Triclosan was selected as a model drug for complexation with titania and further introduction into the nanocellulose based composite. Obtained materials were characterized by a broad variety of microscopic, spectroscopic, and thermal analysis methods. The drug release studies showed long-term release profiles of triclosan from the titania based nanocomposite that agreed with Higuchi model. The bacterial susceptibility tests demonstrated that released triclosan retained its antibacterial activity against Escherichia coli and Staphylococcus aureus. It was found that a small amount of titania significantly improved the antibacterial activity of obtained nanocomposites, even without immobilization of model drug. Thus, the developed hybrid patches are highly promising candidates for potential application as antibacterial agents.Spherical cellulose nanocrystal-based hybrids grafted with titania nanoparticles were successfully produced for topical drug delivery. The conventional analytical filter paper was used as a precursor material for cellulose nanocrystals (CNC) production. Cellulose nanocrystals were extracted via a simple and quick two-step process based on first the complexation with Cu(II) solution in aqueous ammonia followed by acid hydrolysis with diluted H Antibiotic resistance of bacteria and other microorganisms is a serious public health concern and a major cause of morbidity and mortality worldwide ,2. It is2 has gained much attention from both theoretical and practical point of view as a biocompatible nanomaterial with outstanding properties for bioencapsulation and drug delivery 2 Mw 234.18), sodium hypophosphite , triclosan  were purchased from Sigma-Aldrich Sweden AB  and used without further purification. The TiO2 nanosol was produced by CaptiGel AB, Uppsala, Sweden. Munkllerfilter paper (100% cotton linters with an ash content of 0.007%) was used as a precursor material for cellulose nanocrystals (CNC) production.Copper sulfate  were isolated via the two-step process involving initial dissolution of the filter paper into tetraamminediaquacopper dihydroxide  with further regeneration by acid hydrolysis with a 20 wt % sulfuric acid. No chemical pretreatment procedures of the filter paper have been done. The synthesis route is illustrated in +. Then, the precipitate was dissolved in 200 mL of ammonia (25 wt %) giving a deep blue solution of cuam. Then, the desired amount of the filter paper was completely dissolved in the obtained solution. Next, 25 mL of Schweitzer’s reagent solution, containing the dissolved filter paper, were added into 100 mL of 20 wt % of the sulfuric acid solution and stirred vigorously at 70 °C for 1 h. After that, hydrolysis was immediately quenched by adding 500 mL of cold water to the reaction mixture. The resulting nanocellulose slurry was separated from the sulfuric acid by several cycles of centrifuging and washing with distilled water until pH = 6 and stored at 4 °C before further use.In particular, to prepare cuprammonium solution, 5 g of copper (II) sulfate was firstly dissolved in 100 mL of distilled water, and then sodium hydroxide (5 M) was added until precipitation. The copper hydroxide precipitate was thoroughly washed with distilled water to remove Na−4 mol) with SHP (50% by weight of BTCA) aqueous solution at 85 °C during 1 h. To obtain nanocomposite based on cellulose nanocrystals and titania nanoparticles, titania nanosol was added to an aqueous suspension of the BTCA-treated nanocellulose and kept at 70 °C for 2 h. Nanocellulose based nanocomposite grafted with titania and triclosan was prepared by the following procedure  was used as a spacer in the presence of sodium hypophosphite (SHP) as a catalyst. The nanocellulose slurry  obtained from the filter paper was treated by BTCA . For this purpose, 4.74 × 10−5 mol of triclosan powder was dissolved in 1 mL of ethanol and then added to titania nanosol (1.5 × 10−4 mol). The obtained solution was mixed with an aqueous suspension of BTCA-treated nanocellulose and kept at 70 °C for 2 h. The final amount of TiO2 in the obtained nanocomposites was 3.4%. To compare, triclosan loaded nanocomposite was also synthesized without using titania as a binding agent (CNC_TR). In this case, nanocellulose slurry was mixed with TR (4.74 × 10−5 mol) initially dissolved in 1 mL of ethanol and kept at 70 °C during 2 h. Finally, all obtained nanocomposites were dried at 40 °C for 48 h without addition any other polymers and plasticizers. The total amount of TR in the obtained nanocomposites was 3.8 wt %, introduced as the weighted amount of solid drug.Drug grafting was performed in amounts calculated in the assumption of the formation of a uniform, single layer coverage on TiO−1 and 4000 cm−1 in transmittance mode. All spectra were smoothed and baseline corrected. Thermo-gravimetric analysis was carried out in air at a heating rate of 10 °C/min, using a Perkin-Elmer TGA-7 or Pyris 1 device. The X-ray powder diffraction (XRD) studies were carried out at room temperature using a Bruker APEX II CCD diffractometer .Atomic force microscope (AFM) was used to analyze dimensions and compare the surface morphology of the obtained nanomaterials. For this purpose, the AFM measurements were performed by using a Bruker Dimension FastScan Atomic Force Microscope. Image analysis was performed using the in-built particle analysis option of NanoScope Analysis 1.7 software , which generates histograms of particle size distribution. Scanning Electron microscopy (SEM) images of the samples were obtained by a Hitachi TM-1000 scanning electron microscopy . IR spectra of the freeze-dried samples were obtained with a Perkin Elmer FT-IR spectrometer Spectrum-100 . A total of 8 or 16 scans were carried out between 400 cm−1 at room temperature. Prior to analysis, the samples were cut a width of 15 mm and a length of 30 mm. The film thickness was measured by a micrometer. For this purpose, the thicknesses taken from six random positions on the film was detected, and the average values were used in the calculation. Three measurements were performed for each sample, and the average values were calculated. The Young’s modulus (elastic modulus) was calculated from the slope of the initial linear section on the stress–strain curve [Ti, (1). To 0.135 g triclosan (0.46 mmol) in anhydrous acetone, 0.30 mL (1.43 mmol) titanium(IV) ethoxide was added under nitrogen atmosphere. This resulted in a bright yellow clear solution. After heating to ~40 °C, the reaction mixture was stored at −18 °C. After ca. 6 weeks, large brown-orange crystals were obtained in nearly quantitative yield. The mother-solution had also turned orange. IR, cm−1: 3397 w, 1714 sh, 1583 s, 1590 s, 894 s, 816 s, 805 s. NMR 1H δ ppm: 7.54 d (J = 2.15 Hz) 7.28 dd , 7.04 d (J = 2.09 Hz), 6.96 d (J = 8.49 Hz), 6.88 dd , 6.83 d (J = 8.70 Hz), s 6.17, 3.56 q , 2.18 s, 1.18 s. Single-crystal X-ray diffraction data were recorded with a Bruker D8 SMART APEX II CCD diffractometer (graphite monochromator). Data for C62H78O20Cl6Ti4·4(C3H6O): triclinic, P-1, a = 11.985(12), b = 13.177(11), c = 16.930(19) Å, α = 107.40(3), β = 94.90(2), γ = 115.921(12)°, V = 2221(4) Å3. Dcalcd = 1.313 g/cm3 for Z = 1, λ(Mo-Kα) = 0.71073 Å. A total of 7040 (Rint = 0.0851) independent reflections were collected at 296 K up to 2θmax = 50.50° (completeness = 97.6%). The structure was solved by direct methods.5(µ3-O)2(µ2-OEt)5(µ-OEt)8(C9H16O3)(C12H6Cl3O2)Ti, (2). Under nitrogen atmosphere, titanium(IV) ethoxide, 50 µL (0.24 mmol) was added to 20.6 mg  triclosan in 0.40 mL anhydrous acetone. A bright yellow clear solution was obtained. The reaction mixture was heated gently to ~40 °C and subsequently stored in freezer. Small bright yellow crystals of nearly quantitative yield were obtained after some weeks. IR, cm−1, 1712 m, 1582 s, 1593 m, 1488 m, 894, 815 s. NMR 1H δ ppm: 7.47 s, 7.20 d (J = 7.0 Hz), 7.11 s, 3.78 singlet, 7.11 singlet, 6.06 d (J = 8.5 Hz), 4.28 q , 4.07 s, 3.78 s, 2.62 s, 2.61 s, 2.51 d (J = 12.53 Hz), 2.20 s, 2.16 s, 1.93 s, 1.92 s, 1.26 triplet (J = 7.2 Hz), 1.23 d (J = 6.11 Hz). 47H86O20Cl3Ti5, triclinic P-1, a = 11.57(2), b = 14.28(3), c = 21.37(4), α = 79.83(3)°, β = 88.00(3)°, γ = 70.90(3)°. λ(Mo-Kα) = 0.71073 Å. V = 3283(10) Å-3. Dcalc = 1.331 g/cm3 for Z = 2. A total of 7732 (Rint =0.0798) independent reflections were collected at 153 K up to 2θmax = 42.5° (completeness = 96.4%)Single-crystal X-ray diffraction data were recorded with a Bruker D8 SMART APEX II CCD diffractometer (graphite monochromator). Data for CThe structure was solved by direct methods. Data for the structure is based on three data series as the crystals were highly sensitive and degraded under data collection even at low-temperature.1 and 2 are available free-of-charge from the Cambridge Crystallographic data base citing registration numbers CCDC 1534781 and 1832990 respectively using the link http://www.ccdc.cam.ac.uk.The details of structure investigation of compounds 2SO4 solution at 80 °C in a sonicator for 6 h [w/w) and hydrochloric acid (37% w/w) solutions using waste cotton fabrics as starting materials [2 (CNC_TiO2), and nanocomposite based on nanocellulose and TiO2 loaded with triclosan (CNC_TiO2_TR), are presented in At present, one of the most widely used extraction process of cellulose nanocrystals (CNC), nanofibers (CNF) or nanowhiskers (CNW) is acid hydrolysis with 63–65 wt % sulfuric acid concentration, temperature in the range of 40–60 °C, and reaction time of 1–4 h ,38. Howeaterials . The nan2, and CNC_TiO2_TR samples confirmed that the obtained samples have homogeneous topography and feature particles spherical in shape  plane of cellulose II, respectively. Apparently, the modification of nanocellulose by titania nanoparticles has contributed to the increase of the amorphous part of the obtained materials. It is important to note that TiO2 has a core–shell structure with a very small (2–3 nm) crystalline anatase core and an outermost amorphous shell [2 diffraction peaks are difficult to detect, due to fact that the size of titania is smaller than the coherence domain required for the X-ray reflection. It is important to mention that spherically shaped cellulose nanoparticles commonly possess amorphous structure [In particular, the PCNC sample exhibited a sharp doublet peak of cellulose II at 2θ = 8.9° (110) and small peak at 2θ = 16° (004) in addition to the cellulose Iβ peak at 2θ = 4.8° (001), 7.4° (110) and 11.5° (200) . The CNCus shell . In thistructure . Here, X2 (CNC_TiO2), and nanocomposite based on nanocellulose and TiO2 loaded with triclosan (CNC_TiO2_TR). The FT-IR spectra of all the samples clearly show a broad band at 3600–3000 cm−1 region, the peaks at 2892 cm−1 and 1159 cm−1 which are attributed to the O–H stretching vibrations in the cellulose molecules, C–H stretching vibrations, and C–O–C asymmetric stretching vibrations, respectively [−1 indicates water presence due to the presence of O–H bending. Although all FTIR samples were thoroughly dried prior to analysis, it was very difficult to completely eliminate water from cellulose molecules, due to strong cellulose–water interactions [2 as a spacer does not change the chemical composition of the synthesized nanocomposite (CNC_TR). To obtain nanocellulose based nanocomposites grafted with titania nanoparticles via esterification process, 1,2,3,4-butanetetracarboxylic acid was used as a linker (BTCA) and sodium hypophosphite (SHP) as a nucleophilic catalyst. The formation of ester bonds in CNC_TiO2 is confirmed by the appearance of the two characteristic bands at 1727 cm–1 and around 1580 cm−1, corresponding to C=O ester carbonyl stretching mode and the asymmetric carboxyl carbonyl stretching mode, respectively  ethoxide to a solution containing 0.3 equivalents of triclosan in anhydrous acetone after storage at −18 °C for 6 weeks. These substances resulted from alkoxide catalyzed condensation of acetone expected to produce oligonuclear model oxo-complexes. Co-crystallization of the two forms occurred because the chosen solution composition L/Ti = 0.3:1 turned intermediate between the ratios in the resulting products, which are L/Ti = 0.5:1 for compound 1 and L/Ti = 0.2:1 for compound 2. Both species turned helpful in producing insights into ligand binding to titania surface. Isolation of pure individual compounds was not an aim of this study. Compound 1 is a triclinic tetranuclear (Ti4O2) titanium oxo-complex, belonging to the space group P-1. It contains two oxygen bridges (μ3-O) and two alkoxides bridges (μ2-O) (1 was reported earlier in [2 is a triclinic pentanuclear (Ti5O2) titanium oxo-complex , five bridging (μ2-O) ethoxide groups and eight terminal ethoxide groups. Only one triclosan ligand is attached to 2, coordinating via phenoxide bonding just like in 1. Also in 2, there are π–π stacking interactions between the aromatic rings influencing packing of the molecules. To study the bonding between triclosan and titania nanoparticles, titanium oxo-complexes containing triclosan ligands were synthesized and used as models. Both compound s (μ2-O) a. The forlier in . Compoun-complex b, also b1 and 2 in paraffin oil to avoid hydrolysis of the compounds. Vibrations for Ti–O and Ti–O–Ti , belonging to the titanium oxo-core were found. A signal at 894 cm−1 was found for both 1 and 2, belonging to the C–O–C bond in triclosan. Medium to strong absorptions are found around 1712 cm−1, which are carbonyl (C=O) vibrations from the condensation products (and possibly some acetone residues). Several absorptions indicating aromatic carbons from the triclosan rings were also detected. Infrared spectra were recorded for both compound 2, and CNC_TiO2_TR samples have been plotted as a function of temperature and are shown in The thermal stability of the obtained nanocomposites was examined using thermogravimetric analysis. Both the TGA curves and derived curves (DTG) of the PCNC, CNC_TiO2, the initial and maximum degradation temperature occurred at 239 °C and 337 °C, respectively. In case of CNC_TiO2_TR, the main weight loss was obtained at 239 °C with maximum degradation temperature at 334 °C. It could be seen that the degradation process of all samples showed two well-separated pyrolysis processes, where one occurred between 150 °C and 250 °C, and the other between 250 °C and 500 °C. A slight weight loss was observed for all samples at around 100 °C, corresponding to the moisture evaporation . The DTGThe mechanical properties of the nanocellulose based nanocomposite films were investigated by tensile testing at room temperature. The tensile–strain curves of the obtained samples are presented in 2 enhanced the tensile strength and Young’s modulus of the nanocomposite film (CNC_TiO2) to 13.8 MPa and 6.8 MPa, respectively. In this case, the improved mechanical behavior of the film can be related to the reinforcing contribution of titania nanoparticles. Similar effect was observed by Schütz and co-workers [2_TR) to 11.0 MPa and 6.3 MPa, respectively. One possible explanation for this is that triclosan molecules block the surface of titania nanoparticles, weakening, as a result, the interaction between TiO2 and cellulose. This results in poorer mechanical properties. The produced material is rather soft and is scratched even by a 2 M stift in a standard pencil test.It was found that the tensile strength of the neat nanocellulose film (PCNC) was relatively weak (about 1.3 MPa). Compared to it, the addition of TiO-workers , who stu2_TR) in comparison with the CNC_TR sample obtained without using titania as a binding agent are shown in The main objective of the present study was to develop the hybrid nanostructured composites based on spherical-shaped cellulose nanocrystals and titania nanoparticles as potentially highly efficient transdermal drug delivery systems. Triclosan was chosen as a model drug because of its broad antibacterial activity against a wide range of Gram-positive and Gram-negative bacteria, as well as molds, yeasts, and parasites responsible for malaria and toxoplasmosis. Nowadays, a substantial amount of literature has been published on the application of triclosan for oral drug delivery . In this2 exhibited a considerably slower release of triclosan from the nanocomposite CNC_TiO2_TR. This sample displayed a sustained long-term release profile of triclosan with the rapid initial release within the first 10 min and about 83% of the drug in a controlled manner over 3.5 h  according to the various kinetic models. The CNC_TR sample exhibited zero order release kinetic profile with high R2 showing constant rate release behavior. The in vitro release profiles of the triclosan from CNC_TiO2_TR could be best expressed by the Higuchi’s models, as the plots showed highest linearity (R2 from 0.99) indicating diffusion controlled drug release pattern  against E. coli and S. aureus was observed higher than that for the CNC_TiO2_TR sample. The possible explanation for this observation can be attributed to different release rates of triclosan from the nanocomposites. The enhanced speed of triclosan released from CNC_TR may result in increasing the antibacterial properties of the sample. The inhibition zone of the nanocomposite obtained without using TiO2 samples also demonstrated the antibacterial response against both strains. For PCNC, the diameters of the inhibition zone against S. aureus and E. coli were found to be 16 ± 7 mm and 13 ± 4 mm, respectively. This observation is in accordance with earlier studies, where the antibacterial effect of cellulose was obtained by its treatment with polycarboxylic acids [E. coli and S. aureus. They found that the polycarboxylic acids were effective against both bacteria even at lower concentrations. The antibacterial efficiency of pure BTCA with concentration ranging from 7 to 350 μg/mL was confirmed against Pseudomonas aeruginosa and methicillin resistant S. aureus ATCC 33591 by agar well diffusion and micro broth dilution methods [2 sample, the diameters of the inhibition zone of S. aureus and E. coli were found to be 26 ± 6 mm and 19 ± 6 mm, respectively. Compared to PCNC, the increase of the diameter of the inhibition zone can be caused by the presence of TiO2 nanoparticles in the obtained nanocomposite. The neat crystalline titania nanoparticles (4 ± 1 nm) covered with an amorphous layer of triethanolamine used in the present study for the nanocellulose modification, are highly bio-digestible, biodegradable, non-toxic [2 nanoparticles (CNC_TiO2). It was also observed that the PCNC and CNC_TiOic acids ,56,57. Fic acids  examined methods . In caseon-toxic ,62, and on-toxic . However2SO4) and shorter reaction time (1 h). The material is rather susceptible to soft scratching, even by a 2 M stift in a pencil test, but displays considerable tensile strength. The drug release studies showed long-term release profile of triclosan and can be described by Higuchi model. The results of bacterial susceptibility tests displayed that the released triclosan retained its antibacterial activity against E. coli and S. aureus. It was also found that a small amount of titania improved the antibacterial activity of the obtained nanocomposites, even without immobilization of model drug. In summary, we have successfully prepared nanocomposites films based on spherical-shaped cellulose nanocrystals and nano-titania with chemically immobilized model drug triclosan. The methodology developed in this study demonstrates that the spherical shaped cellulose nanocrystals could be produced directly from the filter paper as the model source with usage much lower concentrated acid hydrolysis (20 wt % of H


Tyrosylprotein sulfotransferases (TPSTs) are enzymes that catalyze post-translational tyrosine sulfation of proteins. In humans, there are only two TPST isoforms, designated TPST1 and TPST2. In a previous study, we reported the crystal structure of TPST2, which revealed the catalytic mechanism of the tyrosine sulfation reaction. However, detailed molecular mechanisms underlying how TPSTs catalyse a variety of substrate proteins with different efficiencies and how TPSTs catalyze the sulfation of multiple tyrosine residues in a substrate protein remain unresolved. Here, we report two crystal structures of the human TPST1 complexed with two substrate peptides that are catalysed by human TPST1 with significantly different efficiencies. The distinct binding modes found in the two complexes provide insight into the sulfation mechanism for these substrates. The present study provides valuable information describing the molecular mechanism of post-translational protein modifications catalysed by TPSTs. Although the functional importance of protein tyrosine sulfation is not fully resolved, it has been implicated in altering biological activities of proteins, proteolytic processing of bioactive peptides4, influencing the half-life of proteins in circulation5, and modulates extracellular protein-protein interactions, as observed for inflammatory leukocyte adhesion7. The recent discovery of tyrosine sulfation of chemokine receptors suggests an even broader role in inflammatory responses9. For the chemokine receptor CCR5, sulfated tyrosine residues located in the receptors N-terminal extracellular region have been shown to be crucial in mediating HIV binding/infection13. Furthermore, previous studies have demonstrated the involvement of tyrosine sulfation in the activation of complement component C414, maturation of progastrin to gastrin15 and binding of P-selectin glycoprotein ligand-1 (PSGL-1) to P-selectin6.Tyrosine sulfation, first discovered in bovine fibrinogen, is a major post-translational modificationO-sulfate ester and 3′-phosphoadenosine-5′-phosphate (PAP)  domain and a putative stem region of ~40-residues, followed by a luminally-oriented catalytic domain18. The only two sulfotransferases (human TPST1 and TPST2) function by sulfating a variety of substrate proteins with different efficiencies20.Tyrosylprotein sulfotransferases (TPSTs) are enzymes that catalyse the transfer of a sulfonate moiety from 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to the hydroxyl group of a tyrosine residue to form a tyrosine AP) Fig. . TPSTs a21. In a second example, three tyrosine residues  in complement C4 are sulfated by TPSTs25. In the complement activation cascade, C4 is cleaved into fragments C4a and C4b by MASP-2 protease14. An acidic residue and three sulfotyrosine residues in the C4 α-chain are recognised by the positively charged surface of MASP-2 through electrostatic interactions. Thus, sulfation of tyrosine residues is thought to play important roles in various functional modifications that are controlled by TPSTs.Multiple tyrosine residues in a protein substrate can be sulfated by human TPSTs. For example, a study demonstrated a sequential tyrosine sulfation of CXC chemokine receptor 4 (CXCR4) by human TPSTs26  or a gastrin peptide Fig. . The strKm values of 7.67 µM and 6.52 × 102 µM for C4P5Y5 and the gastrin peptide, respectively. In our previous study, crystal structures of human TPST2 and kinetic constants indicated that Asp residue at the −1 position and the acidic residue at the +1 position are major determinants of the Km values26. In line with this notion, C4P5Y5, which contains these two acidic residues, showed a lower Km value than the gastrin peptide, which does not carry these two acidic residues. Thus, the gastrin peptide is considered to belong to a group, which includes CCR3 and CCR5, with high Km values. Furthermore, the resulting kinetic efficiencies (kcat/Km) of human TPST1 for the gastrin peptide were reduced 633-fold in comparison to that for C4P5Y5. These results indicated that human TPST1 sulfates more efficiently C4P5Y5 than the gastrin peptide.The sulfotransfer efficiencies of human TPST1 toward two substrate peptides, C4P5Y5 and the gastrin peptide, were quantitatively evaluated , and is mediated by three consecutive α-helices  Fig.  and S6. −4, F−3, E−2, D−1, Y°, E+1, F+2, D+3) out of the eleven residues of C4P5Y5 were defined in the electron density map, suggesting that human TPST1 binds strongly to C4P5Y5. The human TPST1 recognizes C4P5Y5 in a manner similar to that of human TPST2  between refined peptide atoms and refined protein atoms  and  out of the twelve gastrin peptide residues were defined. We could model these two patterns with 0.5 occupancies each for all four active sites in asymmetric unit. In one binding pattern, four ordered residues  in the gastrin peptide are thought to constitute a productive form based on the position of Y°  in the gastrin peptide are thought to constitute a nonproductive form  of the gastrin peptide form electrostatic interactions with the active site of human TPST1. Specifically, E−3 of the gastrin peptide interacts with R102, R106 and R123 of human TPST1 through electrostatic interactions, whereas F−3 of the C4P5Y5 peptide interacts with W114 of human TPST1 through a hydrophobic interaction. Collectively, these results suggest that TPSTs recognize substrates through multiple interaction modes and thus are capable of recognizing a wide variety of protein substrates.The backbone of E−3 in C4P5Y5 interacts with W114 of human TPST1 through a hydrophobic interaction  in the original sequence have been mutated to Phe and only one Tyr (Y741) residue is present in the peptide. As described above, the active site of human TPST1 recognizes the residue at the −3 position in the substrate through electrostatic and hydrophobic interactions. The results suggest that the Tyr residue and sulfated Tyr residue can then be recognized by TPSTs through both types of interactions at the −3 position Fig. . Taken tInformation derived from the two crystal structures of human TPST1, in combination with those obtained from enzymatic characterization and interaction energy calculations, has provided insights into how TPSTs recognize various substrate proteins with different efficiencies. The current study provides additional information of molecular mechanism of TPSTs in mediating post-translational tyrosine.26. During the purification of recombinant human TPST1, a spontaneous proteolysis was detected. Since the molecular weight of the isolated protein was estimated to be 35 kDa, the purified domain was predicted to correspond to Lys43-Lys341 of human TPST1. pET15b harbouring the cDNA encoding Lys43-Lys341 of human TPST1 was subsequently prepared using the PCR-based Takara Prime STAR mutagenesis basal kit, with pET15b-human TPST1 (Lys43-Glu370) as the template. The new vector construct was transformed into Origami (DE3) cells (Novagen) carrying the pGro7  plasmid encoding chaperonin proteins GroEL and GroES for the expression of recombinant human TPST1. After overnight induction at 30 °C, the cells were collected by centrifugation at 5,000 × g for 10 min, resuspended in lysis buffer  and disrupted by sonication. The crude homogenate prepared was subjected to centrifugation at 20,000 × g for 30 min and the supernatant was loaded onto a Ni-NTA agarose gel column . The bound proteins were eluted from the column using the lysis buffer containing 500 mM imidazole. DTT  was added to the eluted fraction and the recombinant human TPST1 was concentrated using a centrifugal filter , and further purified using a Superdex 200 16/60 column . The eluted fractions containing recombinant human TPST1 were pooled. The sample was exchanged into 50 mM Tris-HCl (pH 7.0) and 200 mM NaCl buffer using a PD10 column . For crystallization of the human TPST1-PAP-gastrin peptide complex, the N-terminal His-tag of the recombinant human TPST1 was removed by overnight treatment with bovine thrombin (Sigma-Aldrich). Subsequently, thrombin was removed by benzamidine Sepharose 6B . The buffer was exchanged to 50 mM Tris-HCl (pH 7.0) and 200 mM NaCl using a PD10 column.To circumvent potential complications associated with the crystallization of membrane proteins, we opted to overexpress the catalytic domain (Lys43–Glu370) of human TPST1 that lacks the N-terminal tail, the transmembrane domain and part of the stem region. cDNA encoding Lys43–Glu370 of human TPST1 was produced using total gene synthesis and cloned into pUC57 . The human TPST1 cDNA was then subcloned into pET15b , which provides an N-terminal 6 × His tag and a thrombin cleavage site. The prepared expression vector construct (designated pET15b-human TPST1) was sequenced to confirm the reading frame and fidelity of the coding region using the Biostudy Support Division of FASMAC Co., Ltd. The expression and purification of the recombinant human TPST1 was based on the same protocol previously used for the preparation of recombinant human TPST219. The standard assay mixture  consisted of 50 mM MES-NaOH, pH 6.0, 50 mM NaF, 20 mM MnCl2, 2 µg BSA, 0.5 µM [35S] PAPS, a substrate peptide (C4P5Y5 or the gastrin peptide) and 0.6 µg (for C4P5Y5) or 4.0 µg (for the gastrin peptide) human TPST1. The reaction proceeded at 30 °C for 15 min and was terminated by heating at 100 °C for 3 min. The precipitates formed were cleared by centrifugation and the supernatants collected were analysed for [35S] sulfated peptides by a thin-layer chromatography (TLC) procedure, with n-butanol/pyridine/formic acid/water  as the solvent system. Subsequently, the plates were air-dried and analysed using a Fluoro Image Analyzer (FLA-3000G).The sulfotransferase activity assay was performed as described previously with minor modifications29. The crystal was found to belong to the space group P21212. The unit cell parameters were a = 78.6 Å, b = 156.0 Å, and c = 48.9 Å. Data collection statistics are summarized in Table Human TPST1-PAP-C4P5Y5 crystals were prepared by crystallizing purified recombinant human TPST1 2.5 mg/ml) together with 2 mM PAP and 1 mM C4P5Y5 using the sitting-drop vapor-diffusion method at 20 °C. Crystals were grown under a reservoir solution consisting of 0.2 M trimethylamine N-oxide dihydrate, 0.1 M Tris-HCl, pH 8.5, and 20% w/v polyethylene glycol monomethyl ether 2,000 for nine months. For data collection, a crystal was transferred to the cryo buffer . Thereafter, the preparation was flash-frozen using a cryo system (Rigaku). X-ray diffraction datasets were collected on the beamline BL38B1 at SPring-8, Hyogo, Japan. The diffraction data were processed using the program package HKL2000.5 mg/ml P1. The unit cell parameters were a = 51.7 Å, b = 93.3 Å and c = 93.0 Å. Data collection statistics are summarized in Table Human TPST1-PAP-gastrin peptide crystals were prepared by crystallizing purified recombinant human TPST1 (5.0 mg/ml) together with 2 mM PAP and 3 mM gastrin peptide using the sitting-drop vapor-diffusion method at 20 °C. Crystals were grown under a reservoir solution consisting of 0.2 M potassium sodium tartrate trihydrate and 19.5% w/v polyethylene glycol 3,350 for two months. Subsequently, the crystals were soaked for 1 d in a 350 nL solution containing 30 mM gastrin peptide. For data collection, a crystal was transferred to the cryo buffer . Thereafter, the preparation was flash-frozen using the cryo system. X-ray diffraction datasets were collected on the beamline BL38B1 at SPring-8, Hyogo, Japan. The diffraction data were processed using the program package HKL2000. The crystal was found to belong to the space group 30. For TPST1-PAP-C4P5Y5, the structure of the human TPST2-PAP-C4P5Y3 complex (Protein Data Bank accession entry 3AP1) was used as the search model. For the human TPST1-PAP-gastrin peptide complex, the structure of human TPST1-PAP-C4P5Y5 was used as the search model. The two structures were manually modified using the Coot program31. Several iterative rounds of model building were performed in Coot and refined using Refmac532. Refinement statistics are summarized in Table The crystal structures of the human TPST1-PAP-C4P5Y5 and human TPST1-PAP-gastrin peptide complexes were determined by molecular replacement using Molrep33. The atomic coordinates and structural parameters of the human TPST1-PAP-C4P5Y5 complex and human TPST1-PAP-gastrin peptide complex have been deposited in the Protein Data Bank under accession codes 5WRI and 5WRJ, respectively.In the human TPST1-PAP-gastrin peptide structure, Ala64-Pro338, Ala64-Pro338, Ala64-Gly336, and Tyr64-Pro338 were refined for protomer-1A, 1B, 1 C, and 1D, respectively. The quality of the structures was assessed using PROCHECKThe interaction energy between human TPST1 and the substrate peptides was calculated using MOE  and the two crystal structures. Before the calculation, all water molecules in the structure were removed, proton atoms were added to all proteins and energy minimization of the added hydrogen atoms was performed using MOE.Supplementary Information


Complex 1 produced a maximum threefold luminescence enhancement at 587 nm in response to GSH. The linear detection range of 1 for GSH is between 0.2 and 2 M equivalents of GSH, with a detection limit of 1.67 μM. Complex 1 also displays good selectivity for thiols over other amino acids.In this study, we report the use of a cyclometalated luminescent iridium(III) complex for the visualization of thiols. The detection of glutathione (GSH) by complex   The dysregulation of GSH activity has been linked to diseases such as cancer, cystic fibrosis and neurodegenerative diseases.2Cl2 (ppy = 2-phenylpyridine) (0.2 mmol) and 1,10-phenanthroline-5,6-dione (phendione) (0.42 mmol) in a mixture of CH2Cl2:MeOH  was refluxed overnight under N2. The product mixture was then allowed to cool down to 25°C, and was filtered to remove unreacted dimer. To the filtrate, excess amount of NH4PF6 was added and the filtrate was reduced in volume by evaporation until precipitation of the crude product was observed. The precipitate was then filtered and washed by 40 ml water twice followed by 40 ml diethyl ether twice. The product was recrystallized by acetone: diethyl ether vapor diffusion to yield the titled compound as a brown solid.Complex terature. A suspen3. 3.1. 1,, the Research Grants Council , the French Agence Nationale de la Recherche/Research Grants Council Joint Research Scheme , National Natural Science Foundation of China [21575121], Guangdong Province Natural Science Foundation [2015A030313816], Hong Kong Baptist University Century Club Sponsorship Scheme 2015, Interdisciplinary Research Matching Scheme [RC-IRMS/14-15/06], the Science and Technology Development Fund, Macao SAR [098/2014/A2], the University of Macau .


The dose-effects of H2O2 on embryo development were investigated via comparisons of the formation rate at each stage (2- and 4-cell embryos and blastocysts). Experiment 2 was carried out to compare behaviors of embryos in a mild oxidative-stressed status (0.03 mM H2O2) with those in a control (0 mM H2O2). Reactive oxygen species (ROS) levels, variation of mitochondrial membrane potential (MMP), expression of γH2AX, and cell apoptosis rate of blastocyst were detected. We observed a dose-dependent decrease on cleavage and blastocyst rates. Besides, higher level of ROS, rapid reduction of MMP, and the appearance of γH2AX revealed that embryos are injured early in mild oxidative stress. Additionally, γH2AX may involve during DNA damage response in early embryos. And the apoptotic rate of blastocyst may significantly increase when DNA damage repair is inadequate. Most importantly, our research provides embryo models to study cell cycle regulation and DNA damage response under condition of different levels of oxidative stress.Our study aimed to develop embryo models to evaluate the impact of oxidative stress on embryo development. Mouse zygotes, which stayed at G1 phase, were treated with prepared culture medium (containing 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, or 0.1 mM hydrogen peroxide (H Physioltabolism . Howevertabolism , light ctabolism , and gastabolism , 6–8, matabolism . In manytabolism –8. In cotabolism . Hamstertabolism . Besidesolism [2 , 8. But olism [2 , 8. We c2O2 to simulate deleterious effects of ROS on embryo development. H2O2, a form of highly active ROS, readily penetrates the cellular envelope and forms •OH which is a more active form of ROS. Liu et al. chose an intensive (1 mM H2O2 for 1.5 h) and a mild (0.2 mM for 15 min) stimulus to treat zygotes (fertilized in vivo); embryos from both groups do not divide till 96 hpi [2O2 (0–50 μM) for 30 or 60 min; the results showed a negative dose-effect on blastocyst rate [μM H2O2 for 15 min [ROS displays roles of double-edged sword in many physiological and pathological processes in cell . It affel 96 hpi . In anotyst rate . We may r 15 min . ResearcStudies state that accumulation of ROS caused developmental arrest of embryos by altering activities and structures of mitochondria , 11, 14.γH2AX, the marker of DNA damage [2O2-treated sperm [γH2AX provides other molecules such as ATM, Rad 50, and Rad 51 with a recognition site to activate DNA damage response [Another important inducement of embryo developmental retardation is DNA damage. Our previous studies found that A damage –18, appeed sperm . Furthered sperm –21. It hed sperm . Cell cyresponse –18, 23. response . To someresponse .2O2 to simulate effects of oxidative stress on embryonic development. In order to clarify the molecular mechanisms of DNA damage response activated by oxidative stress and cell cycle arrest in early embryos in subsequent experiment, we aim to develop alternative embryo models with oxidative damage in this research.The phenomenon above pushes us to research the molecular mechanisms of developmental retardation in zygotes with oxidative-stressed damage. Because of the limitation of clinical researches on human embryos, we treated mouse zygotes with different doses of exogenous HAdult Kun-Ming mice (3–6 weeks old) were purchased from the Animal Center of Shantou University Medical College and treated in compliance with the Guide for the Care of Use of Laboratory Animal by the US National Institutes of Health  and the rules of the National Animal Protection of China. All experimental protocols were approved by the Laboratory Animal Ethics Committee of our institution (SUMC2014-014). This study was approved by the Institutional Animal Care and Use Committee of Shantou University Medical College.γH2AX; Ser 139) and goat anti-rabbit Alexa Fluor 488 secondary antibody were from Abcam (England). The In Situ Cell Death Detection kit (Fluorescein) was from Roche (Switzerland). PI and DAPI were from Panera (France). Human tubal fluid-HEPES (HTF-HEPES) was from Cooper Surgical (USA). Phosphate-buffered saline (PBS) was dissolved in Milli-Q water: 136.9 mM NaCl, 2.7 mM KCl, 0.9 mM CaCl2, 0.5 mM MgCl2, 7 mM Na2HPO4, 1.25 mM NaH2PO4, 1.5 mM KH2PO4, and 1 g/L PVA . Pancreatin solution was diluted to 0.1% with PBS, the pH adjusted to 3.0 with HCl, filtered, aliquoted, and stored at 4°C. The embryo culture medium was made by adding 0.4% BSA and 10% fetal bovine serum to HTF solution. And the medium of the treated group was made by addition of H2O2 to a final concentration of 0.01, 0.02, 0.03, 0.04, 0.05, or 0.1 mM and then preequilibrated them for 1 hour before use. Stationary liquid was 4% paraformaldehyde in PBS. Membrane liquid was PBS + 0.5% Triton X-100. Sperm capacitation liquid , fertilization liquid (HTF solution + 0.4% BSA), and embryo culture medium were placed at 37°C in a 5% CO2 incubator for 4 h. Blastocysts were cultured in blastocyst medium .All reagents were purchased from Sigma  unless otherwise stated. Rabbit antibody for anti-phospho-histone H2AX . Cleavage and blastocyst formation rates were defined as the total number of embryos from corresponding stage divided by total number of zygotes. The total number of zygotes included in 0.01, 0.02, 0.03, 0.04, 0.05, and 0.1 mM H2O2 was 519, 363, 316, 489, 365, 326, and 50, respectively.According to studies by Liu et al.  and our −3 mol/L in DMSO) was added to the embryo culture medium to a final concentration of 10 μmol/L, and zygotes were incubated at 37°C in 5% CO2 for 30 minutes. Embryos were rinsed three times (5 min each time) in fresh culture fluid to remove the residual dye and then placed on a glass slide with 20 μL culture fluid. Fluorescence staining was observed immediately under a fluorescence microscope with exciting light of 495 nm and emissive light of 520 nm (Nikon Eclipse 90 Ni-E). The fluorescence intensity was recorded at 5 s after exciting the zygotes. Image Pro Plus 6.0 was used to quantify the fluorescence . 34 zygotes from the treated group and 29 zygotes from the untreated group were detected in this part.The intracellular ROS level was detected by 2′, 7′-dichlorofluorescein fluorescence (DCFH-DA). A stock solution of DCFH-DA (1 × 10μmol/L. Zygotes were washed three times with PBS (5 min each time) after staining for 20 min and placed on a glass slide. Images were recorded immediately by using a fluorescent microscope (Nikon Eclipse 90 Ni-E) at an excitation wavelength of 488 nm and beam path control setting at LP 585 nm for Ch1 and BP 505–530 nm for Ch2. Analysis of data was done with Image Pro Plus 6.0 software . The number of treated and untreated zygotes at each hour was shown as follows: 18 and 20 for 0 h, 17 and 25 for 1 h, 22 and 19 for 2 h, 22 and 21 for 4 h, and 19 and 16 for 6 h.MMP was detected at 1-cell embryos with the lipophilic, cationic probe 5, 5′, 6, 6′-tetrachloro-1, 1′ 3, 3′-tetraethylbenzimidazolylcarbocyanine iodide . Then, PBS was added to the solution of JC-1 to a final concentration of 1.25 γH2AX primary antibody (1 : 1000 dilution) for 12 h to 15 h at 4°C, then washed, and incubated with goat anti-rabbit Alexa Fluor 488 secondary antibody (1 : 500 dilution). The immunostained embryos were washed with TPBS, counterstained with propidium iodide, and observed under a fluorescence microscope (Nikon Eclipse 90 Ni-E).Embryos were rinsed with TPBS (PBS + 0.05% Tween 20), and zona pellucidae were removed from embryos with pancreatin (0.1%) for 30 s. Then embryos were fixed with 4% paraformaldehyde in TPBS for 30 min, permeabilized at room temperature for 30 min, and blocked for 1 h at room temperature in blocking solution . Before each step, embryos were washed with TPBS three times, 5 minutes per time. After blocking, embryos were incubated with rabbit anti-TUNEL assay was performed to analyze apoptosis of blastocysts, using the In Situ Cell Death Detection kit (fluorescein) in accordance with manufacturer's instructions. Embryos were withdrawn at 96 hpi to collect blastocysts according to our previous experiment . Zona pet-test. P < 0.05 was considered statistically significant.Results were collected from at least 3 independent experiments and data were analyzed by SPSS 17.0 software . Data showed as percentages were analyzed using Chi square test. Values expressed as mean ± SD of treated and untreated groups was compared, using Student's 2O2 group. And we characterized the dose-response of H2O2 on embryo development. No zygotes divided at 60 hpi when they were treated with 0.1 mM H2O2. H2O2 at 0.01 mM or 0.02 mM had no significant effects on the percentages of two- and four-cell embryos or blastocyst formation at 24, 48, and 96 hpi, respectively  . The mea2O2 at 0, 1, 2, 4, and 6 h. The MMP was higher in control group than treated group at all check points . At the same time, the average apoptotic cell counts was over 2-fold higher in the treated group relative to the untreated group (6.24 ± 2.42 (treated) versus 2.27 ± 0.70 (control)) (P < 0.05). Moreover, the mean apoptotic rate was (15.9 ± 5.8)% for each H2O2-treated embryo versus (4.9 ± 1.3)% for the control. Representative images of normal and apoptotic mouse blastocysts are illustrated in The average cell number per blastocyst in the treated group was 37.77 ± 3.94 and 46.74 ± 3.05 in the control group  have a similar cleavage rate at day 3 but decreasing blastocyst formation rate at day 5 or 6 in 20% O2 atmosphere [2O2 to treat zygotes for 30 minutes according to our preliminary experiment and other researches [2O2 to treat zygotes at 7 hpi as they are in G1 phase [Bain and colleagues reported that effects of Helopment . Anothermosphere , 8. So wsearches , 13, 26,searches . Many emsearches . In orde2O2 at 60 hpi. And there will be a lower percentage of embryo formation rate at each stage with treatment of a higher concentration of H2O2. To put it another way, embryo developmental patterns in 0.01–0.05 mM H2O2 could be used, as alternate models, to research influences of mild oxidative stress on developmental retardation. Developmental conditions of 0.01 or 0.02 mM H2O2 group could be used to study embryo development by Peng et al. No obvious differences were found in cleavage or high quality embryo rate at day 3 [2O2 group may suit studying the human embryo development with low formation rate at day 3 and day 5 or 6 [2O2 in our subsequent studies as we found the developmental style was similar to clinic and other researches [2 concentration, but 5% O2 group have more top-quality embryos on day 3 and more blastocysts on day 5 or 6 [2O2. The concurrent research found G2/M delay of zygotes in 0.03 mM H2O2 group [2O2 group and it may be adverse to the formation of blastocyst. Further investigations will be done to determine the molecular mechanisms about cell cycle arrest. As to reasons for the otherness of human embryo development aforementioned, it may be the inconsistent ovulation induction protocols, different lab conditions, technologist's experiences, technologies and skills, and many other uncontrollable factors. Additionally, we have observed that the shape of embryos in control group is larger than that of the treated group and that outcome may be induced by high apoptotic rate in blastocyst of treated group. Nevertheless, it needs more endeavor to unveil the influences of the above phenomenon on blastocyst quality. Our results showed a higher production of intracellular ROS  two pronuclei were exposed to oxidative stress and both might be damaged; (2) embryonic cytoplasm was affected by ROS, especially mitochondria. Almost the whole cytoplasm is from oocyte and cytoplasm plays a major role in regulating early embryo development [Our previous study revealed that development of zygotes from fresh spermatozoa and sperm treated with 1 mM H similar . But blaelopment . That fielopment , 30.γH2AX. Our results showed that γH2AX appeared at the one-, two-, and four-cell embryos in the treated group. Luo et al. found that mouse embryos exposed to 50-Hz sinusoidal electromagnetic fields (EMFs) for 24 h elicited the presence of γH2AX foci in two-cell embryos or γH2AX expressed in two- and eight-cell embryos with exposure for 48 h [γH2AX at later embryo stage only [γH2AX may be induced by various types of DNA damage or embryos diversely in response to different degree of DNA damage. Further investigations are necessary to better unveil that unsolved puzzle. G2/M checkpoint of zygotes was delayed at 0.03 mM H2O2 group as compared with control group [γH2AX below: firstly, γH2AX may work as it collects other signal or repair proteins to injured sites [γH2AX in cell cycle reminds us of its specific role in the process of mitosis [γH2AX subsequently regulates the cell cycle progression and cell fate in human corneal epithelial cells [γH2AX accumulated beyond its role in DNA damage and as a regulator of cell cycle progression by inhibitor DNA replication [γH2AX may involve in DNA damage response to regulate cell cycle in zygotes with oxidative-stress damage, too. We may learn that normal zygotes mediate G2/M by protein kinase A (PKA), WEE1B, 14-3-3ε, and M-phase promoting factors [γH2AX appeared at one- and four-cell stage [2O2-treated sperm again. In the later stage that did not obviously express γH2AX, we assume that damaged cells in those embryos were removed following either apoptosis or permanently developmental arrest or repairing their damaged DNA. Our results illustrated that ROS gives rise to the formation of blastocysts with less cells. Although DNA damage response may produce positive roles at one-, two-, and four-cell embryo stages, the cell counts of blastocyst and the blastocyst formation rate were still significantly lower than the untreated group, indicating that activation of DNA repair mechanisms and/or cell cycle arrest failed to completely repair the damage. Our results strongly state that adverse effects of ROS on embryo development, at least partly, act through DNA damage.To further delineate the effects of oxidative stress on DNA, we firstly detected the expression of for 48 h . UV irraage only . Those bol group . And DNAol group , but we ol group . Also DNed sites , 17, 23;ed sites ; finally mitosis , 35. Recal cells . Besideslication , 38. Tho factors –41. But ll stage , the aboγH2AX may play an important role in DNA repair of early embryo stages, but cells with unrepaired DNA will reduce the blastocyst formation rate. Therefore, our embryo models should be considered when evaluating the hazardous effects of environmental ROS in human embryos and search for effective approaches for prevention.In brief, effects of oxidative stress on early embryos are dose-dependent and lasting to blastocyst. Also, the decline of MMP caused by oxidative stress in zygotes may forecast the compromised development of embryos.


Thirty to 40 % of non-small cell lung cancer (NSCLC) patients developed higher hypertriglyceridemia in the process of treatment with bexarotene. And bioinformatics studies discovered that the expression of slc10a2 was increased in high-grade hypertriglyceridemia patients. So, we will explore the mechanism which may involve in this process.We constructed slc10a2 overexpressed A549 cells and H1299 cells as cell models, normal A549 cells and H1299 cells as control. Then we explored the effects of slc10a2 on A549 cells and H1299 cells behaviors, including proliferation, invasion and apoptosis. The expression of apoptotic related genes and anti-cancer genes also been detected.We found that the proliferation and migration were inhibited and the apoptosis of NSCLC cells was accelerated by bexarotene. In addition, overexpressed slc10a2 in NSCLC cells can further suppress the proliferation and migration, and promote apoptosis under the treatment of bexarotene. On the contrary, the opposite results were obtained after slc10a2 gene was silenced in NSCLC cells treated with bexarotene. Moreover, the expression of caspase 3, caspase 7, PTEN, P21, P53, LKB1, TSC2 were increased and the expression of Bcl-2, cyclin D1, c-FLIP were declined in NSCLC cells and slc10a2 overexpressed NSCLC cells with the treatment of bexarotene, and the opposite situations were seen after slc10a2 gene was silenced in NSCLC cells. The further studies revealed the increased expression of slc10a2 activated the expression of peroxisome proliferator-activated receptor γ (PPARγ), then up-regulated PTEN expression and down-regulated mTOR expression.These results suggest that bexarotene inhibits the viability of lung cancer cells via slc10a2/PPARγ/PTEN/mTOR signaling pathway.The online version of this article (10.1186/s12885-018-4224-x) contains supplementary material, which is available to authorized users. The incidence of lung cancer is rapidly increasing in the world, and it has become the first leading cause of cancer death, especially in China . Non-smaBexarotene Scheme  is a synSlc10a2 is a member of solute carrier family 10 of the sodium/bile acid co-transporter apical sodium-dependent bile acid transporter (ABST) , which pThe goal of this study is to explore the role of slc10a2 in the treatment of NSCLC with bexarotene. We hypothesis that bexarotene inhibits the viability of NSCLC cells (e.g. A549 cells and H1299 cells) via increasing the expression of slc10a2. In this study, we have successfully constructed slc10a2 overexpressed A549 cells and H1299 cells, and the proliferation, apoptosis, migration behaviors were detected in slc10a2 overexpressed A549 cells and H1299 cells respectively. Moreover, we also explored the expression of apoptosis genes, anti-apoptosis genes, tumor suppressor genes in slc10a2 overexpressed A549 cells and H1299 cells. Furthermore, the possible mechanism which involved in this process was discovered.Bexarotene was obtained from Aladdin . Cell Counting Kit-8 was ordered from Dojindo (Japan). Cell culture plates and Transwell plates were ordered from Corning . Crystal violet was obtained from Beyotime . Annexin V/fluorescein isothiocyanate (FITC) apoptosis detection kit was obtained from Beyotime biotech company (China). Gentamicin, Fetal bovine serum, glutamine, and RPMI 1640 medium were purchased from Thermo Fisher Scientific . The primary antibodies including slc10a2, PPARγ, mTOR and PTEN , GAPDH . The pcDNA3 and pcDNA3-slc10a2 plasmid, slc10a2-shRNA, GW9662 were obtained from Shanghai Funeng Biological Technology, Co., LTD.2.The human NSCLC cell lines A549 cells (CRM-CCL-185™) and H1299 cells (CRL-5803™) was obtained from American Type Culture Collection . Cells were maintained in RPMI 1640 media plus 10% fetal bovine serum, 1% glutamine and 0.05 mg/ml gentamycin sulfate at 37 °C and 5% CO6 cells/well, after cells grow above 80% areas, A549 cells were transfected with 8 mg pcDNA3-slc10a2 plasmid using Lipofect2000 transfection Reagent according to manufacturer’s instructions, pcDNA3 treatment as control. The transfection medium was replaced by regular growth medium after 5 h transfection, and at each time point, the cells were used to observation using inverted fluorescence microscope.Before transfection, A549 cells and H1299 cells were seeded into 6-well plate at a density of 2 × 103 cells per well. After 12 h culture, the cultured medium was replaced by conditional medium, which was added with bexarotene, bexarotene in combination with slc10a2-shRNA, bexarotene in combination with GW9662 respectively. At indicated time point, ten microliter CCK-8 was added each well and continually incubated for 4 h. Then the optical density (OD) value (450 nm) was determined by an enzyme-linked immunosorbent assay plate reader (Bioreader).A549 cells, H1299 cells and slc10a2 overexpressed A549 cells, H1299 cells were seeded in a 96-well plate, 5 × 106 cells/ml. One hundred microliters of A549cells or slc10a2 overexpressed A549 cells were placed in the upper chamber of Transwell plates. Serum-free RPMI 1640 medium with bexarotene, bexarotene in combination with slc10a2-shRNA, was added to the lower chamber respectively, RPMI 1640 medium as control. Prior to the addition of cells suspension, preheated serum free RPMI 1640 medium (300 μl) was added to the upper chamber. After 24 h incubation, the invaded cells were collected from lower chambers, then stained with crystal violet.A549 cells and slc10a2 overexpressed A549 cells were starved for 12 h, then resuspended in serum-free medium, and adjusted to 1 × 105 cells/well) with different medium, including RPMI 1640 medium plus bexarotene, RPMI 1640 medium plus bexarotene in combination with slc10a2-shRNA for 2 days. At indicated times, cells were digested then resuspended in 300 μL binding buffer solution which containing 5 ul Annexin V-FITC and 5 ul PI solution, then incubated in the dark for 20 min at room temperature. Finally flow cytometry  was used to analyzed the apoptotic rate of each kind of cells.The apoptosis of A549 cells, H1299 cells and slc10a2 overexpressed A549 cells, H1299 cells was analyzed using the Apoptosis Detection Kit according to the manufacturer’s instructions. Cells were seeded in 6-well plate . Briefly, total RNA (1 μg) was used as a template to prepare cDNA (Invitrogen), and was amplified by Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). A master mix was prepared for each PCR reaction, which included Platinum SYBR Green qPCR SuperMix-UDG, forward primer, reverse primer, and 10 ng of template cDNA. PCR was performed with the following thermocycling conditions: An initial 5 min at 95 °C, followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s.  The forwA549 cells and H1299 cells were treated with 1 mM, 5 mM, 10 mM bexarotene for 24 h, then the cells were harvested, then total protein from tissue or cell was extracted using radioimmunoprecipitation lysis buffer containing 1 mM phenylmethanesulfonylfluoride and the protein concentration was determined using the Bradford method  according to the manufacturer’s instructions. Proteins (20 μg) were separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking with 5% non-fat milk for 1 h at 4 °C, then membrane was incubated with the primary antibody at 4 °C overnight. Membranes were washed 3 times with 0.25% PBST and then incubated with the peroxidase-conjugated secondary antibody for 2 h at room temperature. After washed 3 times, the specific protein bands were detected using the enhanced chemiluminescence reagents .p-values less than 0.05 were considered statistically significant.All data were expressed as mean ± S.D. Differences between the groups were analyzed using one-way analysis of variance (ANOVA) using SPSS 13.0 . As shown in Fig. As Fig. Transwell invasion test showed that Fig. , the mig), there is no significant difference in apoptosis rate between 0.1 mM bexarotene treated group and control group. However, the apoptosis rate in 1 mM and 10 mM bexarotene treated groups was obviously higher than control group. In addition, the apoptosis rate of slc10a2 overexpressed A549 cells treated with 1 mM and 10 mM bexarotene was significantly increased than in A549 cells, and it distinctly declined in A549 cells after treated with slc10a2-shRNA in combination with bexarotene when compared to the bexarotene single treated group  and the expression of anti-apoptotic genes bcl 2, cyclin D1, c-FLIP were decreased (p < 0.05). Similarly, the same situations were observed in slc10a2 overexpressed A549 cells and H1299 cells. While, the expression of caspase 3, caspase 7, PTEN, P21, P53, LKB1 and TSC2 were reduced (p < 0.05) and the expression of bcl 2, cyclin D1, c-FLIP were increased (p < 0.05) when A549 cells and H1299 cells were co-treated with bexarotene and slc10a2-shRNA.As shown in Fig. According to the aforementioned, we have demonstrated that slc10a2 plays an important role in the proliferation of A549 cells with the treatment of bexarotene, how can the slc10a2 effect in this process. As Fig. p < 0.05) and the expression of anti-apoptotic genes bcl 2, cyclin D1, c-FLIP were reduced (p < 0.05). While, the expression of caspase 3, caspase 7, PTEN, P21, P53, LKB1 and TSC2 were declined (p < 0.05) and the expression of bcl 2, cyclin D1, c-FLIP were increased (p < 0.05) when A549 cells, H1299 cells or slc10a2 overexpressed A549 cells, H1299 cells were co-treated with bexarotene and GW9662.We further explored whether slc10a2 via PPARγ plays an important role in tumor suppressor with the treatment of bexarotene. As shown in Fig. As shown in Fig. Three types of PPARs have been identified: alpha, gamma, and delta, they are a group of nuclear receptor proteins which function as transcription factors regulating the expression of genes . Among tA mass of studies have demonstrated that PPARγ agonists via inhibiting the expression of cyclinD1, cyclinB, cyclinE, CDK4 and CDK2 and increasing the expression of CDKN1A to prevent cell cycle from G1 to S phase to prohibit tumor cells proliferation –26. In tThe activation of PPARγ can induce tumor cell apoptosis via several different pathways. PPARγ agonists was able to up-regulate pro-apoptotic protein BAX and BAD expression, and then induced glioma cells apoptosis through releasing cytochrome C and activating the activation of caspase . Li et aMoreover, the activation of PPARγ can reduce the invasion ability of tumor cells. Thiazolidinedione (TZD) is a synthetic agonist of PPARγ, which contains troglitazone, pioglitazone and rosiglitazone . WillsonPI3K/Akt/mTOR signaling pathway exists in almost all mammals, it regulates cell growth mainly through controlling the protein synthesis. PTEN as a negative regulator of this pathway via suppressing the expression of PI3K and Akt. Patel et al. revealed that PPARγ can combined with peroxisome proliferator responsive element 1 (PPRE1) and PPRE2, the upstream gene of PTEN, and increased the expression of PTEN then induced phosphorylation of Akt decreased, cells differentiation and apoptosis . And it In this study we found that bexarotene can suppress the proliferation, migration, and promote the apoptosis of NSCLC cells. Moreover, we demonstrated that this effects owned to the increased expression of PPARγ via enhancing the expression of slc10a2, then up-regulated the expression of PTEN and down-regulated the expression of mTOR, thus increased the expression of apoptotic genes and anti-cancer genes, and reduced the expression of anti-apoptotic genes to suppress the proliferation of NSCLC cells and promote the apoptosis of NSCLC cells .Additional file 1:Figure S1. (A) The proliferation of H1299 cells treated with bexarotene, bexarotene + shRNA-slc10a2, bexarotene + slc10a2-overexpression respectively, H1299 cells without treatment as control. (B) The apoptosis of H1299 cells treated with bexarotene, bexarotene + shRNA-slc10a2, bexarotene + slc10a2-overexpression respectively, H1299 cells without treatment as control. All experiments were repeated 3 times. (TIFF 739 kb)Additional file 2:Figure S2. (A) The expression of apoptotic related genes Bcl-2, cyclin D1, c-FLIP, caspase 3, caspase 7 in H1299 cells treated with bexarotene, overexpressed slc10a2 in combination with bexarotene, slc10a2-shRNA in combination with bexarotene respectively. (B) The expression of tumor suppressor genes PTEN, P21, P53, LKB1, TSC2 in H1299 cells treated with bexarotene, overexpressed slc10a2 in combination with bexarotene, slc10a2-shRNA in combination with bexarotene respectively, H1299 cells without any treatment as control group. All experiments were repeated 3 times. (TIFF 516 kb)Additional file 3:Figure S3. (A) The expression of apoptotic related genes Bcl-2, cyclin D1, c-FLIP, caspase 3, caspase 7 in H1299 cells when treated with bexarotene, bexarotene in combination with GW9662 respectively. (B) The expression of apoptotic related genes Bcl-2, cyclin D1, c-FLIP, caspase 3, caspase 7 in slc10a2 overexpressed H1299 cells when treated with bexarotene, bexarotene in combination with GW9662 respectively. (C) The expression of tumor suppressor genes PTEN, P21, P53, LKB1, TSC2 in H1299 cells when treated with bexarotene, bexarotene in combination with GW9662 respectively. (D) The expression of tumor suppressor genes PTEN, P21, P53, LKB1, TSC2 in slc10a2 overexpressed H1299 cells when treated with bexarotene, bexarotene in combination with GW9662 respectively. H1299 cells without any treatment as control group. All experiments were repeated 3 times. (TIFF 882 kb)Additional file 4:Figure S4. The expression of slc10a2 in A549 cells treated with 1 mM, 5 mM, 1 0 mM bexarotene respectively, A549 cell without treatment as control. (TIFF 68 kb)


Nuclear factor-kappaB (NF-κB) has a vital role in cell survival and inhibition of NF-κB had proven to be an efficient therapeutic pathway for various cancers though little is known about the underlying mechanism. Previously we identified regulator of calcineurin 1 (RCAN1) as an endogenous inhibitor of NF-κB signaling pathway in lymphoma. In the present study, we have solid data to show that RCAN1 can inhibit the nuclear translocation of NF-κB protein then affect the activity of NF-κB signaling pathway in glioma cells. Overexpression of RCAN1 markedly reduced glioma cells viability. We further found that the suppressing glioma cell growth was closely related to the pro-apoptosis effect, not by inhibiting proliferation by the arrest of cell cycle. Our study implicated a novel therapeutic approach for glioma by RCAN1 through inhibition of NF-κB signaling. People with Alzheimer's disease (AD) may have lower risk of cancer , implyinGlioma, accounted for more than 70% of brain tumors, is the most common neoplasm of central nervous system . AccordiNuclear factor-κB (NF-κB) is a family of transcription factors, which is an crucial regulator of cell survival, proliferation and differentiation . NF-κB sp<0.05, Figure p<0.05). And the ELISA assay showed that the expression of NF-κB was increased in glioma tissue compared with the control. Our data suggested that NF-κB signaling pathway proteins were elevated in glioma tissue.To investigate the levels of NF-κB signaling pathway proteins in glioma, we extracted the proteins of glioma. NF-κB/p65, IKKα, and IKKβ western blot showed that NF-κB/p65, IKKα, IKKβ were significantly increased in glioma rather than the normal controls  compared with negative controls in U251 and T98G cells. And cell colony formation assay showed that RCAN1 overexpression reduced colony forming efficiency compared with negative controls in U251 and T98G cells . RCAN1 overexpression also exacerbated the toxicity of H2O2 on U251 cells, 17.87± 0.88% compared with 11.71± 0.79% in control . And RCAN1 overexpression also exacerbated the toxicity of H2O2 on T98G cells, 21.06 ± 0.93% compared with 14.34 ± 0.41% in control . Tumor necrosis factor TNFα -NF-κB activator could decreased the apoptosis ratio to 0.38±0.12% relative to 1.73 ± 0.27% of control in U251 cells , or 0.8567±0.234% relative to 1.91 ± 0.24% of control in T98G cells . And RCAN1 overexpression exacerbated the glioma cell apoptosis after TNFα treatment . All data suggested that RCAN1 induced apoptosis through NF-κB signaling pathway.Since MTT assay showed RCAN1 decreased cell viability without affecting cell proliferation, we examined if RCAN1 affected glioma cell apoptosis. TUNEL staining was performed in glioma cells infected with lentivirus expressing RCAN1. Compared with the control, overexpression of RCAN1 renders more glioma cells death, a 3.33± 0.41% apoptosis ratio relative to 1.73 ± 0.27% of control by TUNEL staining in U251 cells, and a 3.98± 0.17% apoptosis ratio relative to 1.91 ± 0.24% of control by TUNEL staining in T98G cells  and 118.40±1.62 % (p<0.05) in T98G  was used to knockdown expression of RCAN1. The knock-down efficiency of psiRCAN1 was detected by western blot. Silencing plasmid pSuper was performed as control. Western blot confirmed that RCAN1 expression was decreased with psiRCAN1 to 41.08±0.46% of controls  is associated with a reduced risk of cancer –18. HoweSo we want to explicit if RCAN1 can suppress the growth of glioma and becomes a new therapeutic target. Our study here identified RCAN1 can suppress glioma growth as an inhibitor of NF-κB signaling pathway . The undWe analyzed the archival records from the collection of glioma files of Department of Neurosurgery, Beijing Tian Tan Hospital, Capital Medical University, Beijing, China. All the study protocols involved with patients and healthy donors were approved by the Medical Ethics Committee of BeijingTian Tan Hospital, Capital Medical University, Beijing, China, and written informed consents were obtained from all patients and healthy donors.Cell lysates were extracted with Ripa lysis buffer  supplemented with Roche Complete protease inhibitor cocktail. The protein concentration in the supernatant was measured with a bicinchoninic acid (BCA) assay kit (Thermo). SDS-PAGE was then performed using a 10% or 12% gel to isolate the proteins. After electrophoresis, proteins were transferred onto nitrocellulose filter (NC) membrane (Whatman) and hybridized with primary antibodies. The primary antibodies anti-TBP (T1827) and anti-actin mAb (A2228) were from Sigma-Aldrich . Anti-p65 antibody anti-IKKα, and anti-IKKβ were from Cell Signaling Technology . The Page Ruler Prestained Protein Ladder from Thermo Scientific  was used to indicate the target protein. Detection and quantification were performed with the Li-cor Odyssey imaging system and its software . 1:1000 overnight at 4°C. The secondary antibodies  were all used at a dilution of 1:10000.U251 and T98G cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, 50 unit/ml penicillin G sodium and 50 μg/ml streptomycin sulfate (Invitrogen). All cells were maintained in a 37°C incubator containing 5% CO2. All transfections were carried out with lipofectamineTM2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. The potent NF-κB activation inhibitor  was dissolved in DMSO to 100mM and diluted with complete cell culture medium to the final concentration of 10uM. About 20 ng/ml TNF-α was used as NFκB activator. TNF-α was purchasedfrom Life Technologies .Total RNA was isolated from brains using TRIzol reagent (Invitrogen). The mRNA of NF-κB was quantified by TOYOBO R SYBR Green gene Expression Analysis kit . Primers for real-time quantitative PCR were as follows: human NF-κB, forward, 5’-ATGGCAGACGATGATCCCTAC-3’, and reverse, 5’-TGTTGACAGTGGTATTTCTGGTG-3’; RCAN1, forward, 5’-CGACTGGAGCTTCATTGACT-3’, and reverse, 5’-CCCAAGCTTTCCGCTGAGGTGGATCGGCGTGTA-3’;human β-actin, forward, 5’-GACAGGATGCAGAAGGAGAT-3’, and reverse, 5’-TGATCCACATCTGCTGGAAGGT-3’.7pfu/ml. pSuper-based shRNA plasmid (si-RCAN1) was generated as previously described [The cDNA of RCAN1 was generated as previously described . Then thCell viability was detected with MTT and Colony formation assay. Briefly, 10,000 cells were seeded into 96-well culture plates and infected with lentivirus. When measuring, cells were incubated with 10 μL MTT  (5 mg/mL) at 37°C for 4 hrs, and then 100 μL 10% SDS(PH 4.0) was added to each well and incubated over night. The absorbance was measured at 570 nm (ref: 670 nm) with the Thermo Scientific microplate reader Varioskan Flash . Each experiment was repeated at least three times. Cell colony formation assay: Tumor cells were digested by 0.25% trypsin/0.02% EDTA solution at the logarithmic phase to make a single-cell suspension with culture medium. Then, a cell counting chamber was used to calculate the number of cells in a 10 μl single-cell suspension under an inverted microscope. The cells were then delivered into six-well culture plates containing a sterile glass cover-slip in each well, with 2,000 cells added to each well. The medium was refreshed every 3 days until cell clones could be observed with the naked eye.Cells were harvested and washed twice in PBS, then fixed in 75% ethanol over night at 4°C. After washed in cold PBS three times, cells were resuspended in 1 mL PBS with 40 μg PI and 100 μg RNase A  and incubated for 30 min at 37°C. Samples were then analyzed by BD FACSAria III cell sorter .Luciferase activity was determined as previously described . NuclearEdU staining was performed using EdU Kit  following the manufacturer's instructions. For TUNEL staining, cells were fixed in 4% paraformaldehyde for 30 min and permeabilized with 0.1% TritonX-100 for 10 min. TUNELs taining was performed using the Roche-In Situ Cell Death Detection Kit according to themanufacturers’ instructions. Results were analyzed by fluorescence microscopy (LeicaDMI4000B). And cell nucleus was stained with 1 μg/ml DAPI  at 25°C for 10 min.The flowcytometry was performed using PI and Annexin staining kit , detected by BD FACSAria III cell sorter  and analyzed using FlowJo Software 10.0.t test analysis and two-way ANOVA.All experiments were repeated three to five times. In figures one representative picture was shown; quantifications were from three or five independent experiments. The values represent the means ± SEM. The data were evaluated for statistical significance with Student's


Protein tyrosine phosphatases are not considered to be metalloenzymes. Yet, they are inhibited by zinc cations and metal and non-metal oxyanions that are chemical analogues of phosphate, e.g. vanadate. Metal inhibition is generally not recognized as these enzymes are purified, supplied, and assayed with buffers containing chelating and reducing agents. We screened a series of cations and anions for their capacity to inhibit protein tyrosine phosphatase 1B and discuss the ensuing general issues with inhibition constants reported in the scientific literature. In contrast to zinc, which binds to the phosphocysteine intermediate in the closed conformation of protein tyrosine phosphatase 1B when the catalytic aspartate has moved into the active site, other divalent cations such as cadmium and copper may also bind to the enzyme in the open conformation. Inhibition by both anions and cations, conditions such as pH, the presence of metal ligands such as glutathione, and the existence of multiple conformational states of protein tyrosine phosphatases in the reaction cycle establish a complex pattern of inhibition of these important regulatory enzymes with implications for the physiology, pharmacology and toxicology of metal ions. Ki value of 21 pM was determined for receptor protein tyrosine phosphatase beta (RPTPβ) , copper sulphate (CuSO4), cadmium chloride (CdCl2), lead nitrate (Pb(NO3)2), ferric chloride (FeCl3), ammonium iron(II) sulphate [(NH4)2Fe(SO4)2·6H2O], chromium chloride (CrCl3), lanthanum chloride (LaCl3), lithium chloride (LiCl), manganese chloride (MnCl2), nickel chloride (NiCl2), silver nitrate (AgNO3), ammonium tetrathiomolybdate [(NH4)2MoS4], sodium tungstate (Na2WO4), potassium nitrate (KNO3), ammonium molybdate [(NH4)2MoO4], sodium arsenate, (Na2HAsO4·7H2O), sodium chromate (Na2CrO4·4H2O), boric acid (H3BO3), potassium hydrogen carbonate (KHCO3), histidine, sodium hydroxide, and Triton X-100 were from Sigma-Aldrich; ammonium heptamolybdate [(NH4)6Mo7O24] from BDH Laboratory Supplies, Poole, UK; 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) and 6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU) were from Invitrogen. Recombinant human PTP1B, residues 1–299, was from Millipore, supplied in 50 mM Hepes, pH 7.2, 1 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% (v/v) NP-40. Chromium (III) histidinate was used because it is the least kinetically inert complex. To prepare chromium (III) histidinate, chromium (III) chloride (20 mmoles) and histidine (60 mmoles) were dissolved in 150 mL water and heated to 80 °C for 15 min, sodium hydroxide (50 mmoles) in 50 mL water was added within four minutes. The blood red complex was used for the experiments.Molecular biology-grade HEPES, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), nitrilotriacetic acid (NTA), zinc chloride  Triton X-100, freshly prepared 0.1 mM TCEP, and with different concentrations of metal cations or oxyanions. The reaction was initiated by adding the fluorigenic phosphatase substrate DiFMUP. Assays were performed in triplicates in a total volume of 100 µl in 96-well black optical bottom plates . Product formation (hydrolysis of DiFMUP to DiFMU) was monitored at 460 nm emission and 360 nm excitation with a fluorescence plate reader . Initial rates were determined from the linear portion of the progress curves. Fluorescence intensity was converted to molar concentrations from measurements with a standard of a DiFMUP–DiFMU mixture.Enzymatic activity of PTP1B  was assayed fluorimetrically at 25 °C in a buffer containing 50 mM Hepes/Na2+, Cd2+ and Zn+2 in the buffer   were obtained from fitting semi-logarithmic plots with non-linear regression curves (Sigma plot).The metal buffered solutions were freshly prepared and equilibrated for 15 min before the assay was started by adding enzyme. The reaction was monitored under initial velocity conditions. Inhibition constants .One of the major issues in determining PTP1B activity is the contamination of zinc in the experimental solutions and buffers. Zinc is a ubiquitous contaminant of laboratory chemicals. In order to monitor zinc levels, total zinc concentrations in solutions and buffers were measured using inductively coupled plasma mass spectrometry . Samples were prepared in 5% (v/v) HNOWe investigated a total of 20 metal ions (12 cations and 8 oxyanions) with regard to their capacity to modulate the activity of PTP1B Table . Metal c+2, we determined the inhibition for Cu2+ and Cd2+, two cations with high affinity to ligands according to the Irving-Williams series. Based on free Cu2+, Cd2+ and Zn+2 concentrations and fitting the data to a non-linear regression curve, we obtained inhibition constants of 600 fM, 7.3 nM and 2.4 nM, respectively  serves as a redox and metal buffer in cells. When using 1 mM GSH instead of 1 mM NTA and 0.1 mM TCEP in the assay solution, ZnKi value of 17 mM  values are 1.5 µM (vanadate), 9 µM (heptamolybdate), 200 µM (molybdate) (data collected by Ms. Sherry Sachdeva), 210 µM (tungstate), 200 µM (arsenate), and 54 mM (nitrate)  Fig. a–e. The Ka value of its conjugate acid, is important for inhibition. For example, in the case of phosphate, the dianion binds. Hydrogenphosphate (HPO42−) interacts with the guanidinium group of Arg221 of PTP1B in its closed conformation. For phosphoric acid the pKa values are 2.0, 5.7 and 11.7 while they are 3.8, 7.8, and 13 for H3VO4 . The stereochemistry is also important. In a crystal structure of PTP1B in the presence of nitrate, nitrate is bound in the active site and 50% of the protein molecules are in the closed conformation ) and vanadate (V(V)) are structurally similar to endogenous phosphate (P(V)) and mono-anions due to partial ionization while chromate (Cr(VI)) and molybdate (Mo(VI)) are structurally similar to endogenous sulphate (S(VI)) are fully ionized and hence di-anions Clarkson . Metal oKa values  of molybdic acid  and tungstic acid   at pH 7.5  binding at the active site. The molybdenum atom is six-coordinate with three oxo-ligands in MoO3, two apical water molecules and an S atom from the catalytic cysteine residue, while in the complex with tungstate the WO4 moiety is five-coordinate with four oxo-ligands and an interaction with the S atom of the catalytic cysteine. In contrast to the molybdate complex, the side chain of Gln262 of PTP1B interacts with one of the oxo-ligands in the tungstate complex . Moreover, chromate is a larger oxyanion than molybdate  . The pKa2 of arsenate is 7.03–7.10 and thus between those of phosphate and vanadate. A crystal structure of PTP1B with arsenate has not been reported. However, several crystal structures of arsenate reductase have been solved and shown that the structure of this enzyme is related to the PTP family 3 to H2BO−. Hence it is expected to interact weakly with PTPs.Other oxyanions like borate show no significant effect on PTP1B activity. Borate is a monoanion, such as nitrate and hydrogencarbonate, with a pInhibition of PTP1B by zinc (II) ions has been known for a long time  is both a reducing and a complexing agent and therefore NTA and TCEP are not required when assaying PTP1B. Glutathione forms complexes with zinc, including ternary complexes with ligands such as histidine  ions oxidize the thiol of cysteine in the presence of oxygen, they are irreversible inhibitors. Thus, the highly potent Cu2+ inactivation of the PTPT VHR was discussed as a consequence of the cupric ions oxidizing the active-site cysteine  and/or in the closed conformation of the enzyme . Investigations at varying pH values, lack of knowledge of the type of inhibition and control of anions and cations make it difficult or nearly impossible to compare inhibition data of PTPs in the literature. PTP1B has been and continues to be a major therapeutic target for inhibition by low molecular weight compounds and drug candidates (Heneberg We conclude that a variety of metal cations and metal oxyanions are rather potent PTP1B inhibitors while non-metal oxyanions are generally weak inhibitors. Among cations, CuHeneberg  because


For this purpose, we have used a Prussian Blue analogue as the sacrificial template by reacting with soluble silicate, obtaining particles with nanorod morphology and of small size (75 nm). These nanoparticles present good biocompatibility and higher longitudinal and transversal relaxivity values than commercial Gd3+ solutions, which significantly improves the sensitivity of in vivo magnetic resonance images.We describe the synthesis, characterization and application as contrast agents in magnetic resonance imaging of a novel type of magnetic nanoparticle based on Gd-Si oxide, which presents high Gd T1-positive agents of paramagnetic species and T2-negative agents of super-paramagnetic materials. Due to their high sensitivity and detectability, Gd3+-based soluble chelates are the most representative and preferential T1 CAs in current MRI clinical applications  is used as a sacrificial template by reacting with soluble silicate. By overlapping kinetics of silica condensation with [Fe(CN)6]3− removal, we are able to synthesize particles of an oxide nanocomposite with equal shape and size than former PBA crystallites. The resulting material presents nanorod morphology, small particle size (75 nm average length), and shows good r1 and r2 relaxivity values in a 3 tesla (T) magnetic field, improving positive contrast of in vitro and in vivo MRI images.In order to increase the number of metal centers per particle, without reducing the water accessibility, an alternative is to develop Gd silicate-based MNP, where the high ion density and a disordered structure allow for efficient interaction of most Gdng water . On thisur group , wherein2O)4[Fe(CN)6] has been recently solved 3− species. EDS analysis is consistent with the expected stoichiometry (Gd:Si atomic ratio of about 2:3), and demonstrates almost complete iron removal  , is 83.9T1- or T2-weighted CA for MRI was evaluated by measuring the longitudinal (r1) and transversal (r2) nuclear magnetic relaxation rates of water protons in aqueous suspensions at 3 T and room temperature. Stable colloids were prepared in aqueous xanthan gum (0.1%) solution with Gd3+ concentration in the range 0–1.00 mM. Relaxivity values were determined by using the following expression  precursor following a known recipe [3[Fe(CN)6] and 6.3 g (30 mmol) of Et4NBr were dissolved with 200 mL of methanol in a 500 mL flask connected to a nitrogen line, and stirred at 30 °C for three days under N2 atmosphere. The mixture was filtered off and the filtrate was concentrated to approximately 10 mL by rotary evaporation. The resulting solution was stirred with 100 mL of ethyl ether and the yellow precipitate was collected by filtration. The crude was dissolved in 150 mL of refluxing acetonitrile and the solution was allowed to cool, obtaining (Et4N)3[Fe(CN)6]. For nanosized Prussian Blue analogue preparation, Gd(NO3)3·6H2O  were dissolved in 50 mL of EtOH:H2O (2.5:1 v/v). Then, a solution of the previously-synthesized (Et4N)3[Fe(CN)6]  was added and the mixture was left for a day at room temperature. The precipitate was filtered off, washed with ethanol, and vacuum dried to yield 118 mg of an orange powder (GdFe).Firstly, we prepared the (Etn recipe . Briefly3·3SiO2·xH2O nanoparticles (GdSi) were prepared by modification of a previously reported method [2O)4[Fe(CN)6] , was suspended in 278 mL of EtOH:H2O (2.5:1 v/v) with vigorous stirring. Then, 3-cyanopropiltrichlorosilane  was added and, after 30 min, 3.5 mL ammonia solution and 11 mL of sodium silicate solution  were introduced. The reaction was allowed to proceed for 48 h. Non-reacted silicate ions were removed by subsequent centrifugation , and the obtained white solid was washed repeatedly with milliQ water, centrifuged again , and further freeze dried .Dense, Gd(OH)d method ,41. Gd(H3·3SiO2·xH2O was dried at 80 °C and vacuum (8 torr) for 24 h. Afterwards, 7.4 mL of anhydrous toluene was added and the mixture was heated to reflux. Then, 728 µL (3.13mmol) of 3-aminopropyltriethoxysilane (APTES) was added and the mixture was stirred for 3 h. The obtained product was filtered off, washed with toluene and methanol and freeze-dried . Next, 156 mg of the above material was suspended in 15.6 mL of anhydrous dichlorometane. Then, 195 µL of diisopropyl amine were injected under nitrogen atmosphere. Afterwards, 234 mg of 2,5,8,11-tetraoxatetradecan-14-oic acid succinimidyl ester (PEG4) was added. The reaction was stirred overnight at room temperature. Later, the solvent was removed under reduced pressure and the nanoparticles were suspended in 100 mL of ethanol with stirring. Subsequently, the suspension was filtered off and washed with ethanol (300 mL). Finally, the material (GdSiPEG) was freeze-dried . In order to modify the GdSi surface, we used a method previously described by our group . 375 mg α radiation (λ = 0.1542 nm). Liquid nitrogen adsorption isotherms of 200 mg of sample were measured in a Micromeritics Flowsorb apparatus. Surface area calculations were done by the BET method. Samples for transmission electron microscopy (TEM) were ultrasonically dispersed in a mixture of EtOH:H2O (2.5:1 v/v) and transferred to carbon-coated copper grids. TEM and scanning transmission electron microscopy (STEM) micrographs were collected in a JEOL JEM 2100F microscope operating at 200 kV. The quantitative energy-dispersive X-ray spectroscopy analysis (EDS) was performed in an INCA Energy TEM 250 system from Oxford Instruments, working with a SDD X-MAX 80 detector. Field-emission scanning electron microscopy (FESEM) micrographs were collected in a ZEISS Ultra 55 microscope operating at 2 kV, with a 2 × 10−9 A beam current and 2.5 mm as the working distance. Infrared spectra were recorded at room temperature in the 400−3900 cm−1 region with a Nicolet 205xB spectrophotometer, equipped with a Data Station, at a spectral resolution of 1 cm−1 and accumulations of 128 scans. Samples were outgassed at 400 °C under vacuum during 1 h for complete water removal before acquisition. Finally, water and organic (PEG3) content were calculated from elemental analysis  and thermogravimetric (TGA) measurements (Mettler-Toledo TGA/SDTA851e).Powder X-ray diffraction (XRD) patterns were collected in a Philips X’Pert diffractometer equipped with a graphite monochromator, operating at 40 kV and 45 mA and using nickel-filtered Cu KPEG material stability in physiological medium was monitored by solution assay in phosphate-buffered saline  or glucose aqueous solution (5.5 wt. %). 5 mg of the corresponding material were stirred in 1 mL at 1500 rpm and 37 °C in a Thermomixer® for 24 h. Then, samples were centrifuged  and supernatant metal cation concentration was analyzed by inducted couple plasma (ICP).GdSiv/v). The plates were cultured 24 h at 37 °C and 5% CO2 injection in air. After 24 h cells were treated with the CA at a concentration range of about 0.25–100 µg/mL in RPMI medium. Cells with nanoparticles were incubated at 37 °C for 24 h and 5% CO2. All concentrations were tested in triplicate and negative controls with no nanoparticles were also carried out.The different cell lines were cultured in 96-well cell culture plates with the seeding densities shown below in a final medium volume of 200 µL/well: HeLa 10,000 cells/mL; fibroblast 3T3 and glioblastoma 42-MGBA 50,000 cells/mL and neuroblastoma SH-SY5Y 100,000 cells/mL. HeLa cells were grown in MEM (Earle’s), 3T3 cell line in DMEM, 42-MGBA cell line in MEM (Earle’s) and RPMI 1640 (1:1), and SH-SY5Y cell line was cultured in a DMEM and Ham (F12) Nut MIX (1:1). Cell media were supplemented with 10% FBS and Pen-Strep 1:100 :Cell viability was measured using the 3--2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 200 µL/well of MTT/PBS (1 mg/mL) were added and the plates were incubated at 37 °C for 3 h. Formazan crystals were dissolved with 100 µL DMSO and then absorbance at 595 nm was measured with a 1681130 iMarkData statistical analysis was performed applying arithmetic’s means and error bars of statistical error means (SEM) using MATLAB (MathWorks).T2 and T1 estimation sequences  sharing the same spatial localization. A multi-echo spin echo sequence was used to estimate T2 values acquiring 32 echoes ranging from 14 to 231 ms (ΔTE = 7 ms) with a sequence TR of 2000 ms. T1 values of each tube were estimated using a look-locker inversion recovery acquisition with 107 inversion times ranging from 6.51–5306.51 ms, with an inversion time interval of 50 ms. A new inversion pulse was applied every 6 s to avoid signal saturation due to extremely close inversion pulses. To reduce the influence of readout excitation pulse in the final T1 values, an excitation flip angle of 5° was applied during the TFE shot  (mM), the slopes of these graphs provided the specific relaxivities r1 and r2. Relaxivity determinations and MRI studies were carried out in a clinical 3 T MRI unit  with an eight-channel phased array head coil. The phantom was scanned with a single slice in the coronal orientation, obtaining a transversal view of all tubes filled with variable CA concentration. Geometric parameters remain equivalent between the TFE shot . With reribed at . As in t1H rat body receive-transmitter resonator  was employed. Data were acquired with a Hewlett-Packard console running Paravision software  operating on a Linux platform. Rats were anesthetized in an induction chamber with 3%–4% isofluorane in medical air (0.8–1 L/min) and maintained with 1%–2% isofluorane (IsoFlo) during the MRI experiment. Anesthetized animals were taped down in a custom-made animal holder to minimize breathing-related movement artifacts. The body temperature was kept at 37 °C using a water blanket and animals were monitored using a MRI-compatible temperature control unit . The breathing rate was also measured using a custom designed device. Experiments were carried out in a horizontal 7 T scanner with a 30 cm diameter bore . The system had a 675 mT/m actively-shielded gradient coil  of 11.4 cm inner diameter. A −1) into the catheterized tail vein as a single bolus (0.04 mmol Gd/kg body weight), and acquiring images 30 min afterwards. Acquisition was held for 1 h. T2-weighted anatomical images to position the animal were collected in the three orthogonal orientations using a rapid acquisition relaxation enhanced sequence (RARE), applying the following parameters: field of view (FOV) 40 × 40 mm, 15 slices, slice thickness 1 mm, matrix 256 × 256, effective echo time (TEeff) = 56 ms, repetition time (TR) = 2 s, 1 average and a total acquisition time of 64 s [T1- and T2-weighted images. For the former, FLASH images were acquired with the following parameters: 25 slices, 1.5 mm slice thickness, TR = 197 ms; TE = 2.7 ms; FOV = 6.0 × 5.0 cm; Mtx = 128 × 108; averages = 4; total acquisition time = 90 s. Three images were acquired before the CA administration (baseline) and 20 after it. For the T2-weighted images, RARE was used with the same geometry than the T1-weighted images and the following parameters: TR = 2800 ms; TE = 48 ms; FOV = 6.0 × 5.0 cm; Mtx = 256 × 214; averages = 8; total acquisition time = 600 s. One of these images was acquired at the beginning (baseline) and one at the end of the experiment. Data were analyzed with Image J .Studies were performed by injecting 1 mL of the CA suspension . In this context, future studies will be focused on long-term in vivo stability and potential toxicity of this novel paramagnetic nanocomposite.Gd-Si oxide nanoparticles obtained by alkaline transformation of a Prussian Blue analogue are a novel MRI contrast agent with higher longitudinal and transversal