Parabacteroides goldsteinii was isolated from a blood culture. Genomic DNA was sequenced using a MiSeq sequencer and assembled using the SPAdes genome assembler. The draft genome sequence was 6,851,868 bp, spanning 282 contigs of 5,253 coding sequences, 66 tRNAs, and 5 rRNAs. Several putative novel metallo-β-lactamases were discovered. Parabacteroides goldsteinii is an anaerobic, Gram-negative rod belonging to the microbiota of the human gut , piperacillin-tazobactam (>256 mg/liter), and meropenem (>32 mg/liter) but susceptibility toward metronidazole (0.38 mg/liter). A meropenem-EDTA double-ended gradient strip was positive, indicating the presence of a metallo-β-lactamase , according to protocol.The genomic DNA of the de novo genome assembly was done via SPAdes version 3.0  are 6.49 Mb to 7.09 Mb with a GC content of 43.30% to 43.50%.The final assembly consisted of 282 contigs with an ermF) and tetracycline (tetX and tetQ) resistance, but no genes that account for resistance toward piperacillin-tazobactam or meropenem.Annotation was carried out via the NCBI Prokaryotic Genome Annotation Pipeline, identifying 5 rRNAs, 66 tRNAs, and 5,253 coding sequences (CDS), of which 2,268 of the latter code for purely hypothetical proteins with no obvious homology-inferred function. A search for resistance-associated genes via the ResFinder tool   . Tertiary structure predictions via Phyre2 predict a general β-lactamase-like αββα fold for all nine CDS , 8. ThesLFJV00000000. The version described in this paper is version LFJV01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number


Dasyatis akajei, a cartilaginous fish”  D. akajei, MC1R, MC2R, MC3R, MC4R, and MC5R were aligned with the corresponding melanocortin receptor sequences from the elephant shark, Callorhinchus milii, the dogfish, Squalus acanthias, the goldfish, Carassius auratus, and the mouse, Mus musculus. These alignments provide the basis for phylogenetic analysis of these gnathostome melanocortin receptor sequences. In addition, the Japanese stingray melanocortin receptors were separately expressed in Chinese Hamster Ovary cells, and stimulated with stingray ACTH, α-MSH, β-MSH, γ-MSH, δ-MSH, and β-endorphin. The dose response curves reveal the order of ligand selectivity for each stingray MCR.This article contains structure and pharmacological characteristics of melanocortin receptors (MCRs) related to research published in “Characterization of melanocortin receptors from stingray  Specifications TableValue of the data•These data are valuable for researchers participated in endocrinology of primitive fish and evolution of melanocortin systems.•These could be used as probes to explore orthologs in other cartilaginous fish such as skates, sharks and chimaeras.•The data on ligand selectivity could be useful tools for structure–function relationship studies in endocrinology and pharmacology.1Squalus acanthias) which determined by us Callorhinchus milii, elephant shark and S. acanthias, dogfish), a teleost , and a mammal . Data are also provided for ligand selectivity include effects of stingray Des-acetyl-α-MSH, β-MSH, γ-MSH, δ-MSH, ACTH(1-24) and β-endorphin on MC1R, MC3R, MC4R, and MC5R (Data provided in this article show amino acid sequence comparison of melanocortin receptors (MCRs) in vertebrates and ligand selectivity of stingray MC peptides on these receptors. The amino acids sequences of MC1R , MC2R F, MC3R F, MC4R F, and MC5and MC5R , Fig. 10and MC5R .2D. akajei, the dogfish, S. acanthias, the elephant shark, C. milii, the goldfish, C. auratus, and the mouse, M. musculus, it was essential to identify putative transmembrane domains in each receptor sequence. To this end, the program ‘‘MEMSAT3’’ (http://bioinf.cs.ucl.ac.uk/psipred/) was used. The amino acid sequences where then aligned using the program MEGA 6.0.In order to align the amino acid sequences of the melanocortin receptors for the Japanese stingray, srmcrs were separately synthesized with a V-5 epitope tag at the N-terminal of the receptor, and inserted into a pcDNA3.1 expression vector . Each srmcr cDNA was separately transiently transfected into Chinese Hamster Ovary (CHO) cells. The CHO cells were grown at 37 °C in a humidified 5% CO2 incubator in DMEM/F12 with 5% fetal calf serum. Each sr cDNA was co-expressed with a CRE/Luciferase reporter plasmid −5 cells/well. After 48 h in culture, the transfected cells were stimulated with either synthetic srACTH(1-24), srDes-acetyl-α-MSH, srβ-MSH, srγ-MSH, srδ-MSH, srβ-endorphin or hACTH(1-24), or NDP-MSH at concentrations ranging from 10−6 M to 10−12 M, in serum-free CHO media for four hours at 37 °C. At the end of the incubation period, 100 µl of Bright-Glo luciferase assay reagent  was added to each well, and incubated for 5 min at room temperature. Luminescence was measured with a Bio-Tek Synergy HT plate reader , and the dose response curves were analyzed by using Kaleidagraph software . All experimental treatments were performed in triplicate.To functionally express and determine the ligand selectivity of the stingray (sr) MC1R, srMC2R, srMC3R, srMC4R, and srMC5R paralogs, the nucleotide sequences for the


There is a section missing from the Materials and Methods. The section should appear between the “Manufacturing of the delivery system” and the “Analytical data” subsections. The publisher apologizes for this error. The missing section is:23.5 g (81.2 m mol) Irgasan and 7.78 g (77 m mol) triethylamin  were dissolved in 350 ml tetrahydrofuran , extra dried over molecular sieve, water content < 50 ppm in a 1-l three-necked flask with reflux condenser. The solution was cooled to 0°C and 7.71 g (73.8 m mol) methacryloyl chloride  was dropped thereto within 90 min while stirring. After stirring overnight at room temperature, the reaction mixture was filtered to remove precipitated triethylamin hydrochloride. Phenothiazine (Sigma Aldrich) was added and tetrahydrofuran was distilled off. The crude product was dissolved in 100 ml n-hexane (Sigma Aldrich), washed with 5% sodium bicarbonate solution (3 x 40 ml), then with aqua dest. (4 x 50 ml). The organic phase was filtered over basic aluminium oxide and distilled. Yield: 22.36 g .1H-NMR and 13C-NMR  were performed; 500.13 MHz for proton and 125.77 MHz for carbon in CDCl3. The δ-scale relative to tetramethylsilan was calibrated to the solvent value δ = 7.24 ppm for CDCl3. IR spectra were recorded  with an attenuated total reflection (ATR) unit. Structure analysis was done with gas chromatography-mass spectrometry  and electron ionization (EI).


Background: Magnetic Resonance Imaging (MRI) provides unique diagnostic information yet is contraindicated with conventional pacemakers (PMs) and implantable cardioverter defibrillators (ICDs). MRI with careful monitoring is feasible with PMs/ICDs, but safety continues to be questioned, including heating of device components.Methods: We tested the effect of MRI on local tissue heating over PM/ICD pulse generators (PGs) in a consecutive series of 34 non-thoracic scans in 30 patients (pts) enrolled in an ongoing registry study. MRI was performed with a General Electric 1.5 tesla model Signa HDXT scanner. Temperatures were taken immediately pre- and post-study with an infrared thermometer beamed at the center of the PG pocket and symmetrically on the opposite side of the anterior chest. Ambient temperature was set at 66°F. Pacing modes during MRI were 0D0 for intrinsic rates > 40 and D00 for rates < 40. Diffusion scan sequences were excluded in PM dependent pts. Devices were St. Jude (n = 11), Boston Scientific (n = 12) or Medtronic (n = 11). The primary endpoint was change in device-side versus control-side skin temperature.Results: Pt age was 61 ± 17 y; 23 were males. 28 PMs and 6 ICDs were scanned. 17 pts were PM dependent. Scan location was 16 head/neck/shoulder, 13 lumbar spine, 1 liver, and 2 lower extremity. Scan duration was 41.4 ± 14.95 min. At pre-scan baseline, device and control temperatures were 92.1 ± 1.8 and 91.0 ± 3.2°F, respectively (p = 0.09), and post-scan, 91.7 ± 3.2 and 90.1 ± 3.2° (p = 0.04). Temperature changes (post- vs. pre-scan) were -0.35 ± 2.8°  and -0.87 ± 3.9° (-11.8 to +7.4°), p = 0.56, and were similar for PMs and ICDs. No sensation of warmth was reported. No significant changes in lead or PG parameters were noted.PMs/ICDs cause mild local tissue/skin warming, but MRI did not cause additional, incremental heating. This suggests that heating of PG-can/local tissue with standard non-thoracic MRI scanning with selected contemporary devices appears to be generally mild or negligible. Results of a currently enrolling large registry study (MagnaSafe) will be required to firmly establish patient and device safety. Additional trials will be needed to assess PG heating and safety during thoracic MRI scans.None.


Scoliosis is a 3D deformity of the spine combined with pathological structural changes.Scoliosis tend to progress through growth.BSPTS method based on Schroth principles is a specific physiotherapeutic treatment method for scoliosis. It considers the 3d changes of the spine, the geometrical and axial torsion of the hole spine, thorax and pelvic. Using specific exercise to achieve better muscle balance and corrected posture in order to prevent progression, improve esthetic and function.To present the efficacy of the BSPTS method based on Schroth principles treating progressive moderate AIS in 2 case studies using objective measures.Starting point- age 17.5 years, Risser=2+, Rt thoracolumbar L3-T7 52° cobb, Trace 10/11, scoliometer 16° rt hump, plumb line 2.5 cm rt from center of sacrum.End point- age 19, Risser = 4, Rt thoracolumbar L3-T7 35° cobb, Trace 6/11, scoliometer 12° rt hump, plumb line 1 cm rt from center of sacrum.Starting point- age 14.5 years, Risser=1, Rt Tx 53° cobb, Trace 9/11, scoliometer 17° rt hump, plumb line 2 cm rt from center of sacrum.End point- age 15.9, Risser = 3 Rt Tx 30° cobb, Trace 5/11, scoliometer 9° rt hump, plumb line 1 cm rt from center of sacrum.In spite of the late diagnosed moderate AIS the combined treatment of specific intensive physiotherapy and RSC brace achieved improved Cobb angel, better esthetic and the operation was avoided.Early detection could improve the results in all parameters, school screening or other systematic method for early detection is highly recommendedSpecialized team work of scoliosis rehabilitation - Orthopedic physician, Orthotist, Physiotherapist, and compliant patient can achieve better result even in moderate-severe cases.


Enhanced B cell activity, particularly memory B cells have gained interest in evaluating response during therapies with biologics. CD27-IgD- double-negative (DN) B cells lacking the conventional memory marker CD27 are reported to be part of the memory compartment, however, only scarce data is available for rheumatoid arthritis (RA). We therefore focused on DN B cells in RA, studied their isotypes and modulation during interleukin-6 receptor (IL-6R) inhibition by tocilizumab (TCZ).H gene rearrangements and specific isotypes.DN B cells were phenotypically analyzed from 40 RA patients during TCZ at baseline week 12, week 24 and 1 year. A single B cell polymerase chain reaction (PCR) approach was used to study Ig receptors, VP <0.0001) was observed by in vivo IL-6R inhibition. These changes were observed for all BCR isotypes IgG, IgA and IgM at week 12, 24 and 1 year (P <0.0001). IgA-RF, IgA serum level and IgA+ DN B cells decreased significantly (P <0.05) at week 12 and week 24 during TCZ. Patients with a good European League Against Rheumatism (EULAR) response to TCZ had less DN B cells at baseline as compared to moderate responders (P = 0.006). Univariate logistic regression analysis revealed that the frequency of DN B cells at baseline is inversely correlated to a subsequent good EULAR response (P = 0.024) with an odds ratio of 1.48 .Phenotypic analysis showed a significantly expanded population of DN B cells in RA which contain a heterogeneous mixture of IgG-, IgA- and IgM-expressing cells with a clear dominance of IgG+ cells. DN B cells carry rearranged heavy chain gene sequences with a diversified mutational pattern consistent with memory B cells. In contrast to tumor necrosis factor alpha (TNF-α) inhibition, a significant reduction in mutational frequency of BCR gene rearrangements at week 12, 24 and 1 year  contains supplementary material, which is available to authorized users. In addition to its involvement in immune responses, it also regulates hematopoiesis, the acute phase response and inflammation. Dysregulation of IL-6 production and its pathological role in different autoimmune diseases have been well documented and highlight IL-6 and its signaling cascade as a potential target for autoimmune therapy [Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory, autoimmune disease characterized by inflammation of the joints, which results in their progressive destruction ,2. Recen therapy -13. Cons therapy . TCZ has therapy .Functionally distinct B cell subsets can be defined by the phenotype expression of CD27 and immunoglobulin D (IgD). Human peripheral memory B cells are mainly discriminated from naïve B cells by the phenotypic expression of CD27 (a member of the tumor necrosis factor receptor (TNFR) family) and presence of somatic hypermutation (SHM) in their Ig variable genes ,16. CD27in vivo IL-6R inhibition indicated phenotypic and molecular changes in pre-and post-switch memory B cells [in vivo IL-6R and TNF-α inhibition.Our previous studies of memory B cell subsets during  B cells ,14,25. IPeripheral blood was taken from 44 rheumatoid arthritis patients (RA) with a median age of 54 (range 33 to 79) years and 49 healthy age-matched donors (HD) for B cell phenotype and molecular analysis. All patients met the American College of Rheumatology revised criteria for RA . The patWhole blood staining was used for phenotype studies and peripheral blood mononuclear cells (PBMCs) were used for single-cell PCR approach. Whole blood and PBMCs were stained with the following monoclonal antibodies: anti-CD45-krome orange , cat no. 96416), anti-CD14-PC5.5 , anti-CD19-APC-Alexa Fluor 750 , anti-CD19-APC , cat no. 555415), anti-CD27-PE , anti-CD27-PC7 , anti-CD27-ECD , anti-IgD-FITC , anti-IgA-FITC , anti-IgG-PECy7  and anti-IgM-APC  cat no. 314510). After staining, cells were analyzed by 10-color flow cytometer . B cells were identified by CD19+ cells gated on CD45 + CD14- lymphocytes in combination with forward scatter versus side scatter gating on CD45+ lymphocytes. B cell subpopulations were identified by using CD27 and IgD expression gated on B cells. Expression of IgG, IgA and IgM was analyzed for CD27 + IgD- post-switch and CD27-IgD- DN B cells, respectively. At least 20,000 CD19+ events were collected for each analysis. The total number of B cells of various phenotypes was calculated per microliter of blood, based on the frequency of these cells among the lymphocytes and the absolute number of white blood cells.15 primer, dithiothreitol, RNasin, and RNAse-free double-distilled H2O or RNAse-free H2O.PBMCs were isolated by Ficoll-Paque Plus separation  using the standard protocol. Single B cell sorting from PBMCs was carried out as described previously . BrieflyH3 gene rearrangements comprising the largest VH family were amplified by nested PCRs using family-specific primers as previously described [H1μ-, CH1α- and CH1γ-specific PCRs were carried out using cycle program as described previously [Taq polymerase used in the amplification process was estimated to be 1 × 10-4 mutations/bp [H family-specific PCR products (350 bp) were separated via electrophoresis using 1.5% agarose gel and further purified by using MinElute Gel Extraction kit  according to the manufacturer’s instructions.A mixture comprising RT-PCR buffer, reverse transcriptase and nucleotides from Titan One Tube RT-PCR System  was added into a 96-well plate. cDNA synthesis was carried out at 50°C for 1 hour. Vescribed . Brieflyeviously . The errtions/bp . VH famiH3 and isotype-specific Ig were amplified using BigDye Terminator Cycle Sequencing Ready Reaction kit followed by sequencing in genetic analyzer ABI PRISM 310 . A total of 2,407 sequences were analyzed by matching their closest germline counterparts using the online program JOINSOLVER [Gel extracted PCR products of Ig-VINSOLVER .U test. Univariate logistic regression was used to calculate odd ratios and correlation using Pearson r. The values ≤0.05 were considered to be significant. ***P <0.0001, **P <0.001 and *P <0.01.Statistical analysis was performed using GraphPad Prism 5.0  and SPSS Statistics 22.0 . Values were always compared with baseline levels by using the nonparametric Wilcoxon matched pair test and Mann-Whitney Based on surface expression of IgD and CD27 Figure A, human P <0.0001). The inflammatory parameters erythrocyte sedimentation rate (ESR) and CRP were also significantly reduced (Table P = 0.97) and CRP levels . Univariate logistic regression analysis revealed that the frequency of DN B cells at BL is inversely correlated with a subsequent EULAR good response with a significant (P = 0.024) odds ratio of 1.48  as 1.05 to 2.06) favoring EULAR good response. This indicates that the percentage of DN B cells is elevated in RA and correlated with EULAR response to TCZ.During TCZ therapy, DAS28 of all patients declined significantly from 5.2 at baseline (BL) to 3.3, 2.7 and 1.9 at week 12, 24 and 1 year (P = 0.004) and 20.5% (4.6 to 33.8) at week 24 (P = 0.04), respectively. We did not find any remarkable changes in IgG+ or IgM+ B cells  Additional file In order to reveal the isotypic distribution of DN B cells, we measured the surface expression of IgG, IgA and IgM isotypes and followed them in RA patients (n = 36) during TCZ treatment till week 24. DN B cells showed a heterogeneous mixture of cells containing IgG+, IgA+ and IgM+ expressing B cells with a dominance of IgG isotype. In detail, DN B cells contained a median (range) of 60.6% (40.9 to 86.7) IgG+ isotype of total DN B cells, 24.7% (10.0 to 64.2) of IgA+, and 8.0% (2.3 to 25.8) of IgM+ B cells. For comparison, post-switch memory B cells displayed an almost equal distribution of IgG+ and IgA+ cells; 45.3% (26.2 to 67.3) IgG+ and 42.7% (24.5 to 57.4) IgA+  and from RA patients during TCZ (n = 9) or anti-TNF-α therapy (n = 4). As shown previously,  Ig-VH3 gP <0.0001), 1.98 ± 0.3% at week 24 (P <0.0001) and 1.89 ± 0.3% (P <0.0001) at 1 year respectively during TCZ , 23.1 ± 6.8% at week 24 (P = 0.035) and 17.3 ± 3.9% at 1 year (P = 0.0003) during TCZ therapy. In parallel, a significant increase of unmutated (0 mutation per sequence) sequences from 15.1 ± 3.4% (BL) to 22.7 ± 3.7% at week 12 (P = 0.05), 32.1 ± 4.6% at week 24 (P = 0.005) and 37.1 ± 4.6% at 1 year (P = 0.004) was found (data not shown). In order to evaluate TCZ-specific influences on Ig gene rearrangements, we also studied patients (n = 4) who underwent TNF-α inhibition using adalimumab therapy  compared to IgG+ DN B cells at BL. As expected IgM+ DN B cells showed the lowest mutational frequency. However during TCZ therapy, all three isotypes  of DN B cells showed a significantly reduced mutational frequency. Mutational frequency of IgA+ cells were found to be reduced (mean ± standard error of the mean (SEM)) from 5.42 ± 0.30% (BL) to 3.35 ± 0.22% at week 12 (P <0.0001), 2.41 ± 0.34% at week 24 (P <0.0001) and 2.26 ± 0.33% (P <0.0001) at 1 year, respectively. The mutational frequency of IgG+ cells reduced from 4.45 ± 0.20% (BL) to 2.51 ± 0.22% at week 12 (P <0.0001), to 2.16 ± 0.23% at week 24 (P <0.0001) and 2.00 ± 0.20% (P <0.0001) at 1 year, respectively. Similarly, the mutational frequency of IgM+ cells also decreased from 2.87 ± 0.32% (BL) to 1.55 ± 0.23% at week 12 (P = 0.0016), 0.98 ± 0.21% at week 24 (P = 0.0008) and 0.67 ± 0.17% (P <0.0001) during 1-year TCZ treatment , 48.0 (18.0 to 85.0) bp at week 24 (P <0.0001) and 50 (21.0 to 91.0) bp (P <0.0001) at 1 year during TCZ therapy , 20.7 (14.9 to 23.4) at week 24 (P = 0.004) and 19.2 (12.6 to 24.5) at 1 year (P = 0.004) during TCZ therapy  is considered a signature of antigen contact and T cell help, and it represents imprints of selection. CDR3 lengths of V18.0 to 8.0 bp at 18.0 to 8.0 bp at 18.0 to 8.0 bp at in vivo IL-6R inhibition by TCZ does influence the peripheral pre-switch and post-switch memory B cells in RA [With increased understanding of mechanistic pathways of B cell involvement in autoimmune diseases, a number of studies have provided evidence that B cells play a central role in the pathogenesis of RA and that certain subsets may be exploitable as biomarkers. Memory B cells, in particular, seem to be important in driving chronic inflammation ,34. Targls in RA ,25. Becals in RA ,20, we wls in RA ,36, we fls in RA ,23,37-39We describe a heterogeneous DN B cell population containing IgG+, IgA+ and IgM+ DN B cells with pre-dominance of the IgG clearly different from post-switch memory B cells Figure A. DuringSHM of immunoglobulin gene rearrangements is a hallmark of B cell maturation into memory cells after antigen encounter. Despite their mainly switched phenotype, we also found the mutational frequency of DN B cells in RA to be significantly lower compared to post-switch B cells and similar to pre-switch B cells. This has been previously reported for other diseases and older people ,20,24,25in vivo to a substantial extent. However, at the moment it is not clear how direct and indirect effects on B cells contribute to the observed effects since TCZ is also reported to reduce activated CD4+ T cells as early as 12 weeks after treatment [It is known that SHM favors defined mutational hotspots. In particular, RGYW/WRCY motif mutations are preferentially targeted by T cell-dependent signals including CD40-CD40 ligand interactions . The decreatment . Data streatment . A recenreatment . Furtherreatment . Since mreatment . Since sreatment -39. FurtThe search for predictive biomarkers to individualize treatment strategies in RA is of current interest. For IL-6R inhibition with TCZ, predictive markers for response have not yet been identified. In our study, we were able to relate the level of peripheral DN B cells to the clinical response to TCZ Figure B-C. PatiIn summary, we demonstrate a significantly higher population of CD27-IgD- DN B cells in RA patients. These DN B cells are a mixture of somatically mutated IgG, IgA and IgM isotype-bearing cells with a dominance of IgG isotype. TCZ therapy results in a decrease in the frequency of IgA+ DN B cells in particular. In parallel, a decrease of serum IgA and RF-IgA levels is observed. On the molecular level, TCZ therapy reduces the mutational frequency and RGYW hotspot targeting in all DN B cells over a period of 1 year. Based on these current findings, DN memory B cells may serve as a candidate biomarker for response to TCZ therapy as lower baseline values of these cells were related to higher proportions of EULAR good responders.


N=7, 14 and 21 by on-surface chemical reactions with an employed organoboron precursor. The location of the boron dopant is well defined in the centre of the B-GNR, corresponding to 4.8 atom%, as programmed. The chemical reactivity of B-GNRs is probed by the adsorption of nitric oxide (NO), which is most effectively trapped by the boron sites, demonstrating the Lewis acid character. Structural properties and the chemical nature of the NO-reacted B-GNR are determined by a combination of scanning tunnelling microscopy, high-resolution atomic force microscopy with a CO tip, and density functional and classical computations.Boron is a unique element in terms of electron deficiency and Lewis acidity. Incorporation of boron atoms into an aromatic carbon framework offers a wide variety of functionality. However, the intrinsic instability of organoboron compounds against moisture and oxygen has delayed the development. Here, we present boron-doped graphene nanoribbons (B-GNRs) of widths of  Incorporation of boron atoms into an aromatic carbon framework offers a wide variety of functionality. Here, the authors present boron-doped graphene nanoribbons by on-surface chemical reaction and characterize the structures and properties using scanning probe microscopy at the atomic-scale. However3 Lewis acidic sites at the centre row in the armchair-edge GNR (B-GNR) with a width of seven carbon atoms (N=7). The interval between the boron sites is fixed with a doping density of 4.8 atom%.In experiments, the precursor molecules were deposited on a clean Au(111) surface in ultra-high vacuum and were subsequently annealed at 180 °C . At 4.8 The observed STM contrast is inverted by the polarity of the applied tip bias voltage (−3 and 3 V), indicating the perturbation of the electronic structure by the doped boron atoms as an electron-accepting unit . Pink crN=7 B-GNR, dI/dV spectra were taken with a clean Au tip above four different sites of B-GNR and, for reference, above one Au(111) site , leaving intact the overall electronic structure  site . The conN=7, 14 and 21) were obtained by fusing the armchair edges . To enhance the observed bond visually, a Laplace filterAfter the substrate was further annealed at 510 °C, B-GNRs with different widths  gas. 3 moieties exhibit a Lewis acidic role for the stable gas adsorption. The simulated STM image . 9,10-bis(10-bromoanthracen-9-yl)-9,10-dihydro-9,10-diboraanthracene was deposited on the substrate from a crucible of Knudsen cell, resistively heated at 240 °C. Subsequently, the sample was annealed at 180 and 400 °C to synthesize the N=7 B-GNR. The fused B-GNRs with N=14 and 21 were obtained by further annealing at 510 °C. Further, NO gas was introduced into the ultrahigh vacuum chamber (100 Langmuir) while the sample stayed in the cold microscope. In STM mode, the tip was biased while the sample was electronically grounded. The topographic images were taken in a constant current mode. In AFM mode, the tip apex was terminated by a CO-molecule and all images were taken at a constant height mode.All experiments were performed with an Omicron STM/AFM with a qPlus configuration1, 9,10-bis(10-bromoanthracen-9-yl)-9,10-dihydro-9,10-diboraanthracene, was obtained from compounds 2 and 3 through the synthetic route shown in 1 with extra bulky substituents have been reported1 has not been characterized because of its low solubility in common organic solvents. We successfully characterized the isolated precursor 1 by the NMR analyses in tetrachloroethane at 100 °C and by X-ray crystal structure analysis using chlorobenzene as solvent. The crystalline sample of the precursor 1 can be stored at ambient conditions as a result of the steric protection of the boron atoms by the anthracene groups. Compounds 2  were purchased from Wako Pure Chemical Industries. Dry toluene was purchased from Kanto Chemical.The precursor s 2 ref.  and 3  in ether (13 ml) cooled at 0 °C was added n-BuLi  dropwise. The mixture was stirred at room temperature for 20 min. After removing the solvent in vacuo, the residue was dissolved together with 3  in toluene (8 ml). The resulting mixture was stirred at room temperature for 16 h, and the solvent was removed in vacuo. The crude product was washed with MeOH and hexane and further purified by silica gel column chromatography using CHCl3/hexane (ratio 2:1) as an eluent. Recrystallization from chlorobenzene afforded 1 as an orange solid . Thin layer chromatography : Rf=0.90; 1H-NMR : δ 8.72 ppm , 7.77 , 7.68 , 7.46 , 7.43 , 7.34 . 13C NMR : δ 139.95, 134.19, 133.95, 130.04, 130.00, 128.10, 126.83, 124.74 and 122.69. ; 11B-NMR : δ 69.3; HRMS (m/z):[M]+ calcd for C40H24B2Br2: 686.0425; found, 686.0433.To a solution of 1 were obtained by slow diffusion of octane into a solution of 1 in chlorobenzene (Rint=0.0410). The structure was solved by direct methods (SHELXS-2013) and refined by full-matrix least-squares on F2 for all reflections (SHELXL-2013). All non-hydrogen atoms were refined anisotropically and all hydrogen atoms were placed using AFIX instructions. The crystal data are as follows: Formula C40H24B2Br2; FW=686.03, monoclinic, crystal size 0.14 × 0.02 × 0.02 mm3, C2/c, a=19.259(7) Å, b=17.907(6) Å, c=18.056(6) Å, β=102.721(4) Å, V=6,074(4) Å3, Z=8, Dcalcd=1.500 g cm−3; R1=0.0396 (I>2σ(I)), wR2=0.1006 , GOF=0.987.Single crystals of obenzene . The int−1 under a nitrogen atmosphere . 11B-NMR spectrum was recorded with a JEOL AL-400 spectrometer (128 MHz for 11B). Chemical shifts are reported in δ ppm with reference to residual protons and carbons of 1,1,2,2-tetrachloroethane-d2 (δ 6.00 ppm in 1H-NMR and δ 73.78 ppm in 13C NMR). The external standard of BF3·OEt2 was used for 11B NMR. The mass spectrum was measured with a Bruker Daltonics micrOTOF Focus using a positive-mode Atmospheric Pressure Chemical Ionization (APCI)-time of flight method in a CHCl3 solution. Thin layer chromatography was performed on plates coated with 0.25-mm thickness of silica gel 60 F254 (Merck). Column chromatography was performed using silica gel PSQ 100B .NMR spectra of the precursor k-point convergence was checked for all systems and a 5 × 1 × 1 mesh was found to be sufficient for the large GNR-Au systems. All reference calculations for adsorption energies, charge densities and projected density of states were performed with 4-anthracene GNR on gold, which had a lattice mismatch of only 1.4% (accommodated in the ribbon). When adding boron (B-GNR), this mismatch reduced to 0.3% as the ribbon expands slightly  code394142slightly . To haveet al.et al., and the simulated AFM scan is performed at a resolution of 0.025 Å , with a force tolerance criterion of 4 × 10−6 eV Å−1. The 3D force field is subsequently converted into a frequency shift imagef=24.76 kHz, A=53 pm and k=1,800 N m−1). In Recently, several mechanical AFM models have been proposed46How to cite this article: Kawai, S. et al. Atomically controlled substitutional boron-doping of graphene nanoribbons. Nat. Commun. 6:8098 doi: 10.1038/ncomms9098 (2015).Supplementary Figures 1-11 and Supplementary References


The 1,10-phenanthrolinium cation and 3-hydroxy-2,4,6-trinitrophenolate anion are held together through an N—H⋯O hydrogen bond. In the crystal, cation–anion pairs are connected by C—H⋯O hydrogen bonds, forming a chain structure along [101]. Spectroscopic data also support the formation of a molecular salt. Sensitivity tests and thermal testing indicate that it is an insensitive high energy density material (IHEDM). 12H9N2+·C6H2N3O8−, the cation and anion are connected by an N—H⋯O hydrogen bond. In the anion, an intramolecular O—H⋯O hydrogen bond with an S(6) ring motif is observed. The planes of two of the nitro groups are approximately parallel to the plane of the benzene ring, making dihedral angles of 3.9 (2) and 15.3 (2)°, while the third nitro group is almost perpendicular to the benzene ring, with a dihedral angle of 78.6 (3)°. In the crystal, cation–anion pairs related by an n-glide plane are connected by C—H⋯O hydrogen bonds, forming a chain structure along [101]. Sensitivity tests and thermal testing indicate that the title salt is an insensitive high-energy-density material (IHEDM).In the title molecular salt, C Of the − (acceptor) group of the phenolate anion and the N—H (donor) of the cation form an N—H⋯O hydrogen bond , 9.28–8.19 p.p.m. , 7.0–5.5 p.p.m. ; 13C NMR: δ 156.0, 148.1, 142.2, 138.0, 135.3, 129.9, 127.9, 126.1 and 126.0 p.p.m.].Equimolar solutions of each of styphnic acid  and 1,10-phenanthroline monohydrate  in ethanol were mixed and shaken well for 3 h. On standing at 298 K for two h, the mixture yielded a yellow solid which was ground, washed well with dry ether and recrystallized from a ethanol–water mixture. Shining yellow single crystals were obtained from the mother liquor by slow evaporation . Although the monoprotonated salt is obtained in good yield, several attempts to prepare the diprotonated salt from styphnic acid and 1,10-phenanthroline by mixing them in different concentrations in solvents of different polarity were not successful. The title molecular salt is produced due to a proton-transfer reaction in which one of the two phenolic group hydrogen atoms is transferred to one of the tertiary nitrogen atoms of 1,10-phenanthroline. This type of interaction is also evidenced by the spectroscopic data .Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989015010737/is5402sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989015010737/is5402Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015010737/is5402Isup3.cmlSupporting information file. DOI: 1050845CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Hence, in the current study, higher doses of tocotrienols have used to analyze its effect on plasma pharmacokinetic parameters.Tocotrienols has been known to lower serum lipid parameters below 500 mg/d, while increase lipid parameters at higher dose of 750 mg/d. δ-Tocotrienol has a novel inflammatory property of concentration-dependent inhibition and activation. Therefore, inhibition (anti-inflammatory) property of tocotrienols at low doses is useful for cardiovascular disease, whereas, activation (pro-inflammatory) property using high dose is found effective for treatments of various types of cancer. We have recently described plasma bioavailability of 125 mg/d, 250 mg/d and 500 mg/d doses of δ-tocotrienol in healthy fed subjects, which showed dose-dependent increases in area under the curve (AUC) and maximum concentration  of annatto-based tocotrienols in healthy fed subjects. All four isomers  of tocols (tocotrienols and tocopherols) present in the plasmas of subjects were quantified and analyzed for various pharmacokinetic parameters.An open-label, randomized study was performed to analyze pharmacokinetics and bioavailability of δ-tocotrienol in 6 healthy fed subjects. All subjects (3/dose) were randomly assigned to one of each dose of 750 mg or 1000 mg. Blood samples were collected at 0, 1, 2, 4, 6, 8 h intervals and all isomers of α-,β-,γ-,δ-tocotrienols, and tocopherols in plasmas were quantified by HPLC.0-t8 6621, 7450; AUCt0-∞ 8688, 9633; AUMC t0-∞ 52497, 57199; MRT 6.04, 5.93; Cmax 1444, 1592 (P<0.05), respectively, of δ-tocotrienol isomer. Moreover, both doses also resulted in plasmas Tmax 3.33–4 h; elimination half-life (t1/2 h) 2.74, 2.68; time of clearance  0.086, 0.078; volume of distribution  0.34, 0.30; and elimination rate constant  0.25, 0.17, respectively of δ- tocotrienol isomer. Similar results of these parameters were reported for γ-tocotrienol, β- tocotrienol, α-tocotrienol, δ-tocopherol, γ-tocopherol, and β-tocopherol, except for α- tocopherol.Oral administration of 750 and 1000 mg/d of tocotrienols resulted in dose-dependent increases in plasmas (ng/ml) AUCtmax was 3-4 h for all isomers of tocotrienols and tocopherols except for α-tocopherol (6 h). These higher doses of tocotrienols were found safe in humans and may be useful for treatments of various types of cancer, diabetes, and Alzheimer's disease.This study has described pharmacokinetics using higher doses of 750 mg/d and 1000 mg/d of δ-tocotrienol. These results confirmed earlier findings that T The time to achieve maximum peak (Tmax) varied between 3-4 h for isomers of tocotrienols and 3-6 h for isomers of tocopherols at 125 mg, 250 mg, 500 mg doses, indicating the longer time of excretion for tocopherols compared to tocotrienols that showed better bioavailability of tocotrienols than tocopherols [We have recently reported the pharmacokinetic and bioavailability of various doses  of δ-tocotrienol in 11 fed healthy participants/group (n=33). This was the first study which demonstrated the effect of δ-tocotrienol on several pharmacokinetic parameters of all eight isomers of tocol family  [Tocotrienols have been shown to modulate biological (anti-inflammatory/pro-inflammatory) activity depending on its concentrations . The dosmax was found to be between 3-5 h for both food conditions [max for α- tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol were 2.1∼2.3 h and α-tocopherol was 4.7∼3.1 h [max after administering 2-capsules (300 mg/capsule), which was a mixture of γ-tocotrienol + δ-tocotrienol versus 4-capsules of palm TRF, were 5.64 ± 1.50 h versus 4.73 ± 0.90 h [A number of studies have reported the pharmacokinetics and bioavailability of tocotrienols in humans and different animal models. The bioavailability of 300 mg capsule, a mixture of α-tocotrienol + γ-tocotrienol + δ-tocotrienol was administered to fasted and fed subjects n=8). The plasma Tnditions . In addi. The plain vivo and in vitro in rats after administering in the form of oil solution and aqueous medium respectively [in vitro in the aqueous medium form. The oral bioavailability of α-tocopherol (36%) was significantly higher than γ-tocotrienol (9%). The studies in situ also revealed significantly higher intestinal permeability for α-tocopherol compared with γ-tocotrienol in rats [max for γ-tocotrienol versus α-tocopherol was 2.4 h and 9.7 h respectively [in vivo with δ-tocotrienol also improved radiation-induced alteration in apoptotic and autophagic pathways [The intestinal absorption kinetics and the bioavailability of γ-tocotrienol and α-tocopherol have been compared ectively . There wotrienol %. The stotrienol %. The stpathways .max (2–3 h) and α-tocopherol group had plasma Tmax (3–5 h). None of the published studies have reported the bioavailability of β-, γ-, δ-tocopherol other than our recently published study describing the pharmacokinetic, bioavailability and absorption of all four isomers of tocotrienols and tocopherols [max, Tmax were determined for all isomers  of tocotrienols and tocopherols. The results of this study would be able to establish the safe use of higher doses of tocotrienols for the treatment of cancer, diabetes and Alzheimer's disease.All of these studies using tocotrienols, mixture of tocotrienols, tocotrienol-rich fraction of barley oil (TRF) or tocotrienol-rich fraction of palm oil  were carried out using lower doses 300 ∼ 500 mg), except for pancreatic cancer study in humans [00 mg, exopherols . As repoDeltaGold 125 mg soft gels from annatto seeds (composition 90% δ-tocotrienol + 10% γ-tocotrienol) were supplied by American River Nutrition, Inc. . The HPLC supplies were purchased as reported recently [Six healthy subjects (n=3/dose) were selected for the present study. A small number of subjects were used due to results of our earlier study of 11 subjects/dose were very consistent and there was not much variation (<5.0%) between different subjects of the same group [An open-label, randomized study was carried out to determine the pharmacokinetics and bioavailability of annatto-based tocotrienol after oral administration in 6 healthy male subjects fed condition. All the subjects were fed Pakistani heavy breakfast comprising of fruit cocktail, Halwa, Puri, Paratha, Omlet, orange juice and tea. The volunteers were randomly assigned to one of DeltaGold soft gels 125 mg/capsule dose levels , which they received once after a heavy breakfast. The volunteers were instructed not to consume foods rich in vitamin E to minimize variability in intake. Restricted foods included nuts, cereals and vegetable oils. The food was prepared in “Army Mess” under strict hygienic conditions. All subjects were fed very heavy breakfast comprising of Halwa Puri, Paratha, Spanish Omlet, and Lassi (yugart shake), orange juice, or tea; lunch was chicken biryani, Mutton (Lamb) Qorma, Sheermal/Nan, carrot Halva (desert). Dinner was served at the end of study consisting of fried Basmati rice, Kabab (chicken), Rogni Nan, Shahi Tukray, and Kashmiri tea with pistachios. Fruit cocktail was served with every meal as reported recently [On study day, each subject consumed one dose of δ-tocotrienol with a typical heavy Pakistani breakfast. During the experimental period (0-8 h), subjects consumed good lunch and evening tea as described above. All subjects were allowed to drink water freely. For the determination of the pharmacokinetic profile of tocotrienols in plasma, venous blood samples (2 × 5 ml) were collected in EDTA glazed tubes at pre-dose (0 h) and at post-dose . The samples were then centrifuged at 3000 × g for ten minutes. Processed plasma samples were stored in eppendorf tubes at -80°C till further analysis as reported earlier [The detailed procedure of plasma tocols analyses by high performance liquid chromatography has been described previously . In shorThe working standards solutions were prepared of various tocols 1 μg/ml) in hexane; 1% ascorbic in absolute ethanol, 10 mg/10 ml butylated hydroxyl toluene in absolute ethanol. The prepared working stock solutions were kept at -20°C [ μg/ml inThe peak areas were determined by Shimadzu Integrator- Model C-R3A . The eluting solvent was 0.3% (vol/vol) isopropyl alcohol in hexane. The extracted sample (20 μl) was introduced into the column through the 10 μl loop of Gilson's autosampler-231 injector. All the samples were analyzed in duplicate or triplicate. The retention time of the individual peaks of the unknown tocols were compared against the retention time of the pure standard tocols. The tocols were eluted under these conditions in the sequence: α-tocopherol, α-tocotrienol, β-tocopherol, β-tocotrienol, γ-tocopherol, γ-tocotrienol, δ-tocopherol, δ-tocotrienol [0 – t8, AUCt0-∞, AUMCt0-∞, MRT, Cmax, Tmax, half-life time (t1/2), time of clearance (Cl-T). The values for these parameters were based on mean concentrations of each time point of each isomer  of tocotrienols (T3) and tocopherols (T) and also using the values of each subject in each group. Data of plasma tocotrienols concentration versus time was used to calculate pharmacokinetic parameters. Area under the curve (AUC) from 0 h- time to 8 h-time (AUCt0 – t8 h) for plasma tocotrienols was calculated by trapezoidal rule, where “t” is the last measured time point. The maximum serum concentration (Cmax) and the time to reach peak (Tmax) were obtained directly by inspecting each individual plasma level- time curves. The Ke values were calculated by equation: time of clearance (Cl-T)/Vd/f) [The computer software PK Solver 2.0 was used for calculation of pharmacokinetic parameters through standard two compartmental analysis of each subject in each group .This is the first report describing the quantitative determination of all eight isomers of tocol (four tocotrienols plus four tocopherols) from the human plasma samples obtained after administering higher doses (750 mg and 1000 mg) of annatto-based δ-tocotrienol. The physical characteristics of all participants are reported in HPLC was carried out as reported earlier by using normal phase silica of plasma samples collected at 0 h, 1 h, 2 h, 4 h, 6 h, and 8 h (7 samples) of each subject for the determination of pharmacokinetics of various tocols [Similar pattern of concentrations (ng/ml) of tocols was observed in plasmas obtained from subjects that were fed 1000 mg dose, and tocotrienols (ng/ml) were β-tocotrienol (8337)>γ-tocotrienol (5404)>δ-tocotrienol (4465)>α-tocotrienol (3562), and tocopherols concentrations order was α-tocopherol (13239)>δ-tocopherol (5838)>β-tocopherol (5185)>γ-tocopherol (2212) . None of0 – t8 or ∞), AUMCt0-∞, Mean Residence Time (MRT), plasma maximum concentration (Cmax), time to reach plasma concentration (Tmax), the apparent volume of distribution (Vd/l), time of clearance , half-life time (t1/2), and elimination rate constant (ke).The analyses of important pharmacokinetic parameters for all different isomers were calculated for each time interval/subject/group by PK Solver 2.0, which provided values with standard deviations (SD) for each group. The pharmacokinetics and bioavailability of a compound required analysis of plasma for determination of area under the concentration-time curve (AUCt0 - t8 (ng/ml) for 750 mg (6621 ± 50) and 1000 mg (7450 ± 89) which statistically significant (P<0.05) (0-∞ (ng/ml), 8688 ± 201, 9633 ± 383 (P<0.05); AUMCt0-∞ (ng/ml), 52497 ± 2096, 57199 ± 5007, and MRT (h) 6.04 ± 0.14, 5.93 ± 0.34, were observed for doses of 750 mg and 1000 mg, respectively , 1444 ± 53, 1592 ± 44; time to achieve plasma peak (Tmax), 3.33, 4.0 h; elimination half-life (t1/2 h), 2.74 ± 0.13, 2.68 ± 0.29 h; time of clearance , 0.086 ± 0.002, 0.078 ± 0.003; apparent volume of distribution , 0.341 ± 0.012, 0.300 ± 0.021; and elimination rate constant , 0.253 ± 0.167, 0.260 ± 0.143 for doses of 750 mg, and 1000 mg, respectively, were calculated as shown in The δ-tocotrienol feeding showed dose-dependent increases in plasmas of subjects area under the curve, AUCtP<0.05)  and 5. S  and 5. ectively  and 5. P0 – t8 (ng/ml) 6962 ± 98, 7480 ± 129, (P<0.05), for β-tocotrienol, 11474 ± 316, 11895 ± 231, and for α-tocotrienol, 198 ± 102, 548 ± 19, P<0.05 for doses of 750 mg, and 1000 mg, respectively as shown in max ng/ml) for γ-tocotrienol, 1352 ± 28, 1387 ± 13; β-tocotrienol, 1885 ± 91, 1948 ± 91; and α-tocotrienol, 30 ± 1, 116 ± 4 (P<0.05) were observed for 750 mg, and 1000 mg, respectively (max h) were for γ-tocotrienol, 750 mg (4 h), 1000 mg (4 h); for β-tocotrienol were 750 mg (4 h), 1000 mg (3.33 h), and similarly, for α-tocotrienol were 750 mg (3.33 h), and 1000 mg (4 h) for these isomers, as shown in Similar increases in all the parameters were observed in plasmas for γ-tocotrienol, such as AUCtectively  and 5A. 0 – t8 (ng/ml) 11474, 11892 and Cmax (ng/ml) 1885, 1948 values for both doses were found with β-tocotrienol and lowest values were with α-tocotrienol, AUCt0 – t8 (ng/ml) 198, 548 and Cmax (ng/ml) 1885, 1948 were with 750 mg and 1000 mg doses, respectively. The maximum peak elution time was between 3 h to 4 h for both doses ; AUCt0 – t∞ ; AUMC t0–∞ ; MRT ; Cmax,  as shown in max were 3.33, 4.0 h, for doses 750 mg and 1000 mg, respectively.The values for all these parameters showed dose-dependent decreases and most of them were significantly different at P<0.05–0.01 as reported in 0 – t8 h ; AUCt0-∞ ; AUMC t0-∞ ; MRT ; Cmax  showed dose-dependent increases that were in-significant, while Tmax was 4 h for both doses ; AUCt0-∞  showed significant dose-dependent increases, whereas, values for MRT , Cmax  showed dose dependent increases for 750 mg and 1000 mg and Tmax was 4 h for both doses ; Cmax  were observed with α-tocopherol and Tmax was 6 h for both doses (0 – t8 h (ng/ml) for α-tocopherol was greatest out of all four tocotrienols and tocopherols when compared for 750 mg and 1000 mg doses as shown in The maximum values for AUCtth doses  and 5B. 0 – t8, Cmax, and Tmax. In addition to these three parameters, there were dose dependent increases in AUCt0-∞, AUMC t0-∞, MRT, half-life time (t1/2), time of clearance (Cl-T), the apparent volume of distribution (Vd/f), and elimination rate constant (Ke) for all isomers of tocols except for α-tocopherol. Tmax for all the isomers of tocols was 4 h except for α-tocopherol (6 h). These results are in line with our earlier findings using lower doses of tocotrienols [0-∞ [The present study describes effects of higher doses of annatto-based δ-tocotrienol (without α-tocopherol) on the pharmacokinetic parameters of all eight isomers of tocol family  in healthy fed subjects. The doses 750 mg and 1000 mg of δ-tocotrienol fed resulted in dose-dependent increased levels of all eight isomers of tocols in the plasma and their values of AUCtols [0-∞ .The main difference between our earlier results of lower doses with feeding125 mg, 250 mg, and 500 mg versus present results of higher doses were that there were no peaks for δ-tocotrienol and δ-tocopherol at time point 0 h and 1 h in plasma samples with 125 mg dose. While plasma samples of 250 mg and 500 mg doses there were also any peaks at 0 h time point of δ-tocotrienol and δ-tocopherol [Tocotrienols are lipophilic in nature, therefore poorly soluble and show limited absorption and low bioavailability as reported earlier ,9. Therein vitro and in vivo in rats [in vitro results showed two-fold increase in the solubilization as well as higher cellular uptake while in vivo studies showed two-fold improvement in oral bioavailabity of γ-tocotrienol in SEDDS formulations compared to Tocovid [Although, it is difficult to determine the absolute bioavailability of tocotrienols in humans, but relative bioavailability of γ-tocotrienols increases 3.5-fold when administered with food which has fat as a major component . While,  in rats . The in  Tocovid .In the current study, we have recruited only males to avoid any genetic variability. There are several factors like age, drug-drug interaction, disease, environmental pollutants, and genetics which can contribute to variability in our pharmacokinetics results. In this study, we tried to take care of those factors by selecting the subjects in the same age group, with no past history of major disease, as well as taking no drugs. As food an example of environmental factor contributing to variability in our results, the subjects have to eat large quantities of heavy breakfast or lunch, during eight hour period to have any dietary effect on the total concentrations of tocotrienols and tocopherols.0-8, AUCt0-∞, AUMCt0-∞, Cmax. The Tmax was 3-4 h for all isomers, except α-tocopherol (6 h) for doses of 750 mg and 1000 mg.This study describes the effects of higher doses of 750 mg and 1000 mg of annatto-based tocotrienols on the plasma pharmacokinetic parameters of all eight isomers of tocols in humans. The bioavailability of α-, β-, γ-, δ-tocotrienols and α-, β-, γ-, δ-tocopherols resulted dose-dependent increases in plasma AUCtmax of these higher doses (750 mg and 1000 mg) in humans. Moreover, it is better to take tocotrienols after lunch/dinner (food) to increase their permeability, absorption, and bioavailability [The study participants did not report any adverse effects with higher dose feeding at the end of the study and were safely tolerated by them. Moreover, the novel property of tocotrienols, concentration-dependent inhibition and activation, observed in case of cardiovascular parameters , and cancer patients  had no impact on Tlability . These h


This study investigated the effectsof estradiol and bisphenol A on the proliferation and telomerase activity of humanhepatoblastoma HepG2 cells. The cells were divided into 6 treatment groups: control,bisphenol A, estradiol, anti-estrogen ICI 182,780 (hereinafter ICI), bisphenol A+ICI,and estradiol+ICI. Cell proliferation was measured based on average absorbance usingthe Cell Counting-8 assay. The cell cycle distribution and apoptotic index weredetermined by flow cytometry. Telomerase activity was detected by polymerase chainreaction and a telomeric repeat amplification protocol assay. A higher cell densitywas observed in bisphenol A (P<0.01) and estradiol (P<0.05) groups comparedwith the control group. Cell numbers in S and G2/M phases after treatment for 48 hwere higher (P<0.05), while the apoptotic index was lower (P<0.05) andtelomerase activities at 48 and 72 h (P<0.05) were higher in these groups than inthe control group. The cell density was also higher in bisphenol A+ICI (P<0.01)and estradiol+ICI (P<0.05) groups compared with the ICI group. Furthermore, cellnumbers were increased in S and G2/M phases (P<0.05), while the apoptotic indexwas lower (P<0.05) and telomerase activities at 48 and 72 h were higher(P<0.05) in these groups than in the ICI group. Therefore, bisphenol A andestradiol promote HepG2 cell proliferation  Hepatoblastoma (HB) is the most common malignant, solid liver tumor in children, arisingfrom multipotent stem cells that differentiate into liver and bile duct epithelial cellsin undifferentiated embryonic tissue . ItaccoIt has been suggested that environmental factors and sex hormones play major roles inthe etiology of HB . EstrogeTelomerase is a type of reverse transcriptase consisting of a ribonucleoprotein complexwith a RNA template and various catalytic and regulatory subunits. It is expressed in98% of immortalized cell lines and >90% of malignant tumors . TelomerThis study investigated the effects of physiological and environmental estrogen on HB bytreating human hepatoblastoma HepG2 cells with 17β-estradiol (E2) and bisphenol A (BPA),and then evaluating cell proliferation, apoptosis, and telomerase activity. Themechanism of action of these hormones was examined using anti-estrogen ICI 182,780(hereinafter ICI).The HepG2 cell line was provided by the Medical School of Zhengzhou University.Phenol red-free Roswell Park Memorial Institute (RPMI) 1640 medium and fetal bovineserum (FBS) were obtained from Gibco (USA). ICI was purchased from Santa CruzBiotechnology Co., Ltd. (China). E2 was from IBL International (Germany). The cellcounting kit (CCK)-8 was from Dojindo (Japan). Dimethyl sulfoxide (DMSO) was obtainedfrom Zhengzhou Chengxiang Chemical Technology Ltd. (China). The reverse transcriptionkit was purchased from Invitrogen (USA). SYBR Green supermix was from Toyobo (Japan),and the Telo TAGGG Telomerase PCR enzyme-linked immunosorbent assay (ELISA) kit wasfrom Nanjing KeyGEN Biotech Co., Ltd. (China). Penicillin and streptomycin werepurchased from Beijing BioDee Biotechnology Co. Ltd (China).5′-AATCCGTCGAGCAGAGTT-3′,which was labeled with biotin at the 5′-end. The sequence of reverse primer CX was5′-CCCTTACCCTTACCCTTACCCTTA-3′.The probe sequence was 5′-CCCTAACCCTAACCCTAA-3′ labeled with digoxin at the 5′-end.Primers and probes were synthesized by Invitrogen. The sequence of forward primer TSwas 2and saturatedhumidity. After the cells had attached to the culture dish, the medium was replacedwith phenol red-free RPMI 1640 medium, and the cells were cultured for 24 h. Thecells were examined daily by phase contrast microscopy.HepG2 cells were cultured in RPMI 1640 medium containing 10% FBS, 100 U/mLpenicillin, and 100 U/mL streptomycin at 37°C with 5% CO6 cells/mL and seeded in a 96-well platewith 200 μL each well. After adherence, the culture medium was removed, and cellswere washed twice with phosphate-buffered saline (PBS) before BPA or E2 was added atvarious concentrations . Normal liver cells were similarlytreated with the various concentrations of BPA or E2. ICI was used at1×10−6 M according to a previous report , BPA, E2,ICI, BPA+ICI, and E2+ICI. The volume of DMSO in each group was <0.1% of the totalvolume.5 cells/well in a 96-well plate. After adherence,the culture medium was removed, and cells were washed twice with PBS. CCK-8 solution(10 μL) was added to each well at 0, 24, 48, 72, 96, and 120 h, and the cells werecultured for an additional 3 h before the absorbance at 450 nm (A450nm)was determined using a microplate reader (Bio-Rad). A growth curve was generated fromthe measured values.Cells were seeded at 1×105cells/25 mL culture flask. After 24 h, the cells were washed twice with PBS andsubjected to the various treatments. After 48 h, 1-5×106 cells werecollected by trypsinization and centrifuged at 12,000 g for 5 min at4°C. The cells were then repeatedly washed with PBS and fixed in pre-cooled 70%alcohol at −20°C overnight. After washing with PBS, the cells were treated with RNaseA (10 μL of a 20 μg/mL stock solution in 500 μL PBS) for 30 min at 37°C, followed bycentrifugation at 8,000 g for 5min at 4°C. The cells were thenincubated with 10 μL of a propidium iodide solution (50 μg/mL in 500 μL PBS) for 30min at room temperature in the dark. Cell cycle and apoptosis analyses were carriedout by flow cytometry  using CellQuest software . A total of 10,000 cells was used to analyze and the cell cycle distributionwith FlowJo software (USA).Cells were collected at the logarithmic growth phase and seeded at 3×10gfor 20 min at 4°C, and the supernatant was stored at −80°C until use. Two microlitersof telomerase extraction solution incubated at 65°C for 10 min was used for thenegative control. The TRAP reaction was carried out in a 50 μL volume including 2×substrate buffer (25 μL), TS and CX primers (2 μL or 100 ng each) 2 U Taq enzyme (1μL), TRAP template (1 μL), and sterile diethylpyrocarbonate water. Sterile paraffinoil (40 μL) was used to overlay the reaction solution. Telomeric repeat sequenceswere synthesized by telomerase at 25°C for 30 min, and then the enzyme wasinactivated at 94°C for 5 min. The PCR conditions were as follows: 30 cycles of 94°Cfor 30 s, 50°C for 30 s, and 72°C for 90 s, followed by final extension at 72°C for10 min. To detect telomerase activity, a polyvinyl chloride panel was coated with1:50 biotin-streptavidin (50 μL per well) overnight at 4°C. The following day, thepanel was washed four times with cleaning solution . PCR product (5 μL) was mixed with 20μL denaturation solution (0.5% NaOH), followed by incubation for 10 min at roomtemperature and then addition of 225 μL hybridization solution. A total of 100 μL ofthe mixture was transferred to the panel, and hybridization was carried out at 37°Cfor 3 h. After three washes with washing buffer, 100 μL peroxidase-conjugatedanti-digoxin antibody was added to the panel, followed by incubation for 30 min at37°C. After five washes with washing buffer, 100 μL substrate buffer  was added to the panel. Color development was allowedto proceed for 10-15 min. The reaction was terminated by addition of 2M sulfuricacid. Telomerase activity was determined by measuring the A450nm(reference wavelength: 630 nm) on a microplate reader. Negative and positive controlmeasurements corresponded to A450nm <0.2 and A450nm >1.0,respectively.A PCR-telomeric repeat amplification protocol (TRAP)-ELISA kit ,17 was uData are reported as means±SD. Comparisons between groups were performed by one-wayanalysis of variance. MATLAB software  was used for all statisticalanalyses. P<0.05 was considered to be statistically significant.After 120 h of treatment, the effective concentrations of E2 and BPA to stimulateHepG2 cell proliferation were 2 μg/mL and 10 ng/mL, respectively . For norThe proportions of cells in the various cell cycle phases were obviously different inE2- and BPA-treated cells and control cells P<0.05; . SimilarFlow cytometry showed that the apoptotic index was markedly reduced in cells treatedwith E2 and BPA compared with the control group P<0.05; . SimilarTelomerase activity in HepG2 cells was evaluated by a PCR-TRAP-ELISA. Compared withthe control group, telomerase activity was enhanced in BPA- and E2-treated cells at48 and 72 h P<0.05; . Similarin vitro in time- andconcentration-dependent manners (HB is the most common type of pediatric liver tumor. It is highly malignant, associatedwith poor outcomes, even after treatment, and characterized by occult occurrence,vascular enrichment, rapid growth, and early metastasis ,19. The  manners . Consist manners .The role of telomerase in tumor malignancy has been highlighted by many studies.According to a previous report, the hyperproliferation of tumor cells in 90% of humanmalignancies is linked to inappropriate telomerase activity . A study


Kc=[SiOH]2/[Si-O-Si][H2O]=6±1×10−5 and is helpful in understanding the enhanced hydrolytic stability of surfaces modified with dipodal silanes.Dipodal silanes possess two silicon atoms that can covalently bond to a surface. They offer a distinct advantage over conventional silanes commonly used for surface modification in terms of maintaining the integrity of surface coatings, adhesive primers, and composites in aqueous environments. New nonfunctional and functional dipodal silanes with structures containing “pendant” rather than “bridged” organofunctionality are introduced. The stability of surfaces in aqueous environments prepared from dipodal silanes with hydrophobic alkyl functionality is compared to the stability of similar surfaces prepared from the conventional silanes. In strongly acidic and brine environments, surfaces modified with dipodal silanes demonstrate markedly improved resistance to hydrolysis compared to surfaces prepared from conventional silanes. Pendant dipodal silanes exhibit greater stability than bridged dipodal silanes. The apparent equilibrium constant for the formation of silanol species by the hydrolysis of a disiloxane bond was determined as  Silanes that alter the chemical and physical properties of surfaces have been studied intensively for over sixty years. Silanes The stability of properties associated with surface modification depends on maintaining tight equilibrium conditions in the physical region associated with the transition from the substrate to the matrix. The physical region that extends from the unmodified bulk phases of both the substrate and matrix is referred to as the interphase. The interphase region associated with silane modification is most properly viewed as a dynamic region in which a steep gradient in properties is mediated by the interaction of molecular water with oxane bonds. More specifically, the dynamics are driven by the hydrolysis and reformation of oxane bonds. Plueddeman recognizThe success of silanes in numerous coupling applications is in large part due to the hydrolysis and reformation of oxane bonds between silanes and substrates, but at the same time hydrolysis without oxane bond reformation is a critical factor in their failure. It has been observed that overpolymerization of polymers onto substrates modified with silanes reduces the mobility of hydrolyzed silanes. Long-terThis work explores two issues associated with the long-term failure of silane-modified inorganic substrates: the dynamism of siloxane bond hydrolysis and reformation and organofunctional silanes with dipodal structures, which reduce the probability of diffusion from the inorganic substrate.While the kinetics of siloxane bond formation in aqueous systems have been studied extensively, there exists only sparse data on the equilibrium constant for siloxane bond formation and hydrolysis in aqueous media Scheme .This is presumably primarily because the condensation of silanol species results in species with siloxane bonds that are generally insoluble in water and form bulk insoluble phases that do not readily participate in system equilibria. Silicic acid and trimethylsilanol are examples of species that form bulk phases, silica and hexamethyldisiloxane, respectively, that do not readily participate in equilibrium studies in neutral aqueous media.–7 FurtheOn the other hand, phenomena, such as Ostwald ripening of Stober process silica particles and formation of monolithic structures by sol–gel processing, suggests that the equilibrium is much more balanced.–10 StudiKa of remaining silanol groups. Nevertheless, an understanding of the apparent equilibrium between siloxane and silanol species would be helpful in understanding long-term interphase behavior.Several factors can potentially interfere with a straightforward determination of a silanol–siloxane equilibrium constant. Ionization of silanol groups can take place at near neutral pH, the same conditions of interest for studying the silanol–siloxane equilibrium. Other considerations that may preclude simple determination of the silanol–siloxane equilibrium constant is that most compounds of interest, such as silicic acid and dimethylsilanediol, can form multiple siloxane bonds and undergo polymerization that would influence the pThe objective of this study was to improve the durability of organofunctional silane surface modifications by increasing the number of potentially available sites for an organosilane to form oxane bonds either to a substrate or to form silsesquioxane polymers. It was of further interest to determine whether the proximity of the bonding sites within an organofunctional silane had an effect on the durability of silane surface modification under aqueous conditions. To provide a proper context for the potential of enhancing the hydrolytic stability of organofunctional silane surface treatments, the apparent equilibrium constant for a stable water soluble disiloxane was determined.As probes for the stability of the Si–O–Si bonds, an organofunctional disiloxane and an organofunctional silanol were synthesized by hydrosilylation of tetramethyldisiloxane and dimethylethoxysilane according to methods previously reported. Both comIn independent experiments, both the disiloxane and the silanol were evaluated in neutral aqueous systems and, after equilibration, the relative amounts of silanol and disiloxane were determined Scheme .6–9.Various organofunctional silanes were prepared containing two silicon atoms with alkoxy substituents. Compounds with substitution in the alpha and omega position have sometimes been referred to as “bis(silanes)” and have been the subject of much investigation, particularly in the field of metal coatings and primers.–15 We haTwo synthetic methods were utilized to generate the pendant dipodal silanes evaluated in this study. The pendant silanes with two-carbon separation were synthesized by a disilylation method as reported earlier Scheme .16]16]The syntheses of the pendant silanes with a one-carbon separation were achieved in three steps, by starting with a redistribution reaction, followed by hydrosilylation and then esterification or esterification followed by hydrosilylation Schemes , and 8..Examples of both functional and nonfunctional silanes were prepared. Evaluation of nonfunctional silane surface treatments in aqueous systems was considered to be a better probe than functional silanes for hydrolytic stability. A functional silane anchored by reaction with an organic component in a composite coating would be restrained from diffusing from the interphase and, thus, exhibit extended time to failure. Further, contact-angle studies provide a simple metric that is readily related to the presence of a silane, whereas mechanical properties associated with composites and coatings are less direct.Initial and equilibrium concentrations of 1,3-bis(tetrahydrofurfuryloxypropyl)tetramethyldisiloxane and tetrahydrofurfuryloxypropyldimethylsilanol in solutions of water and THF were determined by GC analysis (with adjustments for response factors) and summarized in the Tables The apparent equilibrium constant for the hydrolysis of one mole of disiloxane to two moles of silanol was calculated for each data set and then averaged, providing the result:−5 is consistent with the dynamism suggested for the siloxane bond in surface modification applications. While long-term hydrolytic stability studies of polymeric siloxane and silica bulk phases are not relevant to the equilibrium solution studies of this investigation, they provided a perspective that suggested that the siloxane bond is less susceptible to hydrolysis than reality. The preservation of the siloxane (Si–O–Si) bond in aqueous systems is significantly more favored than the silicon ester (Si–O–C) bond that has been estimated at 2×10−2.. The mixture was heated to 90 °C and 10 g of 1-decene were added to the mixture by an addition funnel, followed by 0.5 mL of 5 % hexachloroplatinic acid in tetrahydrofuran. An immediate exotherm was observed and the reaction mixture changed from clear to dark brown. An additional 173 g of 1-decene was added at an appropriate rate to maintain the temperature between 90 and 110 °C. Upon completion of the addition, an additional 0.25 mL of 5 % hexachloroplatinic acid solution was added and the mixture was stirred at 100 °C for 45 min. GC analysis of the mixture indicated the presence of some unreacted pentachlorodisilapropane. An additional 10 g of 1-decene were added and the reaction mixture was stirred an additional 45 min at 100°. Distillation provided 242 g (59 %) of the title compound. B.p. 126°/0.1 mmHg; density, 20 °C: 1.178 g/cc; Preparation of 1,1,1,3,3-pentamethoxy-1,3-disilatridecane (9)9. B.p. 123–4 °C/0.1 mmHg, density, 20 °C: 0.955 g/cc; 1H NMR (400 MHz): δ=3.55 , 3.50 , 1.40–1.15 , 0.85 , 0.70–0.60 , −0.05 ppm ; 29Si{1H}: δ −41.85, −4.22 ppm.The product from above was reacted according to the representative esterification procedure to give an 82 % yield of 1H NMR (400 MHz): δ=1.57–1.49 , 1.42–1.35 , 1.34–1.20 , 0.88 ppm .A 2-liter flask equipped with heating mantel, magnetic stirrer, pot thermometer, addition funnel, and a dry-ice condenser was charged with the 1,1,1,3,3-pentachloro-1,3-disilapropane/1,1,1,3,3,3-hexachloro-1,-disilapropane mixture . The reaction mixture was warmed to 90 °C and 5 % chloroplatinic acid in THF (0.5 g) was added followed by the addition of 10 g of 1-octadecene. An immediate exotherm was observed indicating initiation of the hydrosilylation. The remainder of the 187 g (0.72 mol) of 1-octadecene was added at a rate to maintain the reaction temperature between 90 and 110 °C. After the addition was complete, an additional 0.25 mL of the platinum catalyst solution was added and the reaction mixture was heated at 100 °C for an additional 45 min. Distillation provided 229 g (91 %) of the title product. B.p. 192–4 °C/0.2 mmHg; density, 20 °C: 1.059 g/cc; 14 was added to the reaction mixture. The reaction mixture was heated to 85 °C and a 5 % THF solution of chloroplatinic acid (0.25 mL) was added. An immediate exotherm was observed with the solution changing from clear and colorless to dark brown. The balance of the chlorosilane mixture was added at a rate so as to maintain a reaction temperature of 85 to 105 °C. Upon completion of the silane addition, an additional 0.25 mL of the catalyst solution was added and the reaction mixture heated at 95° for 45 min. Distillation provided 80 g (70 %) of the title organosilane. B.p. 170–2 °C/0.5 mmHg; density, 20 °C: 1.262 g/cc; 1H NMR (400 MHz): δ=3.72- 3.56 , 3.65–3.48 , 3.17 , 3.41 , 1.71–1.59 , 0.73–0.68 ppm .Under an atmosphere of nitrogen, a standard-equipped 500 mL flask was charged with allyloxytriethyleneoxymethyl ether . A 46:54 mixture of 1,1,1,3,3-pentachloro-1,3-disilapropane and 1,1,1,3,3,3-hexachloro-1,3-disilapropane  was placed in an addition funnel and ca. 25 g of the mixture containing D20=1.4359; 1H NMR (400 MHz): δ=3.80–3.65 , 3.62- 3.56 , 3.55–3.48 , 3.37 , 3.31 , 1.68–1.59 , 1.19–1.10 , 0.65–0.58 , −0.09 ppm .In a one-pot, two-step process based on the above, allyloxytriethyleneoxymethyl ether  was hydrosilylated and the crude pentachloro derivative converted directly to the ethyl ester to give after distillation 310 g (62 %) of the title organosilane. B.p. 176–8 °C/0.1 mmHg: density, 20 °C: 0.994 g/cc; n1H NMR (400 MHz): δ=3.82–3.71 , 3.65 , 3.48–3.31 , 3.12–3.08 , 2.75 , 3.59–3.54 , 1.69–1.61 , 1.20–1.11 , 0.68–0.61 , −0.07 ppm .A 1-liter flask equipped with condenser, addition funnel, magnetic stirrer, and pot thermometer was charged with the 44:56 mixture of 1,1,1,3,3-pentaethoxy-1,3-disilapropane and 1,1,1,3,3,3-hexaethoxy-1,3-disilapropane  prepared above and phenothiazine (0.04 g). The reaction mixture was heated to 90 °C and allylglycidyl ether  was added keeping the temperature between 85 and 115 °C. The reaction mixture was subjected to distillation through a short Vigreaux column with three successive distillations giving 100 g (64.5 %) of the title product in 96 % purity. B.p. 125–30 °C/0.1 mmHg; A 2-liter flask equipped with magnetic stirrer, condenser, pot thermometer, and addition funnel was charged with the 1,1,1,3,3-pentachloro-1,3-disilapropane/1,1,1,3,3,3-hexachloro-1,3-disilapropane mixture prepared above . The addition funnel was charged with methallyl chloride . The reaction mixture was warmed to 110 °C and to initiate the reaction 20 g of the olefin was added followed by the addition of 0.5 mL of a 5 % solution of chloroplatinic acid in THF. The remainder of the olefin was added a rate such as to maintain a reaction temperature between 110 and 120 °C. After completion of the olefin addition, the reaction was heated for an additional 3 h at 130 °C. Distillation of the product directly from the reaction vessel provided 412 g (60 %) of the title dipodal silane. B.p. 89–91 °C/0.4 mmHg; density, 20 °C: 1.380 g/cc.1H NMR (400 MHz): δ=3.85–3.72 , 3.56–3.50 , 3.39–3.33 , 2.15–2.05 , 1.23–1.15 , 1.07 , 0.9–0.83 , 0.68–0.61 , −0.03 ppm .The product from above was reacted according to the representative esterification procedure to give a 60 % yield of the title organosilane. B.p. 115–117 °C/0.5 mmHg; density, 20 °C: 1.020 g/cc; 1H NMR (400 MHz): δ=3.83–3.69 , 2.76–2.71 , 2.68–2.59 , 2.51–2.45 , 2.40–2.32 , 2.88–2.78 , 1.34 , 1.22–1.11 , 0.95 , 0.79–0.71 , 0.56–0.48 , −0.06 ppm ; 29Si{1H} NMR : δ=−45.62, −8.50 ppm.A 500 mL flask suitably equipped was charged with 1,1,1,3,3-pentaethoxy-6-hexachloro-5-methyl-1,3-disilahexane  and ethylene diamine . The reaction mixture was heated to 110 °C for 16 h and 25 g (0.54 mol) of ethanol added and the bottom layer separated and wiped-film distilled to yield 81 g (89 %) of the title dipodal silane. B.p. (est) 130–140 °C/0.5 mmHg; density, 20 °C: 0.990 g/cc; 2 and treated with the various silanes immediately.Borosilicate glass slides (Schott North America) were acid etched before silane treatment by dipping in 4 wt. % HCl for 45 min before washing with 1) deionized water (5 mL), 2) ethanol (5 mL), and 3) acetone (5 mL). The slides were dried under Nn-decyltriethoxysilane (6) was prepared in cyclohexane and a small amount of deionized water  was added with stirring. The stirred silane solution was allowed to hydrolyze for 5 h prior to dipping the cleaned glass slides into the stirred solution. After 20 h, the glass slides were removed and rinsed with cyclohexane, and dried under N2 before being put into an oven at 110 °C for 30 min. The glass slides were cooled to room temperature in a desiccator and the water contact angles were measured. The contact angle measurements were carried out on a Ramé–Hart 100 goniometer. Multiple sessile drop contact angle (advancing and receding) measurements were carried out for each silane and the average numbers (minimum of 6 measurements) are reported. Glass slides were also prepared for silanes 1,2-bis(trimethoxysilyl)decane (8), 1,1,1,3,3-pentamethoxy-1,3-disilatridecane (9), and 1,10-bis(trimethoxysilyl)decane (7), and the respective water contact angles were measured following the same procedure.A 2 wt. % solution of m HCl, 1 m NH4OH, and 3.5 wt. % aqueous NaCl. Each slide was removed from the solution, rinsed with water, and dried under nitrogen before being put into an oven at 110 °C for 30 min. On cooling to room temperature, the water contact angles were measured. After each contact angle measurement, the treated slides were re-immersed in their parent baths . The process was repeated for all the samples at specified intervals for ca. 140 days or until the coatings were completely delaminated.The durability of the silane coating was assessed by static immersion tests at room temperature in deionized water, 6