Pyrococcus horikoshii OT3 (PhKAT), which is a homodimeric protein, catalyzes the conversion of kynurenine (KYN) to kynurenic acid (KYNA). We analyzed the transaminase reaction mechanisms of this protein with pyridoxal-5′-phosphate (PLP), KYN and α-ketoglutaric acid (2OG) or oxaloacetic acid (OXA). 2OG significantly inhibited KAT activities in kinetic analyses, suggesting that a KYNA biosynthesis is allosterically regulated by 2OG. Its inhibitions evidently were unlocked by KYN. 2OG and KYN functioned as an inhibitor and activator in response to changes in the concentrations of KYN and 2OG, respectively. The affinities of one subunit for PLP or 2OG were different from that of the other subunit, as confirmed by spectrophotometry and isothermal titration calorimetry, suggesting that the difference of affinities between subunits might play a role in regulations of the KAT reaction. Moreover, we identified two active and allosteric sites in the crystal structure of PhKAT-2OG complexes. The crystal structure of PhKAT in complex with four 2OGs demonstrates that two 2OGs in allosteric sites are effector molecules which inhibit the KYNA productions. Thus, the combined data lead to the conclusion that PhKAT probably is regulated by allosteric control machineries, with 2OG as the allosteric inhibitor.Kynurenine aminotransferase from  KYNA is sequentially biosynthesized from l-KYN via a 4-(2-aminophenyl)-2,4-dioxobutanoate (4AD) intermediate by KAT (l-glutamic acid (Glu).Kynurenine-oxoglutarate transaminase (EC 2.6.1.7), also known as kynurenine (KYN) aminotransferase (KAT) e by KAT . KAT traN-methyl d-aspartate) receptor (NMDAR) and plays a key role in the glutamatergic neurotransmission system AedesSaccharomycesTrypanosoma that causes fatal sleeping sickness in human Pyrococcus horikoshii OT3 (PhKAT), is a homolog of human KAT II (HuKAT II) and exhibits low but significant amino acid sequence homology with the KATs of various organisms.In human brain, KYNA acts as a natural antagonist of the glycine site of NMDA . ITC results indicate that PhKAT and PLP and/or 2OG interact with a high affinity. The enzyme kinetic analyses revealed that PhKAT is an allosteric enzyme and that transaminated acceptors for PhKAT are 2OG and oxaloacetic acid (OXA). Furthermore, in the crystal structure of PhKAT complexed with an allosteric effector, 2OG was bound to PhKAT at a rate of 4 molecules per protein. Two 2OG molecules were identified in the binding pocket of PhKAT other than a pocket constructed for active sites. This demonstrates that 2OG is an allosteric effector necessary for the regulation of KYNA biosynthesis.To understand the action mechanisms of PhKAT and compare them with those of their counterparts in humans, other mammals, yeasts, E. coli BL21-CodonPlus (DE3) cells were transformed with pET28a-PhKAT. PhKAT expression was induced with IPTG. PhKAT (48 kDa), which was expressed as a soluble protein, was detected by sodium dodecyl sulfate (SDS)-PAGE (DS)-PAGE . RecombiKd1 = 25.5 and Kd2 = 1.77, and Kd1 = 9.65 and Kd2 = 1.92 µM, respectively (A characteristic of KAT is its ability to form a Schiff-base link with the PLP cofactor (PLP complex). To determine whether PhKAT can form such a complex, it was incubated with PLP, and the changes in the absorption spectrum were recorded. The change in the absorption spectrum upon the formation of the PLP–PhKAT complex is depicted in ectively . The resCrystals of PLP-bound PhKAT belong to space group C2 and have unit cell parameters a, b, and c of 85.927, 71.055, and 136.348 Å, respectively . The refTwo peptide chains (designated chains A and C) were located in an asymmetric unit and form a functional homodimer . Two PLPPreviously, we confirmed the conversion of KYN to KYNA by HPLC Ki) was 20.11 µM  of Kd1 = 238.15 nM and Kd2 = 15.80 µM (as substraes) and Kd3 = 3.12 µM and Kd4 = 1.09 µM , and the negative and positive cooperativities, respectively  of 2OG, PLP, and the PhKAT triple complex at the binding site to the PhKAT subunit with one 2OG substrate. The crystals of 2OG-bound PhKAT belong to space group C2 and have unit cell parameters a, b, and c of 85.817, 70.989, and 136.816 Å, respectively . The refTwo peptide chains were located in an asymmetric unit and form a functional homodimer . The resThe overall structure of the superimposition of the main chain structures of PhKAT–2OG complexes 3AOW and 3ATH, which are 2 different structures with 2 and 4 2OGs as substrates, respectively, with the 2OG-bound PhKAT are shown as cartoon drawings in To verify that PhKAT and 2OG are an allosteric enzyme and allosteric effector, respectively, we cocrystallized PhKAT, PLP, and 2OG in a 1∶1:2 ratio. PhKAT has 4 2OG-binding sites that have different binding characters. In the present study, we determined previously unknown allosteric sites of PhKAT . The allP. horikoshii, and that KYN transamination results in the production of KYNA  is very important, we decided to investigate the complex formation between PhKAT and 2OG. For this purpose, we cocrystallized both intact PhKAT and 2OG molecules, which are required for enzymatic regulation P. horikoshii OT3 may be evolutionarily conserved and related to human KATs localized in the brain. The enzymatic molecular mechanisms for the conversion reaction from KYN to KYNA by PhKAT remain unclear. Therefore, we solved the crystal structures of the PhKAT complexed with KYN or PLP to understand the differences in the enzymatic reaction mechanisms between human KATs and PhKAT in greater detail in vivo and that PhKAT shares a function of 2OG and has 4 pockets with different binding affinities for 2OG. Thus, the allosteric sites of KAT may be a novel drug target for the regulation of KYNA production.Finally, we suggest that the KAT protein from l-kynurenine (SIGMA), kynurenic acid (SIGMA), 2-oxoglutaric acid , oxaloacetic acid , 2-ketobutyric acid , and α-keto-γ-(methylthio) butyric acid  preparations were used as a cofactor, and substrates and/or standards; agar and organic nutrients for Luria–Bertani (LB) medium (Difco) were purchased. PLP, KYN, and KYNA were treated with alkali to dissolve them in water.Pyridoxal phosphate (Wako), NdeI–EcoRI fragment from pET1300Ph NdeI–EcoRI sites of pET28a (Novagen).The pET28a-PhKAT expression vector for the expression of His6-tagged PhKAT was constructed by ligating the E. coli BL21-CodonPlus (DE3) transformed with the pET28a-PhKAT was cultured in 50 mL LB medium containing kanamycin (30 µg/mL) overnight at 37°C. Twenty milliliters of this culture was inoculated into 2 L LB medium and cultivation was continued until the optical density at 578 nm (OD578) reached 0.6. Protein expression was induced by the addition of 0.3 mM isopropylthio-β-galactoside (IPTG) and incubation for 21 h at 17°C; the bacteria were subsequently harvested by centrifugation. The bacterial pellets from 2 L culture were resuspended in 30 mL lysis buffer , and the cells were lysed by 30-s sonication on ice. Cell debris was removed by centrifugation at 43 000 × g for 30 min. The resultant supernatant was loaded directly onto a His TALON Cartridge  equilibrated with 10 column volumes of lysis buffer at 4°C using a syringe. Unbound protein was eliminated by washing the column with 10 column volumes of lysis and wash buffer . PhKAT was eluted with 5 column volumes of elution buffer . Fractions containing PhKAT were pooled and concentrated to 1 mL using an Amicon Ultra with a 30-kDa cutoff (Millipore). This fraction was rebuffered in 50 mM HEPES–NaOH buffer (pH 7.5) containing 100 mM NaCl for the enzymatic assay and ITC or 5 mM HEPES–NaOH buffer (pH 7.5) using prepacked Sephadex G-25 gel filtration columns NAP-10  for crystallization. The protein concentration of monomeric PhKAT was determined spectroscopically with an extinction coefficient of 55,810 M−1⋅cm−1 at 280 nm.A single fresh colony of Kd) was calculated from the difference in absorbance at 360 nm, and the PLP-binding curve for PhKAT was obtained by the 2-site binding model with variable slopes using prism5.PhKAT was added to a 20 µM  PLP solution in a final volume of 500 µL of 50 mM HEPES–NaOH buffer (pH 7.5) containing 100 mM NaCl. Titration of PhKAT with PLP was monitored by absorption spectroscopy. Aliquots of PLP  were added to the cuvette containing PhKAT at 25°C. Spectra were recorded after the addition of PLP, and the PLP-binding constant (PLP-dependent KAT activity was assayed in 500 µL of 50 mM HEPES–NaOH buffer (pH 7.5) containing 100 mM NaCl, 10 µM PhKAT, 15 µM PLP, 20 µM KYN, and 64 µM 2OG. Assays were performed at room temperature (25°C), and the reaction was initiated by the addition of 2OG. KYNA production was monitored spectrophotometrically using a U-2810 UV–Vis spectrophotometer (Hitachi).−1⋅cm−1 at 332 nm.The transaminase abilities of KAT were assayed by measuring the rate of the KYNA production. In a total volume of 500 µL, the reaction mixture contained 10 µM PhKAT (dimer: 5 µM); 12 µM PLP; 0.08, 0.1, 0.4, 0.8, 1, 1.2, 2, 4, 6, 8, 12, 16, 20 and 24 µM 2OG; 0.02, 0.08, 0.1, 0.4, 0.8, 1, 1.2, 2, 4, 6, 8, 12, 16, 20 and 24 µM OXA; 50, or 100 µM KYN; 50 mM HEPES–NaOH buffer (pH 7.5); 100 mM NaCl; 0.4, 0.8, 1, 4, 8, 12 and 16 µM α-KMB, and/or 0.4, 0.8, 1, 4, 8, 12, 16 and 20 µM α-KBA. KYNA production was monitored by the increase in A332 at 25°C. The quantities of KYNA were determined spectroscopically with an extinction coefficient of 9.8 mMKd.The binding affinities of PhKAT with cofactor (PLP) and substrate (2OG) were determined by high-sensitivity microcalorimetry using a VP-ITC device  at 35°C. To avoid air bubbles, we degassed solutions under vacuum before use. Fresh PhKAT, and/or PLP and KYN were filled in the reaction cell at concentrations of 20, 30, or 40 µM  in 50 mM HEPES–NaOH buffer (pH 7.5) containing 100 mM NaCl and titrated in 25 10-µL steps against stock solutions of PLP (0.3 mM), 2OG (0.2 or 0.4 mM) at 5-min intervals. We searched extensively for the crystallization conditions of the PLP and/or 2OG complexes with PhKAT using the sitting-drop vapor diffusion method with Crystal Screen (CS) I and II (Hampton Research) and Wizard (Wiz) I and II . In brief, PhKAT was concentrated to 15 mg/mL (306.6 µM) and mixed with PLP and/or 2OG at 1∶1, and 1∶1:1 or 1∶1:2 ratios, respectively. Crystals of KAT complexes with cofactors and/or substrates and/or allosteric effectors were grown using the sitting-drop vapor diffusion method in CS I No. 14  for the PLP–2OG–PhKAT complex, CS I No. 23  for the PLP–PhKAT complex, and Wiz I No. 12  for the allosteric effector complex at 4°C.The crystals were soaked in crystallization solution containing an additional 18% ethylene glycol (vol/vol) as a cryoprotectant for the allosteric effector complex (PEG400 becomes a cryoprotectant). Radiographic data for the allosteric effector complex were collected at wavelengths of 0.9 or 1 Å at beam line BL38B1 at the SPring-8 synchrotron in Hyogo, Japan. Diffraction images were collected on a CCD-based detector system (ADSC Quantum 210) at liquid nitrogen temperature (100 K). Diffraction data were indexed, integrated, and scaled using the HKL2000 program suite. Crystal data and crystallographic statistics are shown in o|–|Fc| and 2|Fo|–|Fc| maps, we added PLP, water molecules, and/or 2OGs into the model. Subsequent refinement was done at resolutions of up to 1.72, 1.56, and 1.69 Å. The stereochemical geometry of the model was checked with the PROCHECK program.The structures were solved via the molecular replacement method, using the structure of apo-PhKAT (PDB cord: 1×0M) or PLP complex (PDB cord: 3AOV) as a search model with the MOLREP program The atomic coordinates and structure factors for PhKAT in complex with PLP and 2OG as substrates and/or allosteric effectors have been deposited in the Protein Data Bank with the following accession codes: PLP, 3AOV; PLP and 2OGs as substrate, 3AOW; PLP and 2OGs as substrate and allosteric effector, 3ATH.Figure S1Scheme of the KAT-catalyzed reaction showing the conversion of KYNA from KYN. KYNA is synthesized from KYN via a 4AD intermediate.(TIF)Click here for additional data file.Figure S2Spectrophotometric assay ofthe cofactor binding toPhKAT. PhKAT binds with the PLP cofactor as measured by absorbance spectroscopy. (A), PLP only, (B), 10 µM PhKAT, (C), 20 µM PhKAT. PLP performed at concentrations of 1, 2, 4, 8, 16, 32 and 64 µM.(TIF)Click here for additional data file.Figure S3Spectrophotometric assay of the time course of the PhKAT-catalyzed activity of KYN. The spectrum changes were monitored after the addition of 2OG, and PhKAT. 7 µM PLP; 20 µM KYN; 1 mM 2OG; 2 µM PhKAT. The enzymatic activity of PhKAT for KYNA productions cannot measure at this condition.(TIF)Click here for additional data file.Figure S4Comparison between the sequences of PhKAT and HuKAT II by pairwise alignment. The alignment was created using BioEdit and ESPript. Ph, Pyrococcus horikoshii; Hu, human. Black arrows and cylinders indicate the β-sheets and α-helices, respectively. Identical residues are in red boxes. A blue arrowhead indicates a PLP ligand lysine. The horizontal line indicates a possible mitochondrial-targeting peptide (MTP) of HuKAT II predicted by TargetP. PhKAT shares 27% identity with HuKAT II. The GenBank database (GB) accession numbers for PhKAT and HuKAT II are NP_142204 and NP_057312, respectively.(TIF)Click here for additional data file.Figure S5Allosteric regulations for PhKAT. (A) The sigmoid dose-response shows the allosteric inhibition by a second allosteric effector. The region of 8∼24 µM 2OG  in Ki) of 2OG for PhKAT was 20.11 µM (20.11 µM .. (B) Cl(TIF)Click here for additional data file.Figure S6ITC controls for the interaction between the cofactor, substrates and KAT. The ITC profiles include experimental conditions. (A) buffer and PLP; (B) PLP and 2OG; (C) PLP-KYN and 2OG.(TIF)Click here for additional data file.Figure S7Superimposed representation of PLP and/or 2OG as substrate- and allosteric effector-bound complexes structures of PhKAT. Close-up view and Cα-trace ribbon-and-line representation of the α2-helix of PLP complex (PDB code: 3AO V) and 2OG effector complex structures of PhKAT after optimal superimposition. The PLP-bound PhKAT complex is colored gray. Black-labeled numbers indicate the distance (Å) between the Cα traces of PLP complex and allosteric effector complex structures. The figure was generated using PyMOL.(TIF)Click here for additional data file.Figure S8The proposed mechanism for KYNA synthesis from KYN mediated by PhKAT. KYNA is synthesized from KYN via sequential reactions of KAT. PhKAT transaminates KYN to 2OG and/or OXA via PMP. OXA and/or 2OG function as an amino-group acceptors and a naturally allosteric inhibitor that regulates KAT activity, respectively. GLU, glutamic acid; ASP, asparatic acid.(TIF)Click here for additional data file.Table S1Data collection and refinement statistics for PhKAT in complex with PLP and/or 2OG.(TIF)Click here for additional data file.Table S2Comparison of the kinetic parameters of substrates for the transaminase reaction from KYN to the keto-acid group. The transamination abilities of KYN to acceptors  were assayed by measuring the rate of KYNA production.(TIF)Click here for additional data file.Table S3ITC parameters of cofactor and substrate binding to PhKAT. All measurements were performed in 50 mM HEPES–NaOH buffer (pH 7.5) with 100 mM NaCl.(TIF)Click here for additional data file.Table S4ITC control parameters.(TIF)Click here for additional data file.Equation S1Two sites binding with hill slopes .(TIF)Click here for additional data file.Equation S2Sigmoidal dose-response and absolute inhibition constant (Ki). The IC50 value was converted to an absolute inhibition constant Ki using the altered Cheng-Prusoff equation.(TIF)Click here for additional data file.Equation S3Allosteric sigmoidal.(TIF)Click here for additional data file.


The aim of the present study is to compare the already popular oxygenation index (OI) and PaOP <0.05).During a 6-month prospective observational study carried out in a polyvalent 10-bed adult ICU, ABGs were obtained from 145 patients. Two independent measurements were taken from each patient under the same mode of ventilation (SIMVPSV). PFr, SFr, VR and OI were calculated. Demographic data  were also recorded. Kolmogorov-Smirnov test for normality was calculated followed by bivariate nonparametric analysis (statistical significance: A total of 290 measurements were included for further analysis. Mean ± SD values for age and APACHE II score were 61.2 ± 16 years and 15.4 ± 1.8, respectively, while median ± SD values for PFr are 257 ± 135, for SFr 211 ± 58, for OI 4.8 ± 4.46 and for VR 1.19 ± 0.31. Relations between PFr, SFr and OI are displayed in Table Our study identified a strong relation between PFr, SFr and OI but not VR. Thus, these markers may be used interchangeably as bedside indices of oxygenation in critically ill patients. Yet larger studies are needed to come to a safer conclusion.


Although the inhibitory effect of cAMP on mTOR could be mediated by impinging on signalling cascades  that inhibit TSC1/2, an upstream negative regulator of mTORC1, we show that cAMP inhibits mTORC1 in TSC2 knockout (TSC2−/−) MEFs. We also show that cAMP inhibits insulin and amino acid-stimulated mTORC1 activation independently of Rheb, Rag GTPases, TSC2, PKB, MAPK and AMPK, indicating that cAMP may act independently of known regulatory inputs into mTOR. Moreover, we show that the prolonged elevation in [cAMP]i can also inhibit mTORC2. We provide evidence that this cAMP-dependent inhibition of mTORC1/2 is caused by the dissociation of mTORC1 and 2 and a reduction in mTOR catalytic activity, as determined by its auto-phosphorylation on Ser2481. Taken together, these results provide an important insight into how cAMP signals to mTOR and down-regulates its activity, which may lead to the identification of novel drug targets to inhibit mTOR that could be used for the treatment and prevention of human diseases such as cancer.cAMP and mTOR signalling pathways control a number of critical cellular processes including metabolism, protein synthesis, proliferation and cell survival and therefore understanding the signalling events which integrate these two signalling pathways is of particular interest. In this study, we show that the pharmacological elevation of [cAMP] ► cAMP inhibits mTORC1 and mTORC2. ► cAMP causes the dissociation of mTORC1 and 2. ► cAMP inhibits mTORC1 independently of TSC2, Rheb, Rag GTPases, PKB, MAPK and AMPK. ► cAMP inhibits TORC1 via a PKA-dependent mechanism. We provide evidence that this is mediated by a novel PKA-dependent mechanism which is independent of TSC2, Rheb and Rag GTPases. This report provides new insights into how cAMP talks to mTOR and could lead to the discovery of new anti-cancer drug targets.22.1All chemicals were purchased from Sigma-Aldrich , unless specified.2.2+/+/p53−/− MEFs (mouse embryonic fibroblasts), TSC2−/−/p53−/− MEFs ) 2 and 95% air.TSC23, 2.5 mM MgCl2, 2.5 mM CaCl2, 20 mM HEPES, pH 7.4), supplemented with 0.5× MEM amino acids solution (50× stock), 0.5× MEM non-essential amino acids solution (100× stock) and 0.5×l-glutamine (100× stock).Prior to treatments (unless specified in the figure legends) the media were removed and the cells were washed twice and incubated with modified Krebs–Ringer bicarbonate buffer (KRB) . Normalized lysates were stored at − 20 °C until further analysis.After treatment (unless specified in the figure legends), cells were lysed by the addition of ice cold lysis buffer (1% (v/v) Triton, 10 mM β-glycerophosphate, 50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM benzamidine–HCl, 0.2 mM phenylmethylsulfonyl fluoride, 1 μg/ml each of leupeptin and pepstatin, 0.1% (v/v) β-mercaptoethanol and 50 mM NaF). Lysates were then centrifuged for 10 min at 16,0002.3Recombinant adenovirus expressing dominant negative AMPK was kindly provided by Dr. Ian Salt  2.4GTPQ99L and RagCGDPS75L were described previously 2 method as previously described HA tagged S6K1 (HA-S6K1) pRK7 vector was kindly provided by Prof. John Blenis  2.5g. The supernatants were kept, and total protein concentrations were determined by the Bradford assay. Lysates containing 1.2 mg of protein were incubated with anti-myc antibody (Sigma) for 16 h at 4 °C with rotation, followed by the incubation with protein-G sepharose beads for a further period of 2 h at 4 °C with rotation. Beads were washed three times with 0.3% (w/v) CHAPS buffer, and then resuspended in 2× Laemmli sample buffer.The immunoprecipitation of mTORC1 and 2 complexes was essentially performed as previously described 2.6SDS-PAGE and Western blotting were performed as described previously 2.7i was measured by interpolation of a standard curve and related to cellular protein, which was assessed by Bradford assay.Cells were grown in 24-well tissue culture plates. After treatment, buffer was removed and ice cold 0.5 M trichloroacetic acid was added. cAMP was extracted and determined using a radioreceptor assay with binding protein purified from calf adrenal glands as previously described 33.1i, . In these cells, insulin also stimulated mTORC1 as determined by the phosphorylation of its downstream targets S6K1 on Thr389 and 4EBP1 on Ser65 and the phosphorylation of rpS6 on Ser240/Ser244 and a decrease in the electrophoretic mobility of S6K1  MEFs were pretreated with increasing concentrations of forskolin plus IBMX in the absence or presence of insulin  and 4EBP1 (Ser65) and a decrease in the electrophoretic mobility of S6K1 and 4EBP1  were incubated with forskolin and IBMX prior to stimulation with serum, myc-mTOR and associated proteins were then immunoprecipitated from these cells. In serum repleted cells mTORC1 was active as determined by the phosphorylation status of S6K1. In addition, mLST8 and the mTORC1 specific components RAPTOR and PRAS40, and the mTORC2 specific component RICTOR were co-immunoprecipitated with myc-mTOR, indicating the integrity of mTORC1 and mTORC2 under these conditions. As expected, the preincubation of cells with forskolin/IBMX inhibited serum-stimulated mTORC1 activity, as determined by the phosphorylation status of S6K1. Moreover, preincubation of cells with forskolin/IBMX caused the dissociation of the mTORC1 specific component RAPTOR and PRAS40. In addition, forskolin/IBMX caused the dissociation of the mTORC2 specific component RICTOR from myc-mTOR. The association of mLST8 to myc-mTOR was unaffected   During cancer development, the mTOR pathway is often abnormally up-regulated, which favours cancer cell survival, growth, replication, angiogenesis and metastasis i suppresses mTORC1/2 by promoting mTOR complex disassembly and inhibiting mTOR's intrinsic catalytic activity. These observations provide new insights into the crosstalk between cAMP and mTOR, which may also contribute to the design of novel mTOR inhibitors for future strategies in the fight against cancer.In conclusion, we show that elevation of [cAMP]


The PI3-kinase pathway is commonly activated in tumors, most often by loss of PTEN lipid phosphatase activity or the amplification or mutation of p110α. Oncogenic mutants have commonly been found in p110α, but rarely in any of the other catalytic subunits of class I PI3-kinases. We here characterize a p110β helical domain mutation, E633K, first identified in a Her2-positive breast cancer. The mutation increases basal p110β activity, but does not affect activation of p85/p110β dimers by phosphopeptides or Gβγ. Expression of the mutant causes increases in Akt and S6K1 activation, transformation, chemotaxis, proliferation and survival in low serum. E633 is conserved among class I PI3 Ks, and its mutation in p110β is also activating. Interestingly, the E633K mutant occurs near a region that interacts with membranes in activated PI 3-kinases, and its mutation abrogates the requirement for an intact Ras-binding domain in p110β-mediated transformation. We propose that the E633K mutant activates p110β by enhancing its basal association with membranes. This study presents the first analysis of an activating oncogenic mutation of p110β. The PI3-kinase signaling pathway is inappropriately activated in a variety of tumors in vitro. In addition, cells stably expressing this mutation display faster proliferation, enhanced survival in low serum, and increased motility. The region containing this mutation is an acidic patch that is in close proximity to the ABD-RBD linker and the RBD domain of p110β, and it is conserved in all class I PI3Ks. Our data suggests a novel inhibitory interface that can be disrupted in tumors.This study is the first characterization of a tumor-associated p110β mutation. The mutation, E633K, was identified in a HER2-positive breast tumor myc-p110 constructs were mutated using Quickchange site-directed mutagenesis .HEK293T cells were cultured in DMEM/10% FBS. NIH 3T3 cells were cultured in DMEM/10% NCS. Cells were transfected with equal amounts of p85 or p110-myc using Fugene HD (Promega) according to manufacturer’s instructions. For generation of stably-expressing cell lines, NIH 3T3 were transfected with wild type or E633K C-myc p110β in pcDNA3.1 and then selected using G418 (800 µg/ml), and maintained in 200 µg/ml of G418.1gamma2 as described previously 1gamma2 was purified as detailed elsewhere Sf9  cells were cultured and infected with recombinant baculoviruses for expression of Gbeta2, 1 mM CaCl2, 10% glycerol, 1% NP40, containing EDTA-free Protease inhibitor cocktail (Roche) and Phosphatase inhibitor cocktails 1 and 2 (Sigma), and myc-p110β was immunoprecipitated and assayed as described Myc-tagged wild type or E633K p110β was expressed with p85 in HEK 293T cells. The cells were lysed in 120 mM NaCl, 20 mM Tris (pH 7.5), 1 mM MgClwww.tcoffee.org).Sequence alignment of human p110α and human p110β was done using the T-Coffee alignment software .3 cells were plated in 96-well plates in the appropriate media. At various times, the cells were incubated with a 12 mM MTT solution in PBS for 4 h at 37°C. An equal volume of 0.1 g/ml SDS solution in 0.01 M HCl was added, and absorbance was read at 570 nm using a Spectramax M5 plate reader (Molecular Devices). For experiments with TGX-221, the cells were treated with 200 nM of TGX-221 throughout the duration of the experiment.The MTT assay (Invitrogen) was performed as described by the manufacturer. Briefly, 1×105 cells/well) in DMEM/10% NCS for one day and then maintained for 24 hours in DMEM with the specific amount of NCS. Cells were then trypsinized and mixed at 1∶1 volume with 0.4% Trypan Blue Dye. Trypan Blue positive (dead) cells were expressed as a percentage of the total number of cells.Cells were cultured in 6-well dishes (1×10Assays were performed as described in 5 cells/well) in six-well dishes and grown for two weeks, with media (DMEM/10% NCS) being changed every two days. The cells were stained with crystal violet and transformed foci/well counted.Assays were performed as described in 4 cells either in serum free or 10% NCS medium in the upper chamber of tissue culture inserts containing 8.0 µm pores , with DMEM/10% NCS media in the lower chamber. After 5 hours, the cells were fixed in 4% paraformaldehyde. The insert membranes were removed, stained and mounted on coverslips using Dapi Fluoromount . Images were collected at 10x magnification using a Nikon Diaphot inverted fluorescence microscope and a SPOT Idea digital camera, and analyzed using ImageJ software. For experiments with TGX-221, the cells were treated with 200 nM of TGX-221 throughout the duration of the experiment.Stably-transfected NIH 3T3 cells expressing WT or E633K p110β were starved overnight and then plated at 5×10http://faculty.vassar.edu/lowry/VassarStats.html).Statistical significance was determined using student’s t-test  and p110γ


Reduced neutrophil apoptosis plays an important role in the pathogenesis of acute exacerbation chronic obstructive pulmonary disease (AECOPD). The p38 mitogen-activated protein kinase (MAPK) signaling pathway is involved in neutrophil apoptosis. 1α,25-Dihydroxyvitamin D3  can induce tumor cell apoptosis. The aim of this study was to assess the effects of 1α,25VitD3 on peripheral blood neutrophil apoptosis in AECOPD and examine the role of the p38 MAPK signaling pathway.The study enrolled 36 AECOPD patients and 36 healthy volunteers. Venous blood samples were obtained from both groups. Serum 25-hydroxyvitamin D (25-(OH) D) levels in peripheral venous blood were assayed using liquid chromatography-tandem mass spectrometry (LC-MS/MS); the neutrophils were separated and cultured with SB203580 (a p38 inhibitor) and 1α,25VitD3. Neutrophil apoptosis was measured using flow cytometry, and phospho-p38 MAPK protein expression was detected by Western blot. Statistical analysis was performed using analysis of variance. Student's t-test and Pearson's correlation coefficient were used for the between-group differences and correlation analysis, respectively.The 25-(OH) D levels were lower in AECOPD patients than in healthy controls, and the peripheral blood neutrophil apoptosis results were similar. 1α,25VitD3 increased the apoptosis rate and the level of phospho-p38 MAPK in peripheral blood neutrophils of AECOPD patients. SB203580 partly inhibited 1α,25VitD3-induced peripheral blood neutrophil apoptosis and phospho-p38 MAPK overexpression. The 25-(OH) D levels were positively correlated with increased peripheral blood neutrophil apoptosis and phospho-p38 MAPK levels. In addition, expression of the phospho-p38 MAPK protein was also positively correlated with peripheral blood neutrophil apoptosis.Our results suggest that 1α,25VitD3 induces peripheral blood neutrophil apoptosis through the p38 MAPK signaling pathway in AECOPD patients. Chronic obstructive pulmonary disease (COPD), a major cause of morbidity and mortality throughout the world, is characterized by persistent airflow limitation and airway and systemic inflammation responses . The incRecent studies have shown that a substantial proportion of COPD patients have a vitamin D deficiency ,11, whicTherefore, we investigated the effect of 1α,25VitD3-induced neutrophil apoptosis in peripheral blood through the p38 MAPK signaling pathway in COPD patients.Hospitalized patients with documented COPD diagnoses according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria and that presented with an acute exacerbation were included. Acute exacerbation was defined by any combination of the following criteria: worsening dyspnea, increase in sputum volume and increase in sputum purulence . All AECTo investigate the effects of 1α,25VitD3 on the apoptotic behavior of peripheral blood neutrophils in AECOPD patients, 36 AECOPD patients and 36 healthy adult volunteers were enrolled throughout a single year . All patients were investigated upon admission. Peripheral venous blood samples of 25 mL were obtained from the healthy controls and AECOPD patients, Venous blood samples of 5 mL were obtained to detect 25-(OH) D levels, and venous heparin blood samples of 20 mL were obtained and immediately processed for cell culture. On the first day of admission, venous blood was obtained from all AECOPD patients before the administration of drugs. After 3 to 5 days and at discharge , demographic and clinical data, lung function tests, smoking habits, and microbiological findings were collected from the patient’s chart. For hospital stays < 11 days, an ambulatory visit was scheduled.6 cells/mL in RPMI 1640 medium containing 10% fetal calf serum in 5% CO2 and incubated at 37°C with 90% relative humidity.Neutrophils were isolated by Percoll density gradient centrifugation . All neutrophil samples with > 94% viability and purity were confirmed by Trypan Blue staining. The neutrophils were washed and resuspended at a concentration of 1 × 10–7, 3 × 10–7, and 6 × 10–7 M doses of 1α,25VitD3 were used, and a dose of 6 × 10–7 M 1α,25VitD3 was chosen for additional experiments. Neutrophils from the COPD patients were divided into 4 groups: 1) the COPD control group (Group B); 2) the SB203580(+) + 1α,25VitD3 COPD group (Group C) , which was pretreated with 2.5 μM × 10–5 SB203580 for 0.5 h, followed by incubation with 1α,25VitD3 (6 × 10–7 M) (Sigma-Aldrich); 3) the 1α,25VitD3 group (Group D), which was treated with 6 × 10–7 M 1α,25VitD3; 4) the COPD SB203580(+) group (Group E), which was pretreated with 2.5 μM × 10–5 SB203580 as described for group 2. The abovementioned groups of neutrophils were cultured for 24 h at 37°C. The SB203580 (an inhibitor highly specific for p38) doses were chosen based on previous studies and preliminary experiments. Each group was divided into 2 wells, 1 for flow cytometry and another for Western blot analysis. After the 24 h treatment, neutrophil samples were collected for subsequent experiments.All neutrophils were isolated from the healthy controls (Group A) and COPD patients. To pretreat the neutrophils from COPD patients in the preliminary experiments, 1 × 10Serum sample was extracted by adding 1 mL of methyl tert-butyl ether and 20 μL of isotope-labeled internal standard to 200 μL of serum. The mixture was vortexed, centrifuged, dried down and then reconstituted in 100 μL of 80% methanol. Serum 25-(OH) D2 and 25-(OH) D3 were determined using a LC-MS/MS method. LC analysis was performed on a Shimadzu system . Chromatographic separation was achieved on a reverse phase column  at a 1.0 mL/min flow rate with a gradient elution using water and methanol (containing 0.2% formic acid) as the mobile phase. An API4000 tandem mass spectrometer  equipped with an atmospheric pressure chemical ionization source was used for quantitative analysis.2, 140 mM NaCl), incubated with Annexin V-FITC/PI for 10 min in the dark, and analyzed using flow cytometry. Neutrophils in the early stage of apoptosis stained positively for Annexin V-FITC; those in the late stage stained positively for both Annexin V-FITC and PI. The data were analyzed using the Modfit and Cell Quest software programs . Apoptotic neutrophils were measured immediately after obtaining the blood sample (baseline apoptosis) and after culturing for 24 h. The apoptosis rate represents the proportion of apoptotic neutrophils measured minus the baseline value and was considered for further analysis.The percentages of apoptotic neutrophils were determined using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (BioVision) and detected by flow cytometry according to the manufacturer’s protocol. Briefly, after the SB203580 and 1α,25VitD3 treatments, the neutrophils were collected and resuspended in binding buffer . After adding 0.6mL of cold RIPA buffer  and protease inhibitors, the cells were scraped at 4°C. The cell lysate samples were subjected to centrifugation at 14,000 × g for 15min at 4°C. The resultant protein samples were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore). The membrane was stained with Ponceau S to confirm the uniform transfer of all samples and then incubated in blocking solution (PBS with 0.05% tween 20 and 5% non-fat dry milk) for 1 h at room temperature. These samples were then incubated overnight with anti-p38 MAPK and anti-phospho-p38 MAPK antibodies , with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (Sigma-Aldrich) as the loading control. Immunoreactive bands were detected by incubation with a horseradish peroxidase–conjugated secondary antibody and a chemiluminescence substrate . The scanned films were densitometrically analyzed using Quantity One software (Fuji2000).The results are expressed as the mean ± standard deviation (SD). The statistical significance between the different groups was determined by analysis of variance (ANOVA) and Student’s t-test; Chi-square statistics were used to analyze participants’ gender and smoking habits. We used the Pearson's correlation coefficient for the correlation analysis. p < 0.05 was considered statistically significant.1 in percent predicted was 39%. There was no significant difference in the gender, age, and smoking habits between AECOPD patients and healthy controls. The 25-(OH) D levels were significantly higher in the healthy controls than in the AECOPD patients.A total of 36 AECOPD patients and 36 healthy adult volunteers were enrolled in the study. The characteristics of the study population are shown in –7 M) did not significantly affect apoptosis (p > 0.05), However, at higher doses , 1α,25VitD3 increased the apoptosis rate significantly (p < 0.05) in AECOPD patients. Here, we used a 6 × 10–7 M dose of 1α,25VitD3 in culture for further investigation. The peripheral blood neutrophil apoptosis rate was increased in the 1α,25VitD3 AECOPD group compared to the AECOPD control group (p < 0.05)  .To assess whether p38 MAPK was activated in the peripheral blood neutrophil apoptosis induced by 1α,25VitD3, we measured the p38 MAPK expression (phospho-p38 MAPK) by Western blot . The phoTo further elucidate the role of the p38 MAPK signaling pathway in 1α,25VitD3-induced apoptosis, we observed the effects of SB203580, a specific p38 inhibitor, on 1α,25VitD3-induced apoptosis. As shown in Figs As shown in COPD patients are at particularly high risk for developing neutrophil-mediated inflammatory diseases. It has been assumed that the neutrophil half-life in vitro is a reflection of its life span in vivo . Our exp–7 M, which was confirmed by data from Hirsch et al. [–7 M and 6 × 10–7 M doses, which are consistent with the conditions used in NB4 cell lines [–7 M.The possibility that vitamin D insufficiency and deficiency may increase COPD risk has only been recognized recently –13. Seruh et al. . Howeverll lines . This reThe p38 MAPK signaling pathway plays an important role in human neutrophil apoptosis . To explOur data demonstrated that 1α,25VitD3 could induce peripheral blood neutrophil apoptosis in AECOPD and that the mechanism of apoptosis was related to the p38 MAPK pathway.


Thermoplasma acidophilum. We analyzed the modified nucleosides in native initiator and elongator tRNAMet, predicted the candidate genes for the tRNA methyltransferases on the basis of the tRNAMet and tRNALeu sequences, and characterized Trm5, Trm1 and Trm56 by purifying recombinant proteins. We found that the Ta0997, Ta0931, and Ta0836 genes of T. acidophilum encode Trm1, Trm56 and Trm5, respectively. Initiator tRNAMet from T. acidophilum strain HO-62 contained G+, m1I, and m22G, which were not reported previously in this tRNA, and the m2G26 and m22G26 were formed by Trm1. In the case of elongator tRNAMet, our analysis showed that the previously unidentified G modification at position 26 was a mixture of m2G and m22G, and that they were also generated by Trm1. Furthermore, purified Trm1 and Trm56 could methylate the precursor of elongator tRNAMet, which has an intron at the canonical position. However, the speed of methyl-transfer by Trm56 to the precursor RNA was considerably slower than that to the mature transcript, which suggests that Trm56 acts mainly on the transcript after the intron has been removed. Moreover, cellular arrangements of the tRNA methyltransferases in T. acidophilum are discussed.We investigated tRNA methyltransferase activities in crude cell extracts from the thermoacidophilic archaeon  Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at 59 °C and pH 1.9 -AdoMet (2.14 GBq/mmol) was purchased from PerkinElmer . Q-Sepharose Fast Flow, HiTrap Q-Sepharose, HiTrap SP-Sepharose, HiTrap Heparin-Sepharose and HiLoad 16/600 Superdex 200 prep grade were bought from GE Healthcare Japan . DNA oligomers were synthesized by Invitrogen Japan . Other chemical reagents were of analytical grade.-AdoMet were incubated in 40 µL of buffer A at 55 °C for 1 h. The RNA was extracted with phenol-chloroform and then recovered by ethanol precipitation. The RNA pellet was dissolved in 3 µL of 50 mM sodium acetate (pH 5.0), and digested with 2.5 units of nuclease P1 . The sample was separated using 2D-TLC as described previously -AdoMet into the appropriate tRNA transcript. For the reaction, 66 nM enzyme, 4.25 µM transcript, and 17.3 µM [methyl-14C]-AdoMet were incubated in 40 µL of buffer A at 50 °C for 5 min. An aliquot (35 µL) of the reaction was then used for the filter assay. To visualize the methyl-transfer reaction, we used 10% PAGE (7 M urea) and autoradiography. Briefly, tRNA (0.1 A260 units) was incubated with 66 nM enzyme and 17.3 µM [methyl-14C]-AdoMet at 50 °C for 5 min in 40 µL of buffer A, and then loaded onto a 10% polyacrylamide gel that contained 7 M urea. The gel was stained with methylene blue or toluidine blue, and then dried. The incorporation of 14C-methyl groups into the tRNA was monitored with a Typhoon FLA 7000 laser scanner . The 2D-TLC was performed as follows. An aliquot of 1 µg of purified protein, 0.2 A260 units of tRNA transcript and 0.69 nmol of [methyl-14C]-AdoMet were incubated in 40 µL of buffer A at 55°C for 1 h. The RNA was extracted with phenol-chloroform and then recovered by ethanol precipitation. The RNA pellet was digested with 1.5 units of nuclease P1. The sample was separated using 2D-TLC as described previously [14C-methylated nucleotides were monitored with a Typhoon FLA 7000 laser scanner .The transcripts were prepared by using T7 RNA polymerase and purified by Q-Sepharose column chromatography and 10% PAGE (7 M urea). The standard assay for the purified enzymes was to measure the incorporation of eviously . The 14CE. coli RNase P were a gift from Prof. Takashi Yokogawa (Gifu University). The C5 protein was purified as described in the reference [E. coli tRNAMetf was used. The 5'-leader sequence was cleaved by RNase P in accordance with the reference [The plasmid vectors for the C5 protein and M1 RNA of eference . The M1 eference . The 5'-eference .


Post-translational histone modifications play a critical role in genome functions such as epigenetic gene regulation and genome maintenance. The tail of the histone H4 N-terminus contains several amino acids that can be acetylated and methylated. Some of these modifications are known to undergo drastic changes during the cell cycle. In this study, we generated a panel of mouse monoclonal antibodies against histone H4 modifications, including acetylation at K5, K8, K12, and K16, and different levels of methylation at K20. Their specificity was evaluated by ELISA and immunoblotting using synthetic peptide and recombinant proteins that harbor specific modifications or amino acid substitutions. Immunofluorescence confirmed the characteristic distributions of target modifications. An H4K5 acetylation (H4K5ac)-specific antibody CMA405 reacted with K5ac only when the neighboring K8 was unacetylated. This unique feature allowed us to detect newly assembled H4, which is diacetylated at K5 and K12, and distinguish it from hyperacetylated H4, where K5 and K8 are both acetylated. Chromatin immunoprecipiation combined with deep sequencing (ChIP-seq) revealed that acetylation of both H4K8 and H4K16 were enriched around transcription start sites. These extensively characterized and highly specific antibodies will be useful for future epigenetics and epigenome studies. In eukaryotic nuclei, ∼150 bp of DNA is wrapped around the histone octamer, which consists of two copies of four core histones  to form a nucleosome, the fundamental unit of chromatin. Post-translational modifications on these histones play a critical role in genome function, including the regulation of transcription and the maintenance of genome integrity (Bannister and Kouzarides Trimethylation on H4 lysine 20 (H4K20me3) is also involved in heterochromatin formation, correlated with H3K9me3  and carried out according to guidelines for animal experimentation at Hokkaido University, where Mab Institute Inc is located. Animals were housed in a specific pathogen-free facility at Hokkaido University. Humane euthanasia of mice was performed by cervical dislocation by individuals with a demonstrated high degree of technical proficiency.Synthetic peptides . For antibody purification, cells were grown in CD Hybridoma medium (Life Technologies) and the supernatant (250 ml) was filtrated through a 0.45-μm pore filter, as previously described , and Cy5 N-hydroxysuccinimide ester  were dissolved into 100 μl dimethyl sulfoxide  and stored at −20 °C. IgG (100 μg) was diluted into 100 mM NaHCO3 (pH 8.3) in 100 μl. After addition of a dye solution , the mixture was incubated for 1 h at room temperature with gentle rotation. The sample was passed through a PD-mini G-25 desalting column , which was pre-equilibrated with PBS, to remove unconjugated dyes, and dye-conjugated IgG was concentrated up to ∼1 mg/ml using an Ultrafree 0.5 filter . The IgG concentration and dye:protein ratio were calculated from the absorbance at 280 and 494, 552, or 650 nm, using the extinction coefficient of IgG and correction factor at 280 nm provided by the manufacturers. Fluorescent dye-labeled IgG samples that yielded dye:protein ratio ∼3–9 were used for live imaging.Purified IgG was conjugated with a fluorescent dye  and 10 % fetal calf serum (FCS), as previously described  and HeLa cells  were obtained from RIKEN BRC on January 11, 2011  with Hoechst 33342 (100 ng/ml in PBS) in PBS containing 10 % Blocking One-P  for 2 h at room temperature. After washing with PBS, coverslips were mounted in Prolong Gold (Life Technologies). Fluorescence images were collected using a confocal microscope  operated by the built-in software (ver 2.1-4.1) with a PlanSApo 60× (NA = 1.35) or a PlanApoN SC 60× (NA = 1.4) objective lens . Hoechst 33342, Alexa488, Cy3, and Cy5/Alexa647 signals were acquired by sequential scanning using laser lines at 405, 488, 543, and 633 nm, combined with emission dichroic mirror/barrier filter SDM490/BA430-470, SDM560/BA505-525, SDM640/BA560-620, and none/BA650IF, respectively, with a main dichroic mirror DM405/488/543/633.g for 10 min. Supernatant containing 4–8 μg DNA was incubated with a mouse monoclonal antibody against H4K5ac, H4K8ac, H4K12ac, or H4K16ac, pre-bound to magnetic beads at 4 °C overnight with rotation. The immune complexes were washed twice with ChIP buffer and twice with TE buffer. After, cross-links were reversed by incubating in TE containing 1 % SDS at 65 °C overnight with proteinase K, and DNA was purified using a QIAquick PCR purification kit (Qiagen).Cells (ZHBTc4) were cross-linked in 0.5 % formaldehyde and suspended in 250 μl ChIP buffer (5 mM PIPES (pH 8.0), 200 mM KCl, 1 mM CaCl2, 1.5 mM MgCl2, 5 % sucrose, 0.5 % NP-40, and protease inhibitor cocktail; Nacalai Tesque). Samples were incubated for 15 min on ice, sonicated for 5 s three times, and digested with micrococcal nuclease  and Ribonuclease A  at 37 °C for 40 min. The digested samples were centrifuged at 15,000×Sequenced reads of chromatin immunoprecipiation combined with deep sequencing (ChIP-seq) data were mapped onto the mouse genome (mm9) with Bowtie (version 0.12.8) using the parameter “-n 3 -m 2”. Estimation of normalized ChIP-seq signal intensities were calculated as follows. First, we counted mapped reads at 10,000 bp Fig. , 200 bp Sequencing was performed using a Genome Analyzer IIx (Illumina). PolyA+ RNA was enriched from 100 ng of total RNA by two successive rounds of oligo(dT) selection. The polyA+ RNA was fragmented and first-strand cDNA was synthesized by random hexamer priming. Following second-strand cDNA synthesis, dsDNA was repaired using T4 DNA polymerase, Klenow enzyme, and T4 polynucleotide kinase (PNK) (New England Biolabs), followed by treatment with Klenow exo- to add an A base to the 3′ end. After ligation of the adaptor using TaKaRa Ligation Mix (TaKaRa), the adaptor-ligated DNA was amplified using PCR primers for 12 cycles, and the amplified library was isolated using the E-gel Electrophoresis System (Life Technologies). The samples were purified using a QIAquick MinElute Kit (Qiagen). Sequenced reads were mapped onto the mouse genome (mm9) using Tophat (version 2.0.10). Gene expression levels  were estimated by Cufflinks (version 2.1.1) using the parameters “-u -b (on cuffdiff)”. We classified genes into 11 groups based on the expression levels: Silent (FPKM = 0) and 10 groups with different expression levels separated at 10 percentile intervals .For a close look at ChIP-seq results, IGV_2.0.34 was used, as in Fig.We generated mAbs directed against unmodified and specifically modified forms of histone H4, including acetylation at K5, K8, K12, and K16, and three levels of methylation at K20  over the hyperacetylated H4, which harbors acetylation on K5, K8, K12, and K16. Indeed, immnofluorescence revealed that only a fraction of cells were intensely stained with CMA405, and the intra-nuclear distribution resembled replication foci in S phase cells Fig. . To compTo reveal the localization of acetylation at different lysines in H4, we performed ChIP-seq analysis using mouse embryonic stem cells with the specific antibodies. In a genome-wide view, H4K5ac was not enriched in specific loci, but was rather depleted from transcriptionally active regions, complementary to H4K8ac and H4K16ac Fig. . AggregaAmong the H4K20 methylation-specific mAbs, H4K20me1- and H4K20me3-specific mAbs (CMA421 and CMA423) reacted with their targets in the presence of K16ac, and H4K20me2-specific CMA422 hardly reacted with the peptide containing K16ac with K20me2 Fig. , in goodWe showed that mouse mAbs directed against histone H4 and its modifications are highly specific to the targets and are applicable to immunoblotting, immunofluorescence, and ChIP-seq. While histone modification-specific antibodies are now widely available from different vendors, most of them are rabbit polyclonals and therefore associated with lot-to-lot variation of the specificity and affinity. Furthermore, a systematic study revealed that 20–25 % of histone modification-specific antibodies were disqualified for ChIP-seq application (Egelhofer et al. Among the mAbs described here, H4K5ac-specific mAb CMA405 has a unique specificity. It recognizes acetylated K5 with unacetylated K8. This combination is typically found in newly assembled H4, which is diacetylated at K5 and K12, and CMA405 indeed highlights structures that resemble DNA replication foci in S phase cells. Thus, CMA405 will be particularly useful for detecting newly assembled chromatin and also for highlighting cells in S phase by microscopy and flow cytometry.


CSFS-AFM analysis established good correlation between the topography of the developed nanostructures and various features of WO3 nanoflakes synthesized via a two-step sol-gel-exfoliation method. It was determined that β-WO3 nanoflakes annealed at 550°C possess distinguished and exceptional thickness-dependent properties in comparison with the bulk, micro and nanostructured WO3 synthesized at alternative temperatures.The morphology and electrical properties of orthorhombic β-WO Similar to graphene, WO3 can be mechanically or chemically exfoliated to provide fundamental layers. However, unlike graphene, which does not have bandgap, Q2D WO3 has rather large bandgap, making Q2D WO3 nanoflakes more versatile as candidates for thin, flexible devices and potential applications in catalysis [The layered transitional quasi-two-dimensional Q2D) semiconductor oxides MOer limit ,2. AmongD semicono 2.9 eV . It has  470 nm) . Extrinsatalysis , solar catalysis  and variatalysis . It has atalysis .3, nanoscaled Q2D WO3 with thickness less than ~10 nm has received relatively little attention so far compared to its microstructured counterparts and to Q2D transitional metal dichalcogenides MX2 . In addition, last year's reports on alternative transitional semiconductor oxide Q2D MoO3 have exhibited exceptional thickness-dependent properties and the substantial increased of the charge carriers mobility  in Q2D MoO3[2 that the reduction of bandgap can be achieved through decreasing the thickness of Q2D nanoflakes down to monolayer [3 in its Q2D form can further engineer the materials' electrical properties, as quantum confinement effects in 2D form will significantly influence charge transport, electronic band structure and electrochemical properties [3 can enhance the performance of this functional Q2D material revealing unique properties that do not exist in its bulk form [However, despite great interest of the research and industrial communities to the bulk and microstructured WO Q2D MoO3. It was onolayer . Therefo3 nanostructures, such as microwave-assisted hydrothermal [3 is a topic that is yet to be widely explored. For instance, in a recent report, it was demonstrated that one possible way of bandgap reduction in bulk WO3 is to increase its sintering temperature [3 nanoflakes remains largely unexplored.The development of Q2D materials is generally a two-step process, the synthesis of the layered bulk material followed by the exfoliation process . Althougothermal , vapour-othermal , sol-gelothermal , electroothermal  and arc-othermal , synthesperature . However3 obtained for nanoflakes with thickness below ~10 nm developed via two-step sol-gel-exfoliation method. These properties were mapped without damaging the sample by carefully controlling the sample-tip force. This is performed by using current sensing force spectroscopy atomic force microscopy (CSFS-AFM), also known as PeakForce TUNA™ [3 nanoflakes were obtained and compared to the similar properties of the commercially available WO3. The electro-catalytic properties of Q2D β-WO3 were obtained by investigation samples for hydrogen evolution reaction (HER) from water by linear sweep voltammetry (LSV) and a Tafel plot. The obtained results indicate that Q2D β-WO3 nanoflakes are promising electro-catalyst for the HER [In this work, we present for the first time new distinguishing thickness-dependent electrical properties of Q2D β-WOce TUNA™ , which ace TUNA™ . This te the HER ,23,24.3 nanoflakes were obtained via two-step sol-gel-exfoliation process. All of the following precursors including sodium tungstate dehydrate (Na2WO4.2H2O), hydrogen peroxide , ethanol, polyethylene glycol , nitric acid  and perchloric acid (HClO4) were used. Initially, 1 g of Na2WO4.2H2O precursor dissolved in 10 ml de-ionized (DI) water. Then, 6 ml of HNO3 was added drop wise to the solution to obtain a greenish yellow precipitation (H2WO4). After washing with DI water for several times, the remained H2WO4 was dissolved in 2 ml H2O2 and stirred at room temperature for 2 h. The procedure was followed by addition of known amount of PEG to obtain a viscous sol and as a result, adherence and homogeneity of the final transparent films can be improved. Then, 30 ml ethanol was added and the sol was stirred for another 2 h. After 1 day of ageing, the prepared sol was deposited on the Au- and Cr-coated Si substrates by using spin-coating instrument .Ultra-thin sub-10-nm Q2D WO-1. The selection of these temperatures for sintering nanostructured WO3 was based on the fact that orthorhombic β-WO3 phase can be obtained at various annealing temperatures up to 740°C [3 samples was high. The last phase of the process was to apply mechanical exfoliation in order to obtain extremely thin layers for all further analysis. In the mechanical exfoliation method, scotch-tape was used and the similar procedure applied as per exfoliation of the graphene nanosheets. The reproducibility of exfoliated 2D WO3 nanoflakes depended upon the mechanical force applied on the scotch-tape during exfoliation process. Therefore, some of the exfoliated Q2D WO3 nanoflakes were thicker than others.The obtained sol-containing thin films were placed in oven at 80°C for a week to achieve the complete gelation. The dried films were subsequently sintered at 550, 650, 700, 750 and 800°C, respectively, for 1 h at the heating rate of 1°C minto 740°C . Another3 was characterized by RINT 2100VLR/PC, Rigaku X-ray diffractometer  with CuKα radiation (α = 0.1542 Å) at angle step of 1° min-1. XRD intensities and records were collected using a scintillation detector, and each sample was scanned over the 2-theta range 10° to 80°. Spectral analyses were carried out using Bruker ZRD search match programme, EVA™ , and crystalline phases were analysed using the ICDD-JCPDS powder diffraction database. Both the surface morphology and structural configuration of Q2D WO3 nanoflakes were evaluated by a Philips XL30 field emission scanning electron microscopy (SEM). Iridium coating was also applied to the sample to improve the quality of the imaging. All the measurements were completed at room temperature. Meanwhile, the local chemical homogeneity of the WO3 nanoflakes were conducted by Type N energy dispersive X-ray spectrometer (EDX)  equipped with JOEL-JSM 5600 LV SEM. Fourier transform infra-red absorption spectroscopy (FTIR) measurements were performed in air at room temperature by using Nicolet 6700 FTIR Spectrometer . Background gas for this examination was N2. FTIR spectrometer had the following working parameters during the analysis: IR polarization, zero/no polarization; angle of incidence, 90° perpendicular to the sample; analysing material, KBr and type of detector, MCT detector. During each measurement, the background spectrum was registered and consequently subtracted from the sample spectrum captured to obtain the final spectra. These were studied by employing Omnic Spectroscopy Software Suite. All the spectra were acquired in the following range: 4,000 to 400 cm-1. Before experiments, WO3 nanostructures were preheated to 200°C for removal of adsorbed moisture and CO2 and then cooled down to room temperature. For FTIR measurements, Q2D WO3 nanoflakes were also prepared on Au/Si substrate. Ultra-high clean N2 was selected as a background gas. It was flowing through the cell containing WO3 for 10 min with speed 100 ml min-1. After that, WO3 nanostructures were exposed to the air for 10 min before any measurements commenced. At each experiment and evaluation, the background spectrum was recorded and subtracted from the sample spectrum obtained [The crystallinity of the sol-gel-developed WOobtained .3 nanoflakes [2SO4 solution at a room temperature. Lastly, the HER with Q2D WO3 nanoflake as the catalyst was measured using standard three-electrode electrochemical configuration in 1.0 M H2SO4 electrolyte de-aired with Ar, where saturated calomel electrode (Pine Research Instrumentation) and graphite rod  have been used as reference and counter electrodes, respectively. The reference electrode was calibrated with respect to reversible hydrogen electrode (RHE) using Pt wires as working and counter electrodes. In 1.0 M H2SO4, ERHE = ESCE + 0.256 V. Potential sweeps were taken with a 5 mV s-1 scan rate. Electrodes were cycled at least 30 cycles prior to any measurements.CSFS-AFM measurements were performed to construct and identify the surface profile and simultaneously obtain typical topographical, tunnelling and current/voltage properties of the developed and exfoliated Q2D WOnoflakes -29. Thisnoflakes . μSense-3 on Au- and Cr-coated Si substrates, which were sintered at different temperatures. Micrographs of the deposited WO3 thin-films revealed the effect of the annealing temperature on the surface morphology. As shown in Figure 3 nanoflakes annealed at 550°C were in the range of 20 to 50 nm in length with few larger nanoflakes of ~100 nm. However, as the annealing temperature increased, the morphology of WO3 nanoflakes also changed and the average size of the sintered WO3 nanoflakes increased . The results obtained were similar to the previously published data for orthorhombic β-WO3[3 is stable in the temperature range of 330 to 740°C [Figure bic β-WO3. Generalto 740°C ,35. No i3 and their electrochemical properties, such as chemical structure, oxidation states, adsorption properties etc., must be obtained and optimized in order to achieve their best analytical performance in various applications. For this purpose, CSFS-AFM, FTIR and Raman spectroscopy techniques were used.Comprehensive information in relation to the developed ultra-thin Q2D WO3 sintered at 550°C and their characteristics analysed by CSFS-AFM are presented in Figure 3 nanoflakes. Multiple flake morphology of Q2D WO3  and impedance recorded for Q2D WO3 annealed at 650°C (1.8 ohm). Due to very small dimensions, the contribution from the Q2D WO3 working electrode into the total impedance confirmed to be very small. The resistance primarily comes from wiring  and electrolyte, where the resistance of Q2D WO3 nanoflakes is negligible.All impedance measurements were performed on Q2D WOIn situ FTIR spectroscopy of Q2D WO3 nanoflakes was utilized to determine surface chemistry and surface reactions of the developed crystalline nanostructures [3 nanoflakes sintered at 550 and 650°C, respectively, are presented in Figure 3. The higher surface area enables the detection of bands owing to surface OH and adsorbed water in the 3,700 to 3,100 cm-1 region , CO molecules do not adsorb on its surface because of full coordination. The frequency values obtained in spectra of CO adsorbed on Q2D WO3 nanoflakes shifted to the lower values compared to the assignments represented for microstructured WO3[3 nanoflakes, the degree of oxidation on some parts of the WO3 surface has been changed and few WOx3- sites appeared on the surface of nanoflakes causing CO adsorption. It should be noted that some residual hydrated WO3 is most likely present in the sample because hydrated WO3 is formed in the sol-gel process and then converted to β-WO3 during sintering [-1, are assigned to stretching mode of W-O, whereas the stretch at 984 cm-1 is due to W = O vibrations. The W-O stretching modes are less intense, and changes in the low-frequency modes may indicate some modifications in the tungsten-oxide framework. This is possibly owing to the fact that the surface of exfoliated Q2D WO3 itself contains various defects. In general, the majority of experimental phenomena discussed above were associated to adsorption on expected sites of oxide nanoflake surface . However, the appearance of the most active surface centres suggests a connection with defects in nanoflakes [-1 (Si), which directly relates to the substrate platform. The WO3 FTIR spectra also indicated that there were no impurities present in the prepared and exfoliated samples.Considering that WOtured WO3. This isintering ,39. In tnoflakes ,40. The 3, sintered at 550° and 650°C, respectively, and exfoliated ultra-thin Q2D WO3. Raman spectra for sol-gel-developed WO3 and exfoliated Q2D WO3 nanoflakes in the perturbation area of the spectrum are shown in Figure 3 were observed. The bending modes of WO3 are usually located between 600 and 900 cm-1, while the stretching modes can be observed between 200 and 500 cm-1[-1 has been assigned to the symmetric stretching mode of terminal (W6+ = O) groups which may also be vibrationally coupled [3[-1, are influenced significantly by hydration [-1 band can be used as a spectral marker for hydration level of WO3[3) also exhibits bands at these frequencies but is likely to be absent in sample prepared without a thermal annealing step.Raman spectroscopy was employed to determine the vibration and rotation information in relation to chemical bonds and symmetry of molecules in sol-gel-developed WO 500 cm-1. The pro coupled . This peoupled [3. There wydration , and as el of WO3. However3 nanoflakes sintered at 550°C was about two times higher than that the strength of peaks for the same sol-gel-developed WO3. At the same time, the magnitude of the peaks for exfoliated Q2D WO3 nanoflakes sintered at 650°C was just ~0.3 to 0.5 times higher compared to the intensity of peaks for their sol-gel-developed WO3 counterparts. This finding has confirmed the thickness-dependent properties of ultra-thin Q2D WO3. Following sintering at 550°C, there is a reduction in the spectral line width consistent with greater crystalline phase formation. Well-defined bands 712 and 802 cm-1 modes exhibit significant changes, with the mode at 712 cm-1 being particularly sensitive to the cation intercalation [3 as suitable nanomaterial for various sensing platforms [3 than 650°C aiming their further exfoliation and cation intercalation.It is noteworthy that the intensity of the peaks for the exfoliated Q2D WOcalation . Consequlatforms ,46 and r3. I-V curves for the sol-gel-developed WO3 nanostructures sintered at 550°C and for exfoliated ultra-thin Q2D WO3 nanoflakes sintered at 550°C and 650°C are presented in Figure 3[3 nanostructures and exfoliated Q2D WO3 nanoflakes. It is clearly exhibited that the measured current for Q2D WO3 was about from 5 (650°C) to 10 (550°C) times higher than the measured current for the sol-gel-developed WO3 nanostructures. This fact confirms that the CFSF-AFM current originates from the local properties of the material at the tip-sample contact. The higher electrical activity and therefore greater currents for the exfoliated Q2D WO3 nanoflakes appeared to be more related to higher heterogeneous electron exchange rate caused by the quantum confinement effects within the few-layers limit [3 nanoflakes can offer reduced power dissipation because of smaller short channel effects [3 nanoflakes with enhanced properties.Electrical CSFS-AFM measurements revealed and further proved the thickness-dependent properties of ultra-thin Q2D WO Figure 3. Howeverrs limit . Consequ effects . Further3 (surface area = 3 m2 g-1) [3 nanoflakes sintered at 550°C were recorded in a potential region of +0.1 to -0.2 V at a scan rate of 50 mV s-1 in 1.0 M H2SO4 solution. The results are presented in Figure 3 nanowires [3 exhibited very low catalytic activity towards electrochemical reaction for HER in this potential region, whereas Q2D β-WO3 nanoflakes sintered at 550°C displayed improved electro-catalytic activity. The observed electrochemical stability was recorded for 100 consecutive cycles in the solution   and Q2D anowires  in the s3, Q2D WO3 nanoflakes and hexagonal WO3 nanowires are -157, -112 and -116 mV decade-1, respectively [3 nanoflakes indicates that it is a superior material as a hydrogen production electrode of HER compared to hexagonal WO3 nanowires [3[3 nanoflakes developed via two-step sol-gel-exfoliation method can be effective electrode materials with improved HER activity.The Tafel plots Figure B were coowires [3. This coowires [3. These r3 nanoflakes, typically with lengths and widths of the order of 50 to 100 nm and thickness of 7 to 9 nm were produced by a two-step sol-gel-exfoliation method. It was experimentally determined that exfoliation of the ultra-thin Q2D β-WO3 nanoflakes was only possible at nanostructures sintered at 550 and 650°C. Spectral evidence for β-WO3 phase exists in the Raman measurements. This is also consistent with the absence of other crystalline phases in the XRD measurements of this material. CSFS-AFM, FTIR, Raman and electrochemical measurements further confirmed that the annealing temperature of 550°C is the most acceptable sintering temperature for WO3, if ultra-thin Q2D β-WO3 nanoflakes with thickness of ~7 to 9 nm have to be obtained. The results of the conducted research have reassured the thickness-dependent properties of the ultra-thin Q2D WO3 nanoflakes and demonstrated that Q2D WO3 nanoflakes can be excellent electro-catalytic material for HER with high activity and stability in water. The present study also illustrates the fundamental role the nanostructure of WO3 on the catalytic performance. The high surface-to-volume ratio of Q2D WO3 nanoflakes, controllable deposition and compatibility with existing semiconductor fabrication infrastructure suggest that the reported Q2D β-WO3 nanostructures can be utilized in new generation of low-cost oxide semiconductor functional devices including solar cells and various sensing platforms. Moreover, both the fabrication process and its framework have great compatibility with other emerging Q2D semiconductors and conductors such as graphene.Orthorhombic Q2D β-WOThe authors declare no competing interests.3 nanoflakes, characterized them with CSFS-AFM, SEM, FTIR, Raman and electrochemical measurements and analysed the data. S.Z. and E.K organized, wrote and edited the paper. All authors contributed to the discussion and preparation of the manuscript. All authors read and approved the final manuscript.S.Z. conceived the idea, designed the experiments, conducted XRD, EDX and impedance measurements and analysed the data. E.K synthesized Q2D WOFP-011-02 Technical Committee of Standards Australia International and a Head of the Australian delegation in International Standards Organization: ISO TC21/SC8 Technical Committee since 2005. He has published 2 monographs, 6 chapters to books and more than 170 peer-reviewed scientific publications. He is a recipient of the 2007, 2011 and 2013 Australian Academy of Science/Japan Society for Promotion of Science and 2010 Australian Government Endeavour Executive Awards for his work on nanostructured materials.S.Z. obtained his Ph.D. in Materials Science and Engineering in 1991. He has combined experience as Research Scientist working at the different universities in Australia, Japan and Europe and industrial environments for more than 23 years. He is a Principal Research Scientist at Materials Science and Engineering Division of CSIRO. His research interests lie in the area of the development, design and evaluation of new functional nanomaterials for state-of-the-art functional devices. He is also Chairman of E.K. was awarded a BSc (Applied Chemistry) from the University of RMIT, Victoria, Australia (1997). From 1998 until 2004, Eugene worked as a Research Project Officer at Scientific Services Laboratory, Melbourne, Australia. During this period, he was responsible for both technical and management components of Sample and Compliance testing of fire equipment, including detection equipment. Eugene has joined CSIRO Materials Science and Engineering Division in 2004. His current research involves development of nanostructured semiconductor materials for various functional devices.


Thus, either the apatite was less mature or the amount of amorphous calcium phosphate was higher in the composite layer formed at 25 °C. In vivo, the pin tract infection rate by visual inspection for 37F4.0 (45%) was lower than that for 25F1.0 , and the rate of osteomyelitis for 37F4.0 (35%) was lower than that for 25F0.5 . The extraction torque for 37F4.0 (0.276 ± 0.117 Nm) was higher than that for 25F0.5  and that for 25F1.0 . The invasion rate of S. aureus for 37F4.0 (35%) was lower than that for 25F0.5 . On the whole, the FGF-2-apatite composite layer formed at 25 °C tended to be less effective at improving fixation strength in the bone-pin interface and resisting pin tract infections. These results suggest that the chemistry of the calcium phosphate matrix that embeds FGF-2, in addition to FGF-2 content and activity, has a significant impact on composite infection resistance and fixation strength.The purpose of the present study was to fabricate fibroblast growth factor (FGF)-2-apatite composite layers on titanium (Ti) pins in one step at 25 °C using a supersaturated calcium phosphate (CaP) solution, and to evaluate the physicochemical characteristics and biological effects of the coated Ti pins compared with coated Ti pins fabricated at 37 °C. Ti pins were immersed in a supersaturated CaP solution containing 0.5, 1.0, or 2.0 µg/mL FGF-2 at 25 °C for 24 h  or containing 4.0 µg/mL FGF-2 at 37 °C for 48 h (37F4.0). Except for the 25F0.5, the chemical compositions and the mitogenic activity levels of FGF-2 of the composite layers formed by these two methods were similar, except for the Ca/P molar ratio, which was markedly smaller at 25 °C (1.55–1.56 ± 0.01–0.02,  Pin tract infections and loose anchorages are severe problems that can occur during external fixation ,4,5,6,7.Accelerating tissue regeneration around the pin tract is another approach to control bacterial infection. In a previous study, we formed fibroblast growth factor (FGF)-2-apatite composite layers on titanium (Ti) pins by immersing them in an infusion fluid-based supersaturated calcium phosphate (CaP) solution containing FGF-2 at 37 °C for 48 h . FGF-2 iReducing the fabrication temperature and time of the FGF-2-apatite composite layers to enhance their biological activity and practicality is an important challenge. The previous 37 °C and 48 h fabrication temperature and time, respectively, are associated with a significant risk of losing the FGF-2 biological activity ,23. AlthThe purpose of the present study was (1) to fabricate FGF-2-apatite composite layers on Ti pins in one step at 25 °C using an infusion fluid-based supersaturated CaP solution containing different concentrations of FGF-2; and (2) to evaluate the physicochemical characteristics, biological activity, resistance to pin tract infection, and bone fixation strength of the coated Ti pins compared with Ti pins coated at 37 °C.As reported previously, after immersion in a supersaturated CaP solution containing 4.0 µg/mL FGF-2 at 37 °C for 2 days (37F4.0), Ti pins were entirely and homogeneously coated with a fine-structured layer, as confirmed by scanning electron microscopy (SEM) a. Similap = 0.0221) and 37F4.0 (p =0.0229) groups. No significant differences were observed in Ca or P contents among all the groups. However, the layer Ca/P molar ratios of the 25F0.5 , 25F1.0 , and 25F2.0  groups were significantly lower than that of the 37F4.0 group (1.67 ± 0.11)  b.Very broad and small peaks appeared in the X-ray diffraction (XRD) patterns of all the composite layers at approximately 26° and 32° . These p® tape detachment test, the surfaces of the 25F0.5, 25F1.0, 25F2.0, and 37F4.0 pins remained covered with the FGF-2-apatite composite layers, and glue was observed on the surfaces in all the samples  groups, or between the 37F4.0 and 25F2.0 (p = 0.5373) groups.The biological activity of the FGF-2-apatite composite layers on Ti pins coated at 25 °C for 1 day was comparable to that of those coated at 37 °C for 2 days, except for the 25F0.5 group . The mitn = 11/20), 20% , 45% , and 35%  (n = 0/20), 20% , 5% , and 20% . The resistance to pin tract infection (Grade 0v:1v:2v ratio) for the 25F1.0 group was significantly lower than that of the 37F4.0 group (p = 0.0213). It is worth noting that the p value for the difference in Grade 0v:1v:2v ratio between the 37F4.0 and UN1 groups was markedly lower (p = 0.0868) than that between the 25F1.0 and UN groups (p = 0.5361), and between 25F0.5 and UN groups (p = 0.3494). This suggests that there was no improvement in resistance to pin tract infection for the 25 °C groups compared with the UN1 group (uncoated Ti pins). We carried out another set of animal experiments to compare the 25F2.0 and UN2 groups. The 25F2.0 group showed no improvement in resistance to pin tract infection compared with UN2 group (another uncoated Ti pins) , 1v (skin infection without pin loosening), and 2v (infection with pin loosening) after visual inspection of pin tract infections are shown in  = 7/20) a. The raTi pins) b.p = 0.2405–0.7962) (p = 0.5957) , 1s , and 2s (severe inflammation) soft tissue inflammation along the pin tract are shown in –0.7962) a. Simila 0.5957) b.n = 13/20), 45% , 25% , and 30%  (n = 6/20), 40% , 70% , and 55% . The rates of Grade 2b were nearly the same among all groups, varying from 5% to 15%. The resistance to pin tract infection in bone tissue (Grade 0b:1b:2b ratio) of the 25F0.5 group was significantly lower than that of the 37F4.0 group (p = 0.0341). It is worth noting that the p value for the difference in Grade 0b:1b:2b ratios between the 37F4.0 and UN1 groups was markedly lower (p = 0.0801) than that between the 25F1.0 and UN1 groups (p = 0.5846), and between the 25F0.5 and UN1 groups (p = 0.4841), which suggests that there was no improvement in resistance to pin tract infection in bone tissue for the 25 °C groups compared with the UN1 group. In the second set of animal experiments, the 25F2.0 group showed no improvement in resistance to pin tract infection in bone tissue (Grade 0b:1b:2b ratio) compared with the UN2 group (p = 0.6225) , 1b , and 2b (severe osteomyelitis) inflammation in bone tissue along the pin tract are shown in  = 6/20) a. The in 0.6225) b.In the UN, 25F0.5, 25F1.0, and 25F2.0 groups, no or very little bone formed in the medullary cavity region in the Grades 0b and 1b osteomyelitis samples. However, in the 37F4.0 group, a considerable amount of dense bone formed in the cortical and medullary cavity regions in the Grades 0b and 1b osteomyelitis samples .S. aureus detected around the pin tract than the FGF-2-apatite composite layers fabricated at 37 °C for 2 days. The most frequently detected bacterium around the pin tracts was S. aureus. The detection frequency of S. aureus was significantly lower in the 37F4.0 group (35%) than in the 25F0.5 group . Although no significant difference was detected, the detection frequency of S. aureus in the 37F4.0 group (35%) was also lower than in the UN1  and 25F1.0  groups. E. coli were also detected in all groups. Their detection frequencies ranged from 5% to 15%, and no significant differences were detected among the UN1, 25F0.5, 25F1.0, and 37F4.0 groups (p = 0.1489–0.5). The frequency of no bacterial detection tended to be higher for the 37F4.0 group (35%) than the UN1 group  and 25F0.5 group , though no significant differences were detected (p = 0.1468–1.0) (The FGF-2-apatite composite layers on Ti pins fabricated at 25 °C for 1 day were associated with higher frequencies of detected a. In the468–1.0) b.n = 20), which includes all the osteomyelitis cases, was significantly higher than that of the 25F0.5 group  and the 25F1.0 group . The average value of extraction torque from all samples in the 37F4.0 group was higher than that of the UN1 group , although no significant difference was detected (p = 0.2793) (The FGF-2-apatite composite layers on Ti pins fabricated at 25 °C for 1 day were associated with lower extraction torque than the FGF-2-apatite composite layers fabricated at 37 °C for 2 days. The average extraction torque for the 37F4.0 group of 0.276 ± 0.117 Nm (detected a. The av 0.2793) b.FGF-2-apatite composite layers were successfully fabricated on Ti pins in one step at 25 °C for 1 day using an infusion fluid-based supersaturated CaP solution containing FGF-2. FGF-2 molecule, and CaP crystals and their precursors including CaP clusters can have a positive or a negative surface charge depending on solution chemistry and pH . It is eAlthough a correlation is estimated between the FGF-2 concentration of supersaturated CaP solution, and the amounts of FGF-2, Ca and P deposited in the case of 25 °C groups , the corOn the whole, however, the FGF-2-apatite composite layers fabricated at 25 °C for 1 day  were less effective at preventing pin tract infections and loose anchorages than those fabricated at 37 °C for 2 days (37F4.0 group), though these two types of layers had similar microstructure, FGF-2 content, adhesion strength, and characteristics of biological activity of FGF-2. The FGF-2-apatite composite layers fabricated at 37 °C for 2 days demonstrated a preventive effect on pin tract infections, as reported previously . We inteThe differences in pin tract infection prevention between the FGF-2-apatite composite layers fabricated at 25 and 37 °C may be attributed to differences in the CaP matrix that embeds the FGF-2. In general, CaP matrices of signal molecule-CaP composite layers fabricated by similar processes include low-crystalline apatite ,31,32, an = 3) [n = 8) was obtained by the ICP technique in the present study.Two limitations of the present study are that the animal experiments were performed in two sets and different analytical methods were used to determine the Ca/P molar ratios in the present and previously reported 37F4.0 groups. The two different sets of animal experiments make direct comparisons between the 25F2.0 group and other groups difficult. The previously reported method for determining Ca/P molar ratios was X-ray photoelectron spectrometry (XPS) , which in = 3) , while ain vivo efficacy in preventing pin tract infection, and dissolution and resorption of the layers in order to unambiguously clarify the effect of CaP matrix stability. The fabrication conditions to be optimized include solution compositions, and the FGF-2 content of the supersaturated CaP solution. Theoretically, solution compositions that have a higher Ca/P molar ratio and a lower supersaturation can accelerate the crystallization of ACP into apatite [Further optimization of the fabrication parameters is needed to reduce the bacterial infection rate and improve the fixation strength at the bone-pin interface for the FGF-2-apatite composite layers prepared at 25 °C. We plan to explore for another fabrication condition under which FGF-2-apatite composite layers with a Ca/P molar ratio at around 1.67 and having higher chemical stability is fabricated at 25 °C, and to carry out comparative studies on FGF-2 release, biological activity of FGF-2,  apatite ,36,37. T apatite , which i2+: 8.92 mM) was prepared from Ringer’s solution  and Calcium Chloride Corrective Injection 1 mEq/mL . P solution (PO43−: 2.97 mM) was prepared from Klinisalz®  and Dipotassium Phosphate Corrective Injection 1 mEq/mL . FGF-2 solution (100 µg/mL) was prepared by dissolving FGF-2  in the Ca solution. Meylon®  was used as an alkalizer. From these four solutions, three immersion solutions supplemented with 0.5, 1.0, or 2.0 µg/mL FGF-2 were prepared to immerse Ti pins at 25 °C for 1 day.Supersaturated CaP solutions were prepared by mixing clinically available infusion and injection fluids. Ca solution  was used as an alkalizer, following our previous protocol.Another supersaturated CaP solution containing 4.0 µg/mL FGF-2 was prepared in the same manner as previously reported  to immerThe preparation of the immersion solutions was carried out under sterile conditions. The chemical compositions of these CaP solutions are summarized in in vitro and in vivo experiments.The Ti pins were commercially available, gamma ray-sterilized, titanium 4.0 mm diameter, 30 mm length ,21 cance® tape detachment test as previously reported [® tape was affixed and then detached from the coated pin. The resulting surface of the Ti pin was observed using SEM.The surfaces of the Ti pins were observed by SEM  and EDX . The Ti pins were coated with a thin carbon film before observation. To identify the crystalline phase of the surface layer, the layers were scraped off the Ti pin and mounted on a silicon-zero-background plate for analysis using XRD . The strength of adhesion between the composite layer and the Ti pin was qualitatively analyzed by a Scotchreported . A smallTo extract the FGF-2 from the composite layer, the Ti pins coated with the composite layer were immersed in 2 mL of 10 mM citric acid-sodium citrate buffer  for 3 h to completely dissolve the composite layer. Eight FGF-2 extract solutions were used to determine the amounts of immobilized FGF-2, deposited Ca and P on the surface of the Ti pins, and the mitogenic activity of FGF-2. The amount of immobilized FGF-2 was determined by the Bradford method using a protein quantitation kit . The amounts of deposited Ca and P were determined using an ICP . l-glutamine (0.3 mg/mL), bovine serum albumin (1.0 mg/mL), insulin (5.0 µg/mL), and transferrin (1.0 µg/mL) in a well of a 24-well plate in which 1 × 104 fibroblastic NIH3T3 cells  had been precultured for 1 h. After another 72 h of culture, the number of NIH3T3 cells was counted using a cell counting kit .The mitogenic activity of the FGF-2 in the composite layers was evaluated using the proliferation of NIH3T3 cells as described in a previous study . BrieflyThe surgical technique used was the same as that described in our previous studies ,21,24. TAll animal experiments and breeding were performed according to the conditions approved by the ethics committees of both the University of Tsukuba and the National Institute of Advanced Industrial Science and Technology (AIST). All activities were implemented in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals .Four weeks after implantation, the presence of pin tract infections was evaluated using a modified Checketts classification before sacrifice . Grade 0After collection of the exudate from the pin tracts, the proximal tibial metaphyses were fixed in 10% neutral buffered formalin for 7 days and then separated into the soft- and hard-tissue parts. The soft-tissue parts were undecalcified and embedded in paraffin, and the hard-tissue parts were decalcified in an ethylenediaminetetraacetic acid solution and embedded in paraffin. Sample sections were cut 5 μm thick, perpendicular to the tibial longitudinal axis, and parallel to the hole of the pin. Sections were stained with hematoxylin-eosin. The specimens were observed histologically using a light microscope  to evaluate the grade of pin tract inflammation in the soft and bone tissues.For the soft tissue, Grade 0s corresponds to “no inflammation”, in which no inflammation was observed in the surrounding soft tissue along the entire length of the boundary between the pin and soft tissue. Grade 2s corresponds to “severe inflammation,” in which inflammation was observed in the surrounding soft tissue along the entire length of the boundary between the pin and soft tissue ,24. GradSimilarly for the bone tissue, Grade 0b corresponds to “no osteomyelitis,” in which no inflammation was observed in the surrounding bone tissue along the entire length of the boundary between the pin and bone tissue. Grade 2b corresponds to “severe osteomyelitis,” in which inflammation was observed in the surrounding bone tissue along the entire length of the boundary between the pin and bone tissue . Grade 1A single physician who was blinded to the pin identifications evaluated the soft and bone tissues for pin track inflammation.S. aureus and E. coli [After complete removal of the Ti pins, the exudate around each pin was collected with a cotton swab. Those swabs were delivered to a company for clinical laboratory testing  of the major bacterial species present:  E. coli .After sacrificing the rabbits, the peak extraction torque for each Ti pin was measured using a mechanical torque-measuring system  ,24.post-hoc tests. The mitogenic activities of each group were compared using Student’s t-tests. The results of the pin tract infections by visual inspection and histological analyses in each group were analyzed by χ2 test for independence. The bacterial detection data from each group were analyzed by χ2 test for independence. The extraction torque data from each group were compared using Student’s t-test. The significance level was set at p < 0.05 for each analysis.The amount of Ca and P, Ca/P molar ratio, and FGF-2 content for the four groups  were compared using a one-way analysis of variance with Tukey-Kramer in vitro assays confirmed that the FGF-2 immobilized in the composite layers retained its mitogenic activity. The FGF-2-apatite composite layer formed at 37 °C reduced the pin tract infection rate and increased the fixation strength at the bone-pin interface. Moreover, this composite layer reduced the invasion rate of S. aureus into the pin tract. On the other hand, the FGF-2-apatite composite layers formed at 25 °C were less effective at both improving the fixation strength at the bone-pin interface and at resisting pin tract infections. This reduced effectiveness may be attributed to the lower Ca/P molar ratio and resulting chemical instability of the composite layer, or a change in optimum FGF-2 content due to the change in Ca/P molar ratio. Further optimization of the fabrication parameters is needed to reduce the bacterial infection rate and improve the fixation strength at the bone-pin interface for the FGF-2-apatite composite layers prepared at 25 °C. The results of this study suggest that the chemistry of the calcium phosphate matrix that embeds FGF-2, in addition to FGF-2 content and activity, has a significant impact on composite infection resistance and fixation strength.FGF-2-apatite composite layers were successfully fabricated on Ti pins at 25 °C for 1 day in one step, as well as at 37 °C for 2 days. The results of