled Li6PS5Cl powders was performed and the results are presented in 6PS5Cl powder samples, which could be attributed to the organic solvents. Due to incomplete evaporation, residual solvent molecules could be deposited on the surface of the wet-milled Li6PS5Cl powder and reduced to pure carbon during the post-annealing process. These carbon particles located at the grain boundaries of the wet-milled Li6PS5Cl electrolytes work as electron transport carriers, which explains the increased electronic conductivity. As can be seen in 6PS5Cl powder sample had the lowest carbon content (0.19%) and exhibited the lowest electronic conductivity of 5.4 × 10–10 S·cm−1.To examine the effect of impurities on the electronic conductivity of the Li2p, P2p, C1s and O1s XPS spectra of dry- and wet-milled Li6PS5Cl powder samples. The S2p spectra of the dry-milled Li6PS5Cl powder samples exhibited a doublet with a S2p3/2 component at a binding energy of 162.2 eV, attributed to the PS43– tetrahedra (P–S–Li bonds) of argyrodite  or element sulfur (164 eV) were observed  was attributed to the amorphous carbon species  than toluene-processed (181 mAh·g−1), the latter exhibited better retention of the rate capacity than hexane. The ASSC with the heptane-processed Li6PS5Cl electrolyte did not maintain its discharge capacity after the fourth cycle.6PS5Cl powder samples. In other words, high electronic conductivity can lead to a reduced Coulombic efficiency. The ASSCs with xylene- and toluene-processed Li6PS5Cl electrolytes maintained a Coulombic efficiency of 95–96% during the first 10 cycles. By contrast, the ASSC with the hexane-processed Li6PS5Cl electrolytes exhibited Coulombic efficiencies of 90% which gradually decreased with increasing cycle number. In the case of heptane, the ASSC exhibited much lower Coulombic efficiencies than the other ASSCs. The capacity and Coulombic efficiency retention might be closely related to the electronic conductivity of the Li6PS5Cl electrolyte. The Li6PS5Cl electrolyte powders prepared using xylene and toluene exhibited relatively low electronic conductivity on the order of 10–7 S·cm−1, resulting in high discharge capacity and Coulombic efficiency retention after 10 cycles, whereas those prepared using hexane and heptane, exhibited higher electronic conductivities, lower discharge capacity and decaying Coulombic efficiency with cycling.A different trend was observed for the initial Coulombic efficiencies, as is evident in 6PS5Cl powder was synthesized from Li2S, P2S5, and LiCl powder mixture via wet ball milling with various organic solvents and post-annealing. Compared to dry milling, the processing time was significantly reduced. The ionic conductivities of the wet-milled and post-annealed Li6PS5Cl powder samples were in the range of 1.0–1.9 S·cm−1, which was slightly lower than that of the dry-milled Li6PS5Cl powder sample (2.8 S·cm−1). FE-SEM analyses revealed that the particle size was reduced in the wet-milled Li6PS5Cl powder samples, and the increased grain boundary was responsible for the reduced ionic conductivity. The wet-milled Li6PS5Cl powder samples showed three or four orders of magnitude higher electronic conductivity than that of the dry-milled Li6PS5Cl powder sample. In terms of solvent for wet milling, xylene and toluene were found to be better than hexane and heptane, leading to one order of magnitude lower electronic conductivity. It appears that sulfur deficiency or the state of the residual carbon could be responsible for the observed variation in the electron conductivity. The ASSC with the xylene-processed Li6PS5Cl electrolyte exhibited the highest discharge capacity of 192.4 mAh·g−1 and a Coulombic efficiency of 81.3% for the first discharge cycle. The lower discharge capacity and Coulombic efficiency retention observed in the ASSCs with hexane- and heptane-processed Li6PS5Cl electrolytes was attributed to the increased electronic conductivity of these electrolytes. In the future, the strategies to reduce the electronic conductivity of the wet-milled Li6PS5Cl should be presented in order to use it as an all-solid-state lithium battery electrolyte.A single-phase Li


We explored the potential role of zinc (Zn) and zinc transporters in protection against cytotoxicity of cadmium (Cd) in a cell culture model of human urothelium, named UROtsa. We used real-time qRT-PCR to quantify transcript levels of 19 Zn transporters of the Zrt-/Irt-like protein (ZIP) and ZnT gene families that were expressed in UROtsa cells and were altered by Cd exposure. Cd as low as 0.1 µM induced expression of ZnT1, known to mediate efflux of Zn and Cd. Loss of cell viability by 57% was seen 24 h after exposure to 2.5 µM Cd. Exposure to 2.5 µM Cd together with 10–50 µM Zn prevented loss of cell viability by 66%. Pretreatment of the UROtsa cells with an inhibitor of glutathione biosynthesis (buthionine sulfoximine) diminished ZnT1 induction by Cd with a resultant increase in sensitivity to Cd cytotoxicity. Conversely, pretreatment of UROtsa cells with an inhibitor of DNA methylation, 5-aza-2’-deoxycytidine (aza-dC) did not change the extent of ZnT1 induction by Cd. The induced expression of ZnT1 that remained impervious in cells treated with aza-dC coincided with resistance to Cd cytotoxicity. Therefore, expression of ZnT1 efflux transporter and Cd toxicity in UROtsa cells could be modulated, in part, by DNA methylation and glutathione biosynthesis. Induced expression of ZnT1 may be a viable mechanistic approach to mitigating cytotoxicity of Cd. Exposure to cadmium (Cd) is inevitable for most people because this toxic metal is present in virtually all foodstuffs, cigarette smoke, and polluted air from combustion of fossil fuels and biomass ,2,3. Foo2 [2 as compared with blood Cd < 0.18 μg/L. After adjustment for smoking effects, high urinary Cd levels (>0.79 μg/g creatinine) were associated with a 2.48-fold increase in risk of obstructive lung disease as compared with urinary Cd < 0.39 μg/g creatinine [In theory, the absorption rate of Cd increases when the body is in short supply of the elements that share absorption and transport pathway mechanisms with Cd, such as Fe, Zn, Mn, and Ca ,5,6. It 2 . High blIn the present study, our aim was to demonstrate the protection against Cd toxicity by simultaneous Zn exposure. In addition, we examined the putative contribution of zinc and zinc transporters in modulating cytotoxicity of Cd. We used a non-tumorigenic human cell line, named UROtsa, since studies in our laboratory have established that the UROtsa cell line is a suitable cell culture model of urinary bladder epithelium ,20. Our 2 tissue culture flasks in Dulbeco’s modified Eagle’s medium supplemented with 5% vol/vol fetal bovine serum [2/95% atmospheric air. Cells were fed fresh growth medium every three days. At confluence, cells were subcultured at a 1:4 ratio using trypsin-EDTA . For experimentation, cells were grown in 6-well plates containing 2 mL growth media per well (35 mm in diameter). Treatment of cultures with Cd [We used the UROtsa cell line derived from the ureter epithelium of a 12-year-old female donor, immortalized with SV40 large T-antigen . It is ane serum ,21. Cell with Cd , Zn, BSO with Cd , and aza with Cd  were und with Cd . Thus, i2/95% air atmosphere to allow cellular MTT uptake and MTT reduction [Cell viability assay was based on the capacity of cells to reduce a tetrazolium dye, 3--2,5-diphenyltetrazolium bromide (MTT), to formazan . Brieflyeduction . The cel® . A 2 µL aliquot of RNA sample containing 40 ng of total RNA was subjected to cDNA synthesis using the iScript™cDNA synthesis Kit  in a 20 μL total volume. The cDNA was stored at −20 °C for later analysis. Real-time PCR was performed in triplicate with iQ™SYBR® Green Supermix  in the iCycler Real-Time Detection System (Bio-Rad Laboratories), using 2 μL of cDNA  in a 20 μL final volume, containing 0.2 μM each of the primers. Primer pairs and PCR conditions for ZIP and ZnT genes assayed were detailed previously [Total RNA was extracted from cells using TRI Reagenteviously . Target P-values ≤ 0.5 were considered to identify statistically significant differences.Expression levels of zinc transporter genes and cell viability data were analyzed with SPSS 17.0 . A Kruskal–Wallis test was used to determine differences in expression levels of an individual zinc transporter gene across four groups of cell cultures, exposed to varied Cd concentrations (five cultures for every Cd concentration and control). The Spearman’s rank correlation test was used to evaluate a correlation between expression levels of an individual zinc transporter gene and Cd exposure concentrations. Differences in means for pairs of variables such as untreated control versus treated cells were determined by a Mann–Whitney test. Expression levels of ZIP and ZnT zinc transporters by UROtsa cells 24 h after exposure to 0, 1, 2, and 4 µM Cd concentrations are shown in At basal state (0 µM Cd), ZnT7 was most abundantly expressed among the ZnT family members, followed by ZnT5 and ZnT1 with on average of 758, 510, and 365 transcript copies per 1000 transcripts of β-actin, respectively. On-average transcript copies of ZnT4, ZnT6, and ZnT10 were 65, 51, and 11 and 1 per 1000 transcripts of β-actin while ZnT2 and ZnT3 were expressed between 6 and 15 transcripts per 100,000 β actin transcripts. For ZIP family, ZIP7 was most abundantly expressed at basal state followed by ZIP14, ZIP6, ZIP1, ZIP10, and ZIP3A with on average of 204, 146, 92, 82, 54, and 19 transcripts per 1000 β-actin transcripts, respectively. ZIP2, ZIP3B, and ZIP8 were expressed between 1 and 4 transcripts per 1000 β-actin transcripts. ZIP 4 and ZIP5 were expressed between 1 and 6 transcripts per 10,000 β-actin transcripts.Exposure to varying Cd concentrations for 24 h affected expression levels of multiple zinc transporters. Shown in r = −0.28, p = 0.03) as were ZnT4 transcript levels . Likewise, ZIP1, ZIP3A, ZIP5, and ZIP7 gene expression levels showed an inverse correlation with Cd exposure levels with r and p-values of −0.29 (p = 0.02), −0.33 (p = 0.01), −0.27 (p = 0.05), and −0.42 (p = 0.001), respectively. A particularly strong inverse correlation was seen between Cd exposure levels and transcript levels of two ZIP zinc transporters, ZIP2  and ZIP10 . Distinct from all other zinc transporters, ZnT1 gene transcript levels showed a positive correlation with exposure Cd concentrations .For most zinc transporters, their transcript levels showed an inverse relationship with concentrations of Cd to which cells were exposed. For the ZnT family, transcript levels for ZnT3 were decreased with increasing Cd concentrations  of Cd in UROtsa cells. Exposure to Cd at the LD50 level (2.5 µM) for 48 h together with Zn at 10, 25, and 50 µM concentrations prevented loss of cell viability caused by Cd. This was evident from the viability of 80% among those with concurrent Zn exposure as compared with 14% of those exposed to Cd alone with equivalent Cd molar concentrations  was 19% as compared with 54% in untreated cells. BSO pretreatment affected ZnT1 by Cd. In untreated cells, 1 µM Cd increased ZnT1 expression by 7.5, and it fell to 4.2, 3.7, and 2.7 in cells treated with 100, 500, and 1000 µM BSO, respectively . At 5 µM Cd2+, the viability of untreated cells was 27% only, compared with 80%, 56.6%, and 57.1% in cells pretreated with 2.5, 5, or 10 µM aza-dC, respectively (p < 0.05). For aza-dC-treated UROtsa cells, LD50 for Cd was increased by two-fold (from 2.5 to 5 µM Cd). Aza-dC treatment did not affect expression of ZnT1 nor the extent of ZnT1 induction by 2.5 or 5 µM Cd (Inhibition of DNA methylation by 5-aza-2’-deoxycytidine (aza-dC) treatment 24 h prior to Cd exposure produced results opposite to BSO treatment. Tolerance or resistance to Cd toxicity was seen among cells treated with aza-dC at all three aza-dC concentrations tested  C. At 2.5 5 µM Cd D.Cd at 1 µM did not affect expression of ZnT5, ZnT7, ZIP8, or ZIP14 in untreated control cells A. HoweveExpression levels of individual zinc transporter genes, in the basal state, varied widely between the two gene families of zinc transporters, as did expression levels among members in each family . ZIP8, ZIt has been shown in a previous study using a rat liver epithelial cell line (TRL 1215) that pretreatment of cells with cyproterone, a synthetic steroidal antiandrogen with a structure related to progesterone, decreased sensitivity to Cd through a decreased accumulation of Cd . Howeverr = −0.69, p < 0.001) and ZIP10 genes . A positive correlation was seen only between ZnT1 gene expression and Cd exposure concentrations . We considered it as initial evidence for a selective induction ZnT1 by Cd, which may represent a defense mechanism of UROtsa cell use to prevent Cd and Zn accumulation to toxic levels. Time-course and dose-response analyses have confirmed that Cd concentrations between 0.1 and 1.5 µM increased expression of the ZnT1 gene. Of note, at Cd concentrations between 0.05 and 0.5 µM concentration, cell viability was not affected. The highest fold ZnT1 induction of 8.8 occurred within 24 h of exposure to 1.5 µM Cd (In a correlation analysis , an inve.5 µM Cd A,B. AmonEvidence for the protective role of ZnT1 upregulation has emerged from a series of experiments involving simultaneous Zn exposure, BSO, and aza-dC pretreatments. Simultaneous exposure to Zn at 4-, 10- and 40-times higher molar concentrations than that of Cd prevented loss of cell viability, indicative of Cd cytotoxicity A,B. UndeBSO treatment caused a reduction in ZnT1 inductive responses coincidental with an increase in sensitivity to Cd B. Cd at BSO treatment is known to perturb both GSH biosynthesis and DNA methylation . Thus, iUnlike BSO treatment, pretreatment of UROtsa cells with aza-dC for 24 h prior to Cd exposure resulted in resistance to Cd cytotoxicity C. Aza-dCExpression of Cd cytotoxicity in UROtsa cells and the expression level of ZnT1 zinc transporter, known to medicate efflux of both Zn and Cd, could be modulated, at least in part, by DNA methylation state and glutathione biosynthesis. A diminished ZnT1 induction may account for an increased sensitivity to Cd cytotoxicity in cells treated with BSO prior to Cd exposure. Conversely, ZnT1 induction that remains impervious in aza-dC-treated cells may account for resistance to Cd cytotoxicity.


Background: Spindle and kinetochore-related complex subunit 3 (SKA3), a member of the SKA family of proteins, is associated with the progression of multiple cancers. However, the role of SKA3 in gastric cancer has not been studied.Methods: The expression levels of SKA3 and dual-specificity phosphatase 2 (DUSP2) proteins were detected by immunohistochemistry. The effects of SKA3 and DUSP2 on the proliferation, migration, invasion, adhesion, and epithelial-mesenchymal transition of gastric cancer were studied in vitro and in vivo.Results: Immunohistochemical analysis of 164 cases of gastric cancer revealed that high expression of SKA3 was negatively correlated with DUSP2 expression and related to N stage, peritoneal metastasis, and poor prognosis. In vitro studies showed that silencing SKA3 expression inhibited the proliferation, migration, invasion, adhesion and epithelial-mesenchymal transition of gastric cancer. In vivo experiments showed that silencing SKA3 inhibited tumor growth and peritoneal metastasis. Mechanistically, SKA3 negative regulates the tumor suppressor DUSP2 and activates the MAPK/ERK pathway to promote gastric cancer.Conclusion: Our results indicate that the SKA3-DUSP2-ERK1/2 axis is involved in the regulation of gastric cancer progression, and SKA3 is a potential therapeutic target for gastric cancer. Gastric cancer (GC) has a high morbidity and mortality rate worldwide. Although GC patients can benefit from surgery and chemoradiotherapy, the 5 years survival rate is only 40% . The pooSpindle and kinetochore related complex subunit 3 (SKA3), which is encoded by the SKA3 gene on chromosome 13q12.11, is a newly discovered important factor involved in the formation of the spindle and kinetochore related complex (SKA), which controls mitosis together with the NDC80 complex . Recent in vitro  is a member of the tyrosine-specific phosphatase family , which cin vitro . Howeverin vitro and vivo. SKA3 may be the initiating factor to promote the progression of gastric cancer. The amplification of SKA3 through its downstream cascade signal transduction may be an important reason for the progress of GC. SKA3 may mediate EMT and promote the progression of GC through the DUSP2-ERK1/2 axis. Our results identified the SKA3-DUSP2-ERK1/2 axis as a potential therapeutic target for GC.Here we studied the expression and progression of SKA3 in GC Our study included two independent experimental cohorts. The first group included 35 GC cases with fresh frozen tumors and adjacent tissues selected from January to March 2018 in the Second Hospital of Jilin University, these samples were analyzed by quantitative real-time PCR (qRT-PCR) and western blot analysis. The second group included 164 GC tissue samples from patients who received radical gastrectomy at the Gastrointestinal Cancer Surgery Department of the First Affiliated Hospital of China Medical University from March 2009 to June 2012; these samples were analyzed by immunohistochemistry for SKA3 and DUSP2. All included patients had a single lesion as confirmed by pathological verification and complete postoperative follow-up data; the patients did not receive chemoradiotherapy before surgery. The study protocol was approved by the hospital ethics committee, and all patients signed informed consent.2O2 solution for 20 min at 37°C to eliminate endogenous peroxidase followed by incubation in sodium citrate buffer (pH 6.0) at 121°C for 2 min. Sections were then incubated with primary antibodies against SKA3 , or DUSP2  at 4°C overnight. Sections were then incubated with secondary antibodies at 37°C for 30 min and then stained with 3, 3′-diaminobenzidine, followed by counter-staining with hematoxylin for 1 min. Sections were dehydrated in a gradient ethanol series followed by xylene. Samples were scored according to the staining intensity and proportion of positive cells. Staining intensity was scored as follows: 0 for colorless, 1 for yellow, 2 for brown, and 3 for dark brown. The proportion of positive cells was scored as follows: < 5% was 0, 5–25% was 1, 25–50% was 2, 50–75% was 3, and >75% was 4. The final score was the product of the staining intensity and proportion of positive cell scores; samples with scores ≥5 were considered positive and samples with scores ≤4 were negative. Two authors evaluated and scored, and were blinded to the patient’s pathological information. The controversial cases were re-scored and finally reached an agreement.After dewaxing and hydration, paraffin-embedded sections were immersed in 3% H2.The immortalized gastric epithelium cell line GES-1 and GC cell lines SGC-7901, MGC-803, HGC-27, and MKN-45 were purchased from the Cell Bank of the Chinese Academy of Sciences . Cells were cultured in DMEM or RPMI 1640 medium containing 10% serum. The human peritoneal mesothelial cell (HPMC) HMrSV5 cell line was provided by Youming Peng  and cultured in RPMI 1640 medium containing 10% serum. Cells were cultured in an incubator at 37°C in 5% COSKA3 silencing lentiviruses (sh-SKA3), DUSP2 silencing lentiviruses (sh-DUSP2), DUSP2 overexpressing lentiviruses (oe-DUSP2) and empty vector virus (control) were produced by Genechem . GC cells were seeded in 12-well plates and when cells achieved 60–70% confluence, an appropriate amount of virus was added according to the cell line MOI value. Stably infected cell lines were selected in 5 μg/ml puromycin or neomycin. The sequences used to contrast sh-SKA3 are follows: sh-1, CTTATGAGAATCTGCTCAGAA and sh-2, GCATAGCTTTGGTATCCACAA. The sequence used for sh-DUSP2 is CATCTGTCTGGCATACCTCAT and the control sequence is TTCTCCGAACGTGTCACGT.Total RNA was extracted from GC tissues and cells by Trizol reagent . cDNA was generated by reverse transcription reaction using a reverse transcription kit , and the SYBR Premix Ex Taq ™ kit  was used to conduct qRT-PCR. The experiment was repeated three times. GAPDH mRNA was used as the internal reference gene. Data expression was determined by the −ΔΔCt method The primer sequences for qRT-PCR are as follows.SKA3 forward: 5′-GCCATAGATACAGAATCCAGGCT-3′SKA3 reverse: 5′-CCAAAGGAGAGTTGGTATATTCGG-3′DUSP2 forward: 5′-TGTGGAGGACAACCAGATGGTG-3′DUSP2 reverse: 5′-GAGGTATGCCAGACAGATGGTG-3′GAPDH forward: 5′-GTCTCCTCTGACTTCAACAGCG-3′GAPDH reverse: 5′-ACCACCCTGTTGCTGTAGCCAA-3′Total protein was extracted from tissues and cells using lysis buffer. Samples were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked in a 5% skim milk for 1 h. The membrane was then incubated overnight at 4°C with primary antibody, followed by incubation in secondary antibody for 1 h. ECL luminescent solution was used to visualize protein bands. Antibodies against SKA3 (ab118560), DUSP2 (ab137640), and GAPDH (ab181602) were purchased from Abcam and antibodies against ZEB1 (#70512), E-cadherin (#14472), N-cadherin (#13116), Vimentin (#5741) Erk1/2 (#4695), and p-Erk1/2 (#4370) were obtained from Cell Signaling Technology, antibodies against PCNA (10205-2-AP), c-MYC (67447-1-Ig), Cyclin D1 (26939-1-AP) were obtained from proteintech group.HGC-27 cell lysates were incubated with primary antibody (or IgG as control) and A/G agarose beads at 4°C for 6 h. After three washes with IP buffer, samples were analyzed by western blot.3 cells/well in 96-well plates. After culture for 24, 48, 72 and 96 h, 10 μl CCK8 solution was added to each well. After incubation for 2 h, the absorbance value was measured at 450 nm using a Biotek microplate reader.Cell viability was measured using the Cell Counting Kit-8 . GC cells were seeded at 2 × 103 cells/well). After culture for 14 days, the cells were fixed with paraformaldehyde for 20 min and stained with 0.4% Trypan blue for 15 min for clone counting.For colony formation assays, cells were seeded in 6-well plates  was added to the top well, and the lower chamber was filled with 600 μl medium containing 10% serum. After 16 h of culture, the Transwell chamber was removed, fixed with paraformaldehyde, and stained with 0.4% trypan blue.For migration experiments, a Transwell chamber  was placed in a 24-well plate and cells were seeded into the upper chamber at a density of 2 × 104/well cells were also seeded into the upper chamber. The subsequent steps were the same as for the migration experiment.For invasion experiments, 90 μl of diluted Matrigel  was added to the upper chamber and 2 × 104) were then added to each well of the plated HMrSV5 cells. The cells were incubated in the dark for 1 h and then washed. The number of GC cells that adhered to HMrSV5 cells was counted under a fluorescence microscope.HMrSV5 cells were seeded in a 12-well plate 1 day in advance and GC cells were seeded in a 6 cm Petri dish. Next, 2 μl Calcein-AM  was added to GC cells and cells were incubated in the dark for 1 h. The stained GC cells . The MGC-803 cells infected with lentivirus for SKA3 silencing or controls were injected into 3 × 10n = 10 per group), and 5 × 106 MGC-803 cells infected with lentivirus for SKA3 silencing or controls were injected into the abdominal cavity. After 6 weeks, mice were killed and dissected. The number of tumors on the peritoneal tissue was observed.An intraperitoneal metastasis model was established in 20 BALB/c nude mice. Mice were assigned to two groups   .SKA3 promotes proliferation, migration, invasion and adhesion of GC cells.Our results showed that high SKA3 expression was detected in MGC-803 and HGC-27 cells. Therefore, these cell lines were selected to generate cells with lentivirus-mediated stable knock-down of SKA3 (MGC-803-shSKA3 and HGC-27-shSKA3). Cells infected with lentivirus expressing control were designated as shNC. qRT-PCR and western blot were used to confirm knock-down efficiency .p < 0.05) (CCK-8 cell proliferation assay showed that the viability of cells silenced for SKA3 was significantly lower than that of the control cells . Colony  < 0.05) . Flow cy < 0.05) . Wound-h < 0.05) , and Tra < 0.05) . Our res < 0.05) . These rSKA3 regulates epithelial-mesenchymal transition (EMT) through the MAPK/ERK pathway.Since abnormal activation of EMT is an important biological process in tumor progression, we examined the EMT-related markers ZEB1, E-cadherin, N-cadherin and Vimentin in GC cells. Western blot showed that silencing SKA3 n GC cells resulted in the up-regulation of E-cadherin and down-regulation of ZEB1, N-cadherin, and Vimentin. The level of phosphorylated ERK1/2 (Thr202/Tyr204) was also decreased in SKA3 silenced cells, while there was no change in overall ERK1/2 level. Proliferation-related markers, PCNA, c-MYC and Cyclin D1 were also decreased in SKA3 silenced cells. These results suggest that silenced SKA3 inhibited proliferation, also through ZEB1 inhibited EMT and the MAPK/ERK pathway .We next established a subcutaneous tumor model and examined the effects of SKA3 knockdown on tumor growth . We founin vivo using an intraperitoneal metastasis model  (We established two stable cell lines overexpressing DUSP2 (MGC-803-oe-DUSP2 and HGC-27-oe-DUSP2), as well as a control (oeNC). Western blot was used to confirm overexpression efficiency in the DUSP2 overexpressing cell lines . CCK-8 c < 0.05) . Cell cy < 0.05) . Wound-h < 0.05) , and Tra < 0.05) . In adhe < 0.05) . Western < 0.05) . We perf < 0.05) , therebyKnockdown of DUSP2 partially rescued the effects on proliferation and invasion caused by SKA3 knockdown.To verify the SKA3-DUSP2-ERK1/2 signal axis, we performed functional rescue experiments by co-infecting GC cells with shSKA3 and shDUSP2. First, we verified the DUSP2 silencing efficiency in co-infected cells . The co-The progression of GC is the result of the joint action of a variety of factors. The progression from the initiation of cancer to invasion and metastasis takes a certain amount of time. Tumor invasion and metastasis are extremely complex dynamic processes and the most essential feature of malignant tumors. Therefore, research in GC has focus on exploring the mechanism of invasion and metastasis with the aim to identify potential targets .SKA3 is highly expressed in malignant tumors such as colon cancer, liver cancer, cervical cancer, and prostate cancer and plays an important role in cancer occurrence and development . HoweverEMT is generally regulated by upstream transcription factors. When EMT occurs, cell adhesion ability is reduced and migration ability is improved. EMT therefore plays a key role in cancer progression, as it results in tumor cell detachment from the primary focus and infiltration to distal sites . The MAPin vitro (Our study identified DUSP2 as a downstream key gene of SKA. DUSP2 is expressed at low levels in a variety of tumors, such as prostate cancer, pancreatic cancer, colon cancer, and leukemia, and it participates in the regulation of tumor development through various signaling pathways . A previin vitro . Our resin vitro , our stuin vitro . HypoxiaIn conclusion, our study found that SKA3 activates the MAPK/ERK pathway by regulating DUSP2, promoting proliferation and EMT in GC and leading to invasion and peritoneal metastasis. As an independent prognostic factor for GC, SKA3 may be a potential therapeutic target and predictive indicator in GC.


Background: Fullerenols (water-soluble derivatives of fullerenes), such as C60(OH)36, are biocompatible molecules with a high ability to scavenge reactive oxygen species (ROS), but the mechanism of their antioxidant action and cooperation with endogenous redox machinery remains unrecognized. Fullerenols rapidly distribute through blood cells; therefore, we investigated the effect of C60(OH)36 on the antioxidant defense system in erythrocytes during their prolonged incubation. Methods: Human erythrocytes were treated with fullerenol at concentrations of 50–150 µg/mL, incubated for 3 and 48 h at 37 °C, and then hemolyzed. The level of oxidative stress was determined by examining the level of thiol groups, the activity of antioxidant enzymes , and by measuring erythrocyte microviscosity. Results: The level of thiol groups in stored erythrocytes decreased; however, in the presence of higher concentrations of C60(OH)36 (100 and 150 µg/mL), the level of -SH groups increased compared to the control. Extending the incubation to 48 h caused a decrease in antioxidant enzyme activity, but the addition of fullerenol, especially at higher concentrations (100–150 µg/mL), increased its activity. We observed that C60(OH)36 had no effect on the microviscosity of the interior of the erythrocytes. Conclusions: In conclusion, our results indicated that water-soluble C60(OH)36 has antioxidant potential and efficiently supports the enzymatic antioxidant system within the cell. These effects are probably related to the direct interaction of C60(OH)36 with the enzyme that causes its structural changes. Oxidative stress is a consequence of an increase in the level of reactive oxygen species (ROS) generated by physical or chemical agents, such as ionizing and UV radiation, ultrasound, peroxides, heavy metal ions, herbicides, and several drugs . An addiπ bonds , 0.1% NaHCO3, 0.005% KCN), 20 µL of hemolysate (study sample), and 20 µL of distilled water (blank) were added. The absorbance at λ = 541 nm was measured.The hemoglobin concentration was determined in 96-well plates. To 280 µL of Drabkin’s reagent (0.03% K60(OH)36 in PBS was added to a 2% suspension of erythrocytes in PBS at a final concentration of 50, 100, and 150 µg/mL, respectively, and thoroughly mixed. The samples were incubated at 37 °C. The determinations were made after 3 and 48 h of incubation.A 5 mg/mL solution of Ck = 6.5 × 10−9 s/mTW0 = width of the spectral center lineh0 = height of the spectral midfield lineh–1 = height of the highfield line of the spectrumA 50% hematocrit was prepared by centrifuging 1.6 mL of the incubated erythrocyte suspension and collecting 1.54 mL of the supernatant. To the remaining 60 µL, 0.6 µL Tempamine (spin tracer: 10 mM of Tempamine dissolved in PBS) was added and stirred for 30 min at room temperature. The samples were then washed with 1 mL of 80 mM potassium ferricyanide and centrifuged, and the EPR spectra of the samples see , as wellτR = rotational correlation time of the tracer inside erythrocytesBτ = rotational correlation time of the tracer dissolved in a solution of known viscosityRη = erythrocyte inner viscosityBη = viscosity of known solutionThe viscosity of the interior of the erythrocyte was calculated from the following equation:Incubated erythrocytes with 2% hematocrit were diluted to a hematocrit of 0.75% with a final volume of 160 µL. Then, 0.45 µL of tracer was added so that the final concentration in the sample was 80 µmol. After 3 min of incubation with the tag, EPR spectra were obtained similarly as described previously .To a spectrophotometric cuvette, 3 mL of 0.018 M hydrogen peroxide in 0.05 M phosphate buffer (pH 7.0) at 25 °C was added so that the absorbance measured at 240 nm was approximately 1. Then 15 µL of hemolysate was added, and the decrease in absorbance at λ = 240 nm was measured for 1 min. The activity was referred to 1 mg of hemoglobin protein.3 solution, 50 µL of hemolysate to which 2 µL of 100 mM sodium azide was added to block catalase, and 2 µL of a mixture of 60 mM K3Fe(CN)6 and 48 mM KCN solutions were added to convert hemoglobin to cyanmethemoglobin. The reaction was initiated by adding 50 µL of 12 mM t-butyl hydroperoxide. The decrease in absorbance at λ = 340 nm was measured for 3 min. The activity was referred to 1 mg of hemoglobin protein.Glutathione peroxidase activity was determined by an indirect method. In the spectrophotometer cuvette, 250 µL of phosphate buffer (0.1 M pH 7.0 with 0.1 mM EDTA) equilibrated at 37 °C, 50 µL of glutathione reductase (2.4 units/mL), 50 µL of 10 mM GSH, 50 µL of 1.5 mM NADPH in a 0.1% NaHCOEight hundred fifty µL of phosphate buffer (0.1 M pH 6.5) equilibrated at 37 °C was added to the spectrophotometer cuvette followed by 50 µL of 0.02 GSH solution, 50 µL of CDNB solution , and 50 µL of hemolysate. The absorbance at λ = 340 nm was measured for 1 min. The activity was referred to 1 mg of hemoglobin protein.One thousand µL of 2.2 mmol/L oxidized glutathione solution at 37 °C was added to a spectrophotometer cuvette followed by 40 µL of hemolysate. It was thoroughly mixed and 200 µL of 0.17 mmol/L of NADPH was added. The decrease in absorbance was measured at λ = 340 nm for 2 min. The activity was referred to 1 mg of hemoglobin protein.All experiments were carried out 3 to 8 times. Values were expressed as mean ± standard deviation (SD). Data were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test, all using GraphPad 4.0 software .60(OH)36 can modulate the redox balance of human erythrocytes subjected to oxidative stress induced during a prolonged incubation in PBS at 37 °C. Apart from direct antioxidant action, fullerenol interacts with the antioxidant enzymes and stimulates them to more efficient and longer activity. A 3 and 48 h treatment with C60(OH)36 (50–150 µg/mL) maintains the proper function of the antioxidant system (CAT and GPx). However, we hypothesize that changes in its activity are not caused directly by the involvement of ROS, but conformational changes induced by C60(OH)36 itself. These effects are related to the concentration of C60(OH)36 and to the kind of parameters examined that characterize cell damage.The results presented here reveal that C


After that, the as-prepared SBA-15/Fe3O4 functionalized with 3-minopropyltriethoxysilane (APTES) via post-synthesis approach. Then, the guanidinylated SBA-15/Fe3O4 was obtained by nucleophilic addition of APTES@SBA-15/Fe3O4 to cyanimide. The prepared nanocomposite exhibited excellent catalytic activity in the synthesis of dihydropyranopyrazole derivatives which can be related to its physicochemical features such as strong basic sites (presented in guanidine group), Lewis acid site (presented in Fe3O4), high porous structure, and high surface area. The characterization of the prepared mesoporous nanocomposite was well accomplished by different techniques such as FT-IR, EDX, FESEM, TEM, VSM, TGA, XRD and BET. Furthermore, the magnetic catalyst was reused at least six consequent runs without considerable reduction in its catalytic activity.In this study, a novel mesoporous nanocomposite was fabricated in several steps. In this regard, SBA-15 was prepared by the hydrothermal method, next it was magnetized by  Based on the IUPAC definition, the porous materials are classified in three main groups depending on their pore size (or pore width): microporous (pores width < 2 nm), mesoporous (2 < pores width < 50 nm), and macroporous (pores width > 50 nm) materials3. In 1998, Stucky and et al. synthesized a novel kind of hexagonal array of pores named SBA (Santa Barbara Amorphous). After that, different types of SBA materials such as SBA-1, SBA-16 and SBA-15 have synthesized. The SBA-15 is a highly ordered mesoporous materials with striking properties for instance high surface area, straight cylindrical pores, thick framework walls, adjustable pore size (4–30), large pore diameter (which provide a facilitated diffusion of reactant molecules), and excellent ability to be modified/functionalized. It has been extensively utilized in various application such as catalysis4, removal of pollutants form wastewater5, hyperthermia6, drug delivery8, and chromatographic techniques9. To obtain high performance SBA-15-based catalysts, the surface functionalization or modification needs to be performed. There are two main approach to modify/functionalize the silica-based mesoporous materials including one-pot synthesis or co-condensation and grafting technique or post-synthesis. In the first approach, the active phase is added to the reaction mixture which subsequently co-assembles into the inorganic framework for the construction of the mesoporous material in single step, but in the second approach siliceous support is prepared followed by the modification with active moieties or their precursors11. Incorporating Fe3O4 MNPs as a superparamagnetic material into the silica-based mesoporous materials is a practical way to achieve retrievable and reusable nanocomposite with enhanced surface area12. The chemical modification of SBA-15 gives high efficiency porous catalyst by more active sites to interact with reactants. In recent years, a great attention has been dedicated to prepare amine functionalized SBA-15 to enhance its potential catalytic application. For instance, several types of amines functionalized SBA-15 were fabricated via grafting technique by using three different aminosilane reagents then, it was applied as heterogeneous catalysts for Michael addition13. One of the most widely used materials to modify the SBA-15 through both co-condensation or and post-synthesis is the 3-aminopropyltriethoxysilane (APTES)15. Guanidine, the nitrogenous analogue of carbonic acids, is a fascinating group of basic organic compounds. They are exceedingly strong Brønsted and Lewis bases, their basic strength more than amines, pyridines, diamines and amidines. This strong basicity is ascribed to great delocalization of positive charge on the guanidinium cation above the three nitrogen atoms. After protonation of guanidine group, the highly stable guanidinium ion act as a bidentate hydrogen bond donor which able to activate different hydrogen bond acceptors species such as carbonyl groups17. Therefore, guanidine and their derivatives can be considered as appropriate candidates in base-catalyzed organic reactions. The guanidinylation reaction has been used to convert primary amine groups in different materials for example chitosan19 and Poly(2-guanidinoethylmethacrylate)20 into guanidine groups. Multicomponent reactions (MCRs) are one of the most significant methods for the synthesis of heterocyclic compounds because of their outstanding properties such as high atomic economy, short reaction time, straightforward reaction model, high selectivity, and great compliance with principals of green chemistry23. Among the product of MCRs, the pyranopyrazole derivatives have received much interest of researchers due to their extensive application in pharmacology and medicine27. In continuation of research on heterogeneous nanocatalysts30, in this study a novel SBA-15 based nanocomposite prepared in four steps as illustrated in Fig. Porous materials with remarkable characteristics such as high surface area, well-defined porous structure, uniform pore size, and processability have attracted great interest from researchers in scientific fields1HNMR and 13C NMR nuclear magnetic resonance spectra were recorded on a Bruker DRX-500 spectrometer at 500 and 125 MHz, respectively. Analytical thin-layer chromatography (TLC) was done by Merck silica gel GF254 plates.All the required chemical reagents and solvents were purchased from the chemical international companies including Merck and Sigma Aldrich. Several analyses were carried out to demonstrate the construction of the catalyst. Fourier-transform infrared (FT-IR) spectra were carried out by applying a Shimadzu IR-470 spectrometer by using KBr pellet. Elemental analysis of the prepared samples was performed by energy dispersive x-ray analysis (EDX) recorded on Numerix JEOL-JDX 8030 . X-ray diffraction (XRD) pattern of fabricated samples were obtained by X-ray diffractometer (Bruker D8 Advance). The morphology and surface of samples were studied by a field emission scanning electron microscope (FESEM) and transmission electron microscopies (TEM) using VEGA2 TESCAN instrument and Zeiss-EM10C-100 kV, respectively. The magnetic behavior of samples was measured using VSM analysis , and thermal stability of samples was studied by thermogravimetric analysis (TGA) using a BAHR-STA 504 instrument. Melting points of synthesized products were measured with an Electrothermal 9100 apparatus. The specific surface area of prepared samples was determined using the Brunauer–Emmett–Teller . 31, the SBA-15 was synthesized by the hydrothermal method. First, 3.5 g of P123 surfactant as a structure-directing agent was dissolved in 120 mL of 1.6 M hydrochloric acid. Next, the obtained solution stirred at 40 °C for 24 h to get a homogenous mixture. After that, 8 mL (7.5 g) of TEOS (tetraethyl orthosilicate) as an alkoxysilanes reagent was added into the above clear solution and stirring was continued at 40 °C for 24 h to complete hydrolysis of silica source. Then, the resulting mixture was transferred into the teflon-lined autoclave, and heated at 150 °C for 24 h. After the autoclave temperature gradually decreased, the resulting mixture was filtered, washed several consecutive times with distilled water (15 × 25 mL), and then dried at room temperature. The final white powder was calcined at 550 °C for 4 h with a heating rate of 2.3 °C min-1 for removal of surfactant and forming the ordered mesoporous channels.According to the previously reported studiesin-situ co-precipitation method was performed. For this, 2 mmol (0.4 g) of FeCl2·4H2O and 4 mmol (0.54 g) of FeCl3.6H2O were dissolved in 50 ml distilled water. The obtained homogeneous solution was transferred to a 2-necked round-bottom flask containing 0.5 g dispersed SBA-15 in 50 mL distilled water. The final mixture was kept stirring at room temperature for about 45 min under N2 atmosphere. Then, the temperature was gradually raised to 85 °C. After that, 10 mL of ammonium hydroxide (25%) was added dropwise to the stirring solution. The reaction was kept under this condition for 2 h to form Fe3O4 MNPs on the mesoporous SBA-15 support. The synthesized magnetic product was collected by a magnet, washed with distilled water and acetone, and then dried in an oven at 90 °C for 5 h.To magnetize SBA-15, the 3O4 by APTES was carried out by the post-synthesis approach. At first, 0.5 g of SBA-15/Fe3O4 and 2 mL of APTES were dispersed in 35 mL of toluene by stirring. Then, the obtained mixture was refluxed at 115 °C for 48 h. It is expected that free silanol groups (Si–OH) of SBA-15/Fe3O4 covalently react with the silica group on the APTES to form Si–O-Si bond (siloxane bond) through condensation reaction. The dark brown precipitate was isolated by a magnet, washed several times with ethanol and acetone and then dried in oven at 100 °C.The functionalization of SBA-15/Fe3O4 was obtained by nucleophilic addition between APTES @SBA-15/Fe3O4 and cyanamide. Initially, 0.5 g of cyanamide was dissolved in 5 ml distilled water and added to 0.1 g APTES@SBA-15/Fe3O4 which dispersed in 35 mL acetone; next the mixture was kept stirring for 24 h at 85 °C. Then, the final product collected by a magnet, washed several times with distilled water and acetone, and dried in oven at 100 °C.The guanidinylated SBA-15/Fe3O4 mesoporous catalyst in the presence of 1 ml EtOH/ H2O (1:1) at 80 °C. The reaction completion process was investigated by thin layer chromatography (TLC). After completion of the reaction, hot ethanol was added to dissolve the product, then undissolved magnetic mesoporous catalyst was separated from the reaction mixture by a magnet and filtration. The crude products were recrystallized from EtOH to obtain pure dihydropyrano pyrazole derivatives.The catalytic performance of fabricated mesoporous catalyst was evaluated in one-pot synthesis of the pyranopyrazoles. For this, four components including ethyl acetoacetate (1 mmol), hydrazine hydrate (1.2 mmol), aromatic aldehyde (1.0 mmol) and malononitrile (1.0 mmol) were mixed and reacted by adding 0.01 g of the guanidinylated SBA-15/Fe5b): 1H-NMR : δH (ppm) = 1.79 , 4.63 , 6.91 , 7.19–7.20 , 7.37–7.38 , 12.12 ; 13C NMR ; δC (ppm) = 10.18, 36.04, 57.30, 97.66, 121.06, 128.91,129.82, 131.69, 136.12, 143.95, 155.19, 161.38.6-Amino-4-(4-chlorophenyl)-3-methyl-2,4-dihydropyranopyrazole-5-carbonitrile (5e): 1H-NMR : δH (ppm) = 1.81 , 4.88 , 7.05 , 7.65 , 8.02 , 8.11 , 12.20 ; 13C NMR ; δC (ppm) = 9.74, 35.66, 56.17, 96.65, 120.49, 121.83, 121.93, 130.22, 134.36, 135.89, 146.8, 147.88, 154.69, 161.13.6-Amino-3-methyl-4-(3-nitrophenyl)-1,4-dihydropyrano  pyrazole-5-carbonitrile (5k): 1H-NMR : δH (ppm) = 1.78 , 4.47 , 6.68–6.69 , 6.76 , 6.94–6.96 , 9.25  12.03 ; 13C NMR ; δC (ppm) = 10.2, 35.9, 58.3, 98.5, 115.6, 121.3, 128.9, 135.2, 135.9, 155.2, 156.5, 161.1.6-Amino-4-(4-hydroxyphenyl)-3-methyl-2,4-dihydropyranopyrazole-5-carbonitrile 34. IR spectrum of SBA-15/Fe3O4 in addition to similar absorptions which observed in spectrum a showed a new absorption bands at 577 cm−1 which assigned to Fe–O stretching vibration. After the functionalization of the SBA-15/Fe3O4 with APTES, two new absorption bands have emerged in 1562 and 2925 cm-1 which are related to the bending vibration of NH2 and the stretching vibrations of the C-H bond in the propyl chain of APTES, respectively (spectrum b) Furthermore, in this spectrum the intensity of absorption of the Si–OH bands at 958 cm-1 was decreased, signifying that the surface silanols are substituted by aminosilane groups after functionalization36. Moreover, guanidinylation of APTES @SBA-15/Fe3O4 was evaluated by comparing IR spectra of guanidinylated SBA-15/Fe3O4 (c) and APTES@SBA-15/Fe3O4 (b). Nucleophilic addition of APTES@SBA-15/Fe3O4 to cyanimide resulted in disappearing the bending vibration of NH2 at 1560 cm-1 and intensification the absorption band at 1627 cm-1 which can be attributed to the stretching vibrations of the imine bond of guanidine group formed during this final step modification37.FT-IR analysis was used to detect different functional groups in the samples fabricated in each step. As is observed in Fig. 3O4 (spectrum b), observing two Fe peaks are related to the existence of Fe3O4 MNPs in the structure of the SBA-15/Fe3O4. By modification of the SBA-15/Fe3O4 with APTES, the peaks of C and N elements were added to the previous peaks in the spectrum c. The guanidinylated SBA-15/Fe3O4 can cause the emerging of Fe, O, Si, C and N peaks in the EDX spectrum d. Moreover, the distribution of elements in this mesoporous nanocomposite is shown in the EDX mapping images (e).Detection of the organic and inorganic elements in the prepared samples was carried out by the EDX analysis as a qualitative method. As is shown in Fig. 3O4 and SBA-15 are presented in three scales: 1 µm, 500 and 200 nm. The SBA-15 images presented a porous structure, but in the nanocomposite images in addition to the porous structure, the distribution of spherical Fe3O4 MNPs on the SBA-15 as a mesoporous support was also can be observed. Therefore, fabrication Fe3O4 MNPs onto SBA-15 mesoporous matrix and subsequent modification resulted in change the its morphology. The average particle size for 35 spherical particles in the nanocomposite was determined to be about 26 nm using Digimizer software. TEM analysis was performed to more accurately study the morphology and particle size of the mesostructured guanidinylated SBA-15/Fe3O4 catalyst. As can be seen in Fig. 3O4 MNPs onto SBA-15 support was observed, but formation of the magnetite NPs onto the certain amount of SBA-15 channels and subsequent functionalization lead to hide some part of ordered pore arrangement.Field emission scanning electron microscopy was employed to observe particle size distribution, surface morphology and particle aggregation mode in prepared samples. As is observed in Fig. 3O4, SBA-15/Fe3O4 and guanidinylated SBA-15/Fe3O4 were about 52.1, 30.1, and 12.16 emu/g, respectively. The reduction in Ms of the SBA-15/Fe3O4 compared to bare Fe3O4 is due to the presence of SBA-15 as a non-magnetic component in the SBA-15/Fe3O4 structure. Then, by further chemical modification on SBA-15/Fe3O4 and the addition of more non-magnetic segments, the amount of Ms in guanidinylated SBA-15/Fe3O4 was decreased to about 12.16 (emu/g), which seems reasonable. However, the nanocomposite is sufficiently magnetized to be effortlessly separated from the reaction mixture by a magnet and reused for several consecutive times. Besides, considering that the values of coercivity (Hc) and remanence (Mr) in the VSM curves of the prepared samples are zero, their superparamagnetic behavior is confirmed.To evaluate the magnetic properties of the prepared samples, vibrating sample magnetometer (VSM) was employed at room temperature and in the field range of -10 < kOe <  + 10. As indicated in Fig. 3O4 and guanidinylated SBA-15/Fe3O4 was performed by TGA analysis in the temperature range 50–800° C with a heating rate of 10 ºC min-1 under air atmosphere. For a more detailed study, the thermal behavior of SBA-15 and Fe3O4 which reported in literature was also studied. According to the reported information, SBA-15 has high thermal stability; it has maintained above 90–95% of its weight up to 700 ºC and a continuous slight weight loss was attributed to dehydrogenation or dehydroxylation of its surface39. Moreover, the thermal behavior of Fe3O4 displayed that with increasing the temperature to 800 °C, a small weight loss of about 5–6% was occurred, which was ascribed to the evaporation of water molecules absorbed in it40. As can be observed in Fig. 3O4 (a) exhibited a gradual gentle weight loss between 50 and 800° C which may be related to the evaporation of adsorbed water molecules in the cavities of this sample, and dehydrogenation or dehydroxylation of its surface. Therefore, high thermal stability of this composite was demonstrated with just 9% weight loss until 800 °C. In the thermogram of guanidinylated SBA-15/Fe3O4 nanocomposite (b), the first observed weight loss (~ 2%) in the temperature range of 50–160 °C is attributed to the evaporation adsorbed water molecules in the cavities and surface of the mesoporous nanocomposite. Next, with increasing temperature to 550 °C weight loss of about 10% occurs, which can be due to the separation and thermal decomposition of organic parts  that covalently bounded to the SBA-15/Fe3O4, and the continuation of weight loss with increasing temperature up to 800 °C can be ascribed to the condensation of silanol groups of guanidinylated SBA-15/Fe3O441. The residual weight of these mesoporous nanocomposites up to 800 °C is about 85%, which is only 6% more weight loss than the SBA-15/Fe3O4 compound. Therefore, it can be referred that the two steps chemical modification of SBA-15/Fe3O4 did not have a significant effect on its thermal resistance. Furthermore, considering the difference in residual weight of the two samples, it can be calculated that just about 6% of the total weight of the guanidinylated SBA-15/Fe3O4 nanocomposite composed of the organic part. The differential thermogravimetric analysis (DTGA) of guanidinylated SBA-15/Fe3O4 (spectrum c) showed main endothermic peaks at 123 and two weak peaks at 411 and 510, the first one is attributed to the loss of adsorbed water molecules in mesoporous nanocomposite and the others related to decomposition of the organic part which constitute a very small weight percent of nanocomposite and the condensation of silanol groups of SBA-15. It can be seen from these results that the prepared mesoporous nanocomposite has structural stability at high temperatures and can be used for catalytic reactions at high temperature.Thermal analysis of the SBA-15/Fe42.The diffractogram of guanidinylated SBA-15/Fe3O4 (c) exhibited the relatively broad peak at 2Ө: 20–30° with attributed to the presence of SBA-15, which was lower intensity compared to unmodified SBA-15, this reduction can be ascribed to three steps of modification of SBA-15. Moreover, there are several peaks at 2Ө: 30.38°, 35.74°, 43.40°, 58°, 63.22° correspond exactly to the index peaks in the XRD pattern of Fe3O4 confirming the presence of these nanoparticles in the guanidinylated SBA-15/Fe3O4 structure. It can be concluded that formation of Fe3O4 MNPs on SBA-15 matrix even with modifications has enhanced its crystallinity. The average crystallite size of neat Fe3O4 and the guanidinylated SBA-15/Fe3O4 nanocomposite by the Scherer equation was calculated to be about 19 and 37 nm, respectively. The increase in average crystallite size of mesoporous nanocomposite in compared with neat Fe3O4 can be mainly due to the SBA-15 matrix Furthermore, the X-ray diffraction patterns of all prepared samples at low angles in the 2Ө: 0–5° are shown in Fig. 3O4, APTES@SBA-15/Fe3O4 and guanidinylated SBA-15/Fe3O4 samples showed the first peak corresponding to the (100) reflection which intensity decreases with each step modification, but two minor peaks corresponding to (1 1 0) and (2 0 0) planes are became weaker or almost disappeared in these samples which can be attributed to partial disruption of the structure by incorporation of Fe3O4 MNPs and the lowering of the local order.The XRD analysis was used to investigate the crystalline nature of prepared samples. As is depicted in Fig. 2 adsorption–desorption isotherm of the SBA-15, SBA-15/Fe3O4 and guanidinylated SBA-15/Fe3O4 are presented in Fig. 2/g which is more than the BET surface area of the SBA-15/Fe3O4 and guanidinylated SBA-15/Fe3O4. This observation can be mainly related to blocking of a certain amount of SBA-15 pores by the embedding of Fe3O4 MNPs on the SBA-15 pores and its subsequent functionalization, which resulted in less available surface area for gas adsorption. BJH pore size distributions of samples are depicted in Fig. 3O4 and guanidinylated SBA-15/Fe3O4 samples has slightly more pore width (size) and also relatively broader pore size distributions compared to their precursor matrix.The N3O4 investigated in the synthesis of dihydropyrano  pyrazole derivatives. To obtain the best result, different experimental conditions such as temperature, solvent, amount of catalyst and the type of catalysts was examined in the one-pot four components reaction of ethyl acetoacetate (1 mmol), hydrazine hydrate (1.2 mmol), 3-nitrobezaldehyde (1 mmol) and malononitrile (1 mmol). First, the model reaction was performed without catalyst and solvent in two different temperatures, the yield of products was trace , Lewis acid site (Fe3+ in Fe3O4), high porosity and large surface area acted as an excellent catalyst to accelerate the synthesis of dihydropyrano  pyrazole derivatives via one-pot four-component reaction.The catalytic activity of the guanidinylated SBA-15/Feropyrano , 3-c pyrropyrano , 3-c pyrropyrano , 3-c pyrropyrano , 3-c pyrropyrano , 3-c pyrropyrano , 3-c pyrropyrano , 3-c pyr3O4 mesoporous nanocatalyst in short reaction times.To assess the generality of the optimum conditions, a wide range of benzaldehydes bearing both electron-donating and electron-withdrawing substitution were tested. As is observed in Table 3O4 nanocomposite as an appropriate catalyst to promote the dihydropyrano, its catalytic activity in the synthesis of 5e derivatives was compared with some other previously reported methods. As indicated in Table To assess the catalytic efficiency and advantages of the guanidinylated SBA-15/Fe3O4 mesoporous nanocomposite with dual active sites: base (primary and secondary amines in guanidine groups), Lewis acid site (Fe3+ in Fe3O4), high porous structure and large surface area, play an important role in all steps of this four-component reaction as illustrated in Fig. 47, several main steps give the final product. First, the carbonyl groups of ethyl acetoacetate are activated by both amine group through hydrogen bonding and the Lewis acid site in the guanidinylated SBA-15/Fe3O4 catalyst and then subjected to the nucleophilic attack of hydrazine hydrate with two nucleophilic sites. In this step, a pyrazolone ring is formed (intermediate I) by removing water and ethanol molecules, respectively. On the other hands, intermediate II  was produced through the Knoevenagel condensation reaction between catalysts-activated malononitrile  and activated aromatic aldehyde (by hydrogen bond and Lewis acid site). Then, the Michael addition reaction between catalyst-activated the intermediate I and II, resulted in the compound III. The subsequent enolization and cyclization of intermediate III provided compound IV. Eventually, by tautomerization of molecule IV, dihydropyranopyrazole derivatives (5a-p) were obtained.As mention earlier, guanidinylated SBA-15/Fe55. The reusability of prepared mesoporous catalysts in the synthesis of pyranopyrazoles derivatives was evaluated in several runs. For this, after completion of the reaction, the catalyst was separated from the reaction mixture by a magnet, eluted by ethanol and dried in an oven at 60 °C for 6 h in order to be ready for the next catalytic run. Later, the recovered catalyst in a constant amount was used for the subsequent runs. The presented results in Fig. The recovery and reusability of catalysts in catalytic reactions are important issues in term of the green chemistryin-situ construction of Fe3O4 MNPs, consequent functionalization by APTES and finally guanidinylation reaction. This fabricated nanocomposite exhibited great catalytic performance in the synthesis of dihydropyrano pyrazole. The corresponding products were obtained in high yields without a complicated work-up procedure. The result of TGA analysis indicated that this nanocomposite has a very high thermal stability and has lost only about 15% of its weight up to 800 °C. The XRD pattern of nanocomposite in comparison with SBA-15 revealed that fabrication of Fe3O4 MNPs with crystalline nature on SBA-15 porous support, even with two consecutive chemical modifications has enhanced its crystallinity. Moreover, presented peaks in diffractogram of guanidinylated SBA-15/Fe3O4 mesoporous were in good agreement with characteristic peaks of SBA-15 and standard Fe3O4. The VSM analysis demonstrated the superparamagnetic property of nanocomposite with a Ms of about 12.2 emu/g. The FESEM images of SBA-15 and mesoporous nanocomposite showed the porous structure of SBA-15 and distribution of spherical shaped Fe3O4 MNPs on SBA-15 support with an average size of about 26 nm. The FESEM of SBA-15 and mesoporous nanocomposite showed the porous structure of SBA-15 and distribution of spherical shaped Fe3O4 MNPs on SBA-15 support with an average size of about 26 nm, and TEM images of nanocomposite exhibited both a regular mesoporous arrangement with two-dimensional hexagonal honeycomb structure and the Fe3O4 MNPs onto SBA-15 support. Based on the suggested mechanism, the guanidinylated SBA-15/Fe3O played a vital role in conducting the synthesis reaction of dihydropyrano pyrazole derivatives with beneficial features such as strong basic sites, Lewis acid site, porous architecture and high surface area.A novel mesoporous SBA-15 based nanocomposite prepared by modification of SBA-15 through Supplementary Information.


Here, we have performed a detailed side-by-side comparison of the ion selectivity of TMEM16F using the whole-cell and inside-out excised patch configurations to directly compare the results. In inside-out configuration, Ca2+-dependent activation was fast and the TMEM16F-mediated current was activated in a few milliseconds, while in whole-cell recordings full activation required several minutes. We determined the relative permeability between Na+ and Cl¯ (PNa/PCl) using the dilution method in both configurations. The TMEM16F-mediated current was highly nonselective, but there were differences depending on the configuration of the recordings. In whole-cell recordings, PNa/PCl was approximately 0.5, indicating a slight preference for Cl¯ permeation. In contrast, in inside-out experiments the TMEM16F channel showed a higher permeability for Na+ with PNa/PCl reaching 3.7. Our results demonstrate that the time dependence of Ca2+ activation and the ion selectivity of TMEM16F depend on the recording configuration.TMEM16F is involved in several physiological processes, such as blood coagulation, bone development and virus infections. This protein acts both as a Ca K1/2 slightly decreased with membrane depolarisation from 22.2 μM at +60 mV to 17.4 μM at +100 mV, while the Hill coefficient ranged from 1.6 to 3.8 , where c is the Ca2+ concentration, K1/2 is the Ca2+ concentration producing half-maximal current activation and nH is the Hill coefficient. Similar to the data obtained in whole cell recordings, K1/2 slightly decreased with membrane depolarisation from 43 ± 6 μM at +60 mV to 28 ± 1 μM at +100 mV  calculated with the Goldman–Hodgkin–Katz equation was 0.52 ± 0.03 , very different from the value of 0.52 ± 0.03 (n = 7) calculated from whole-cell recordings , showing that also in these experimental conditions Na+ was more permeant than Cl¯. Thus, the significant difference between PNa/PCl measured in whole-cell or in the inside-out configuration was not due to an asymmetric channel pore , as expected for channels more permeable to cations than to anions. Pcordings F. Thus, urations C,F, we rurations E. The avnel pore F.Na/PCl from the Goldman–Hodgkin–Katz equation [Na/PCl obtained with solutions containing 280 and 70 mM NaCl were 1.98 ± 0.09 and 2.3 ± 0.2 respectively, significantly different from the value of 3.7 ± 0.1 measured in 14 mM NaCl . However, although PNa/PCl changed with the variation of ionic strength, the TMEM16F wt channel in inside-out patches remained more permeable to Na+ than to Cl¯.Since using the dilution method could cause significant deviations from the expected value of Pequation ,39, we tNa/PCl of TMEM16F wt in inside-out patches may depend on Ca2+ concentration [2+ from 100 to 13 µM, a concentration that produced about 10% of the maximal current at +100 mV . The large anion MeS¯ was also permeant, although at different extents depending on the recording method. Indeed, substitution of NaCl with NaMeS in whole-cell shifted the reversal potential of 21.5 ± 0.5 mV (n = 3), indicating that MeS¯ was much less permeant than Cl¯, whereas in inside-out the change was only −1.6 ± 0.7 mV (n = 14) showing a very small difference between MeS¯ and Cl¯ permeability  in Q559K , indicating that the pore of the Q559K mutant was not asymmetric, similarly to the wt pore . Furthermore, PNa/PCl  for the constitutive scramblase mutant Y563A was not significantly different from the wt value, whereas PNa/PCl for the scrambling-deficient D703R mutant  was significantly higher than the wt value  in excised patches. Both the fast Ca2+ activation and the rundown in inside-out patches resemble those measured in TMEM16A [Our data confirm that the kinetics of activation by Calication D in agree et al. , who sho TMEM16A  and TMEM TMEM16A ,9.2+-activated current in whole-cell recordings slowly and progressively developed after a delay of some minutes following membrane breaking and did not show rundown. The time necessary to obtain the maximal current was about 2 min with 100 μM Ca2+ in the patch pipette and increased to about 20 min with a reduction in intracellular Ca2+ to 3.8 μM  was not different from that measured at 100 μM Ca2+  when NaCl concentration was reduced from 140 to 14 mM at the intracellular (or extracellular) side of the patch membrane and channels were activated by high Ca2+ concentrations. PNa/PCl increased to 1.9 when TMEM16F Q559K was activated by 13 μM Ca2+  measured in whole-cell was not different from the value of TMEM16F wt (0.52), showing that the mutation did not modify selectivity in the whole-cell configuration.When the permeability ratio was measured in whole-cell, P2+-activated scramblase activity of TMEM16F could play a role in ion selectivity. Indeed, previous work [Aspergillus fumigatus) reconstituted in lipid bilayers were reported to strongly depend on the lipid composition, suggesting that different lipid environments generated by the scramblase activity may affect the properties of the TMEM16F pore. Le et al. [Na/PCl increased in the mutant  supplemented with 10% foetal bovine serum , 100 IU/mL penicillin and 100 μg/mL streptomycin  at 37 °C in a humidified atmosphere of 5% COpEGFP-N1 plasmids containing the cDNA of mouse TMEM16F wt (version 1 as in ) or Q559TMEM16F-transfected HEK-293 cells were identified by EGFP fluorescence using an Olympus IX70 microscope  equipped with the appropriate filter. TMEM16F currents were recorded in whole-cell or inside-out configurations in voltage-clamp mode using an Axopatch 1-D amplifier controlled by Clampex 9.2 via Digidata 1322A . The data were acquired at a rate of 10 kHz, and the signals were low-pass filtered at 5 kHz. Patch electrodes were made of borosilicate glass  and pulled with a PP-830 micropipette puller . Pulled patch electrodes had a resistance of 2–3 MΩ for whole-cell recordings and 0.5–1 MΩ for the inside-out configuration when filled with pipette solution.The bath was ground with a 3 M KCl agar bridge connected with an Ag/AgCl reference electrode. In experiments with pipette solution without Cl¯, a 3 M KCl agar bridge was used. All experiments were conducted at room temperature. Solutions were rapidly changed close to the cells or excised patches using multibarrel glass tubes. Each tube composing the multibarrel had an internal diameter of 0.9 mm . Changes between different solutions were performed using the Perfusion Fast-Step SF-77B .2+-containing solution for 1 s at +100 mV and then applied a ramp from +80 to −80 mV at 0.36 mV/ms. Leak currents measured in nominally 0 Ca2+ solution were subtracted. The reversal potential (Erev) was estimated by using a fit with the polynomial function I = a1 + a2V + a3V2 + a4V3 + a5V4 + a6V5 + a7V6, where I is the current, V is the voltage and ai are numerical parameters.For IV relations in the inside-out configuration, we exposed the patches to Ca1 exp (−t/t1)), was fitted to tail currents to extrapolate the tail current value at the beginning of the step. Tail current values were plotted as a function of voltage, and Erev was estimated using a fit with a polynomial function.For whole-cell recordings of tail currents, the protocol consisted of voltage steps of 500 ms duration from a holding potential of 0 mV to + 80 mV followed by voltage steps ranging from −20 to +50 mV. A single-exponential function , and applied voltages were corrected off-line.2, 1 MgCl2, 10 glucose and 10 HEPES, pH 7.4 with NaOH.Cells were kept in mammalian Ringer’s solution composed of (in mM) 140 NaCl, 5 KCl, 2 CaCl2 to obtain 3.8, 13 or 30 µM free Ca2+, respectively. For intracellular solutions containing Ca2+ concentrations >30 µM HEDTA was omitted and an appropriate amount of CaCl2 was used. For permeability experiments, cells were bathed with solution containing (in mM) 140 NaCl and 10 HEPES, pH 7.4. NaCl was reduced to 14 mM or replaced with N-Methyl-D-glucamine chloride (NMDG-Cl), sodium methanesulfonate (NaMeS) or sodium isothiocyanate (NaSCN). The osmolarity in the low NaCl solution was adjusted with sucrose.For whole-cell recordings, the pipette solution contained (in mM) 140 NaCl, 10 HEPES and 10 HEDTA, pH 7.2 and 5.86, 8.26 or 9.26 mM CaCl2+ solution containing (in mM) 140 NaCl, 10 HEDTA or 5 EGTA and 10 HEPES, pH 7.2. For dose-response relations, the bath solution was the same as that used in the pipette for whole-cell recordings. For permeability experiments using the dilution method, the bath solution contained (in mM) 140 NaCl, 10 HEPES and 0.1 or 1 mM CaCl2 and NaCl was changed to 280, 70, 28, or 14 mM. The osmolarity in the low-NaCl solution was adjusted with sucrose. In some experiments, NaCl was replaced with NMDG-Cl, NaMeS, NaSCN, NaBr, NaNO3 or NaI. All chemicals, unless otherwise stated, were purchased from Sigma-Aldrich .For inside-out recordings, the pipette solution contained (in mM) 140 (or 14) NaCl, 5 EGTA and 10 HEPES, pH 7.2 with NaOH. The patches were maintained in nominally 0 Ca1 exp (−t/t1) + A2 exp (−t/t2), where t1 and t2 are the time constants. The normality of the data was tested with the Shapiro–Wilk test, while the homogeneity of variance was tested with Levene’s test. For normally distributed data, statistical significance was determined using paired unpaired t-tests or ANOVA as appropriate. When a statistically significant difference was determined with ANOVA, a post-hoc Tukey test or Dunnett test was performed to evaluate which data groups showed significant differences. For non-normally distributed data, we used the Wilcoxon–Mann–Whitney test. For multicomparison of the data we used Dunn–Hollander–Wolfe test after Kruskal–Wallis analysis. Data in p values < 0.05 were considered significant.Data analysis and figures were made with IgorPro software . Data are presented as the mean ± sem (standard error of the mean), with n indicating the number of cells or excides patches. The double exponential function used to fit the data in


A miniature tyrosinase-based electrochemical sensing platform for label-free detection of protein tyrosine kinase activity was developed in this study. The developed miniature sensing platform can detect the substrate peptides for tyrosine kinases, such as c-Src, Hck and Her2, in a low sample volume (1–2 μL). The developed sensing platform exhibited a high reproducibility for repetitive measurement with an RSD (relative standard deviation) of 6.6%. The developed sensing platform can detect the Hck and Her2 in a linear range of 1–200 U/mL with the detection limit of 1 U/mL. The sensing platform was also effective in assessing the specificity and efficacies of the inhibitors for protein tyrosine kinases. This is demonstrated by the detection of significant inhibition of Hck  by the Src inhibitor 1, an inhibitor for Src family kinases, as well as the significant inhibition of Her2  by CP-724714 through the platform. These results suggest the potential of the developed miniature sensing platform as an effective tool for detecting different protein tyrosine kinase activity and for accessing the inhibitory effect of various inhibitors to these kinases. Protein tyrosine kinases are one of the phosphotransferase families that transfer γ-phosphate of adenosine triphosphate (ATP) to the tyrosine residues of the target proteins . Tyrosin32P-phosphate labeling [To detect the activity of protein tyrosine kinases, several direct methods, i.e., labeling , fluoreslabeling  and masslabeling , and indlabeling  and the labeling , were deElectrochemical methods have been developed for the detection of interactions between protein–protein ,24,25, pRecently, a novel label-free protein tyrosine kinase biosensor was reported by oxidizing the tyrosine residue(s) on the substrates of protein tyrosine kinases by tyrosinase to generate L-DOPA quinone, which was then reduced on the electrode to give a reductive response. The tyrosinase-mediated tyrosine oxidation can be blocked by the phosphorylation and suppress the reductive responses . Thus, tYTEIL, a Hck peptide substrate) [YFYV, a Her2 peptide substrate) [YGSFK, a c-Src peptide substrate) [2 was purchased from Yakuri Pure Chemical Co. . Sodium dihydrogen phosphate, sodium hydrogen phosphate and MnCl2 were supplied by SHOWA . DMEM (Dulbecco’s Modified Eagle’s medium) and fetal bovine serum were obtained from HyClone Laboratory Inc. .Carbon fiber paper  was purchased from AvCarb . Tyrosinase, CP-724714, Src inhibitor-1 (src-I1), HEPES, chitosan, N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich . Hck and Her2 were bought from SignalChem . ELMO-Y511 (QNLSbstrate) , FLT3 (Dbstrate) , c-Src sbstrate) , and kem2) of a CFP strip (0.3 × 1.0 cm2) was first cleaned with oxygen plasma using a plasma cleaner . The plasma treatment was performed under the oxygen pressure of 0.4 N and a power of 75 watts for 15 s. After plasma treatment, the CFP strip was rinsed once with double deionized water (d.d. H2O), followed by spreading 2 μL of 0.4% chitosan/1% acetic acid solution on the working area and then air-dried. The immobilization of tyrosinase (200 U) was performed by first mixing with the mixture of 10 mM NHS and 10 mM EDC in 50 mM phosphate buffer, pH 6.6. After incubating at room temperature for 15 min, the tyrosinase/EDC/NHS mixture (6 μL) was then spread on the working area of CFP under the room temperature for at least 2 h.The working area (0.3 × 0.3 cm3) with a cavity of 1.8 × 1.9 × 5.7 cm3. It is composed of two parts: (a) the desk-like top part (~5.5 cm height) contains a hole of 0.8 cm in diameter to place the reference electrode; (b) the C-shaped bottom part (~2 cm height) contains a rectangular cleft of 0.3 × 0.05 cm2 to fix the working electrode. Once the holder was assembled, the counter (Pt wire) and reference electrodes  were fixed on top of the working electrode at a vertical distance of 2 mm (The miniature tyrosine kinase sensing platform consists of a polylactic acid (PLA) holder, a working electrode, a platinum counter electrode and a Ag/AgCl reference electrode . The PLA of 2 mm .2, 3 mM MnCl2, 100 μM substrate peptide and 0.5 mM ATP) in a microfuge tube and incubated at 30 °C for a period of time.The kinase reaction of Hck and Her2 was performed by mixing 2 μL of protein kinase stock solution with 18 μL kinase reaction mixture . The space between working and counter/reference electrodes  was firs2 of 0.998. The developed miniature tyrosinase-based tyrosine kinase sensing platform exhibited a repeatability of 6.6% RSD (Relative standard deviation) for a repetitive measurement of 50 μM c-Src substrate I (n = 6) (A miniature protein tyrosine kinase sensing platform  with a t (n = 6) .The specificity of the miniature sensing platform to recognize peptide with tyrosine residues was further demonstrated by alternately adding 10 μM FLT3, a Her2 peptide substrate, and 100 μM kemptide, and a PKA peptide substrate into the phosphate buffer . The ele2 of 0.997 and 0.991, respectively.Previously, the miniature tyrosine kinase sensing platform was shown to be able to detect peptide substrates of protein tyrosine kinase, i.e., ELMO-Y511 and FLT3. To further elucidate the capability of miniature sensing platform in detecting the tyrosine kinase activity the dose-dependent responses and time-dependent phosphorylation of ELMO-Y511 and FLT3 were investigated. As shown in −7 ± 6.70 × 10−9 A and 4.48 × 10−7 ± 3.03 × 10−9 A, respectively. Upon the phosphorylation with tyrosine kinases, e.g., Hck and Her2, the reductive current of ELMO-Y511 and FLT3 peptides  reduced about 25% after 10 min reaction and reduced over 50%  after 30 min reaction. After 90 min phosphorylation, the electrochemical responses of both peptides reached plateau with a reductive current of 2.78 × 10−8 ± 4.26 × 10−9 for ELMO-Y511 and 4.52 × 10−8 ± 3.06 × 10−9 for FLT3. This result suggests that the activity of Hck and Her2 can be detected on the miniature sensing platform in a kinase reaction for as short as 10 min.A time-dependent phosphorylation of ELMO-Y511 and FLT3 peptides by Hck (10 U/mL) and Her2 (10 U/mL), respectively, was also determined at 30 °C for 0, 10, 30, 60, 90, and 120 min . The red2 = 0.972) and Her2 (2 = 0.941) with the lowest detection limit of 1 U/mL (S/N ≥ 3) to Hck and Her2. The phosphorylation of ELMO-Y511 and FLT3 peptides by various activities  of Hck and Her2, respectively, was carried out at 30 °C for 30 min prior to the electrochemical measurement. As shown in Protein tyrosine kinase inhibitors are widely used in the laboratory to elucidate the signaling pathway as well as in the clinic to treat cancers. The effect of inhibitors can be accessed by the decrease in the protein tyrosine kinase activity after treatment. Therefore, the capability of the developed miniature tyrosine kinase sensing platform in studying the inhibitory effect of Src-I1, the inhibitor of Src family kinases , and CP7−7 ± 2.66 × 10−8 A (closed bar) and 4.57 × 10−7 ± 3.25 × 10−8 A (open bar), respectively  was used to prepare the kinase reaction mixture that contains peptide substrate, i.e., ELMO-Y511 or FLT3, and protein tyrosine kinases, i.e., Hck or Her2. The reductive current of ELMO-Y511 and FLT3 alone was 4.15 × 10−8 ± 5.31 × 10−9 A and 8.51 × 10−8 ± 8.39 × 10−9 A, respectively; while in the presence of culture medium, the responses of peptides (kinase + culture medium) changed to 1.63 × 10−7 ± 2.44 × 10−8 A and 1.97 × 10−7 ± 2.88 × 10−8 A, respectively. This result indicates that the kinase activity of Hck and Her2 was suppressed 33% and 43%, respectively, by the culture medium. Although the exact mechanism underlying the medium-mediated inhibition of kinase activity is not clear, the presence of kinase inhibitors, phosphatases, proteases and/or thiol compounds, in the culture medium is postulated. Phosphotyrosine phosphatases are known to remove the phosphate group from the phosphotyrosines; while proteases contaminants can degrade protein kinases. The contamination of both substances may result in the underestimation of kinase activity. Thiol compounds, such as cysteine and glutathione, could block the reaction of tyrosinase by forming the inactive conjugates from the intermediates [In contrast, the activity of Hck and Her2 was moderately affected by the culture medium. This is demonstrated by the finding that the phosphorylated form of ELMO-Y511 (dark bar) and FLT3 (bright bar) decreased in the kinase reaction mixture containing the culture medium . The ELMmediates . To avoiIn this work, a simple and label-free miniature sensing platform for detecting the activity of protein tyrosine kinases was developed and characterized. Compared to the previous experimental setup  and publ