Multilayer Hydrophilic Poly(phenol-formaldehyde resin)-Coated Magnetic Graphene for Boronic Acid Immobilization as a Novel Matrix for Glycoproteome Analysis

Capturing glycopeptides selectively and efficiently from mixed biological samples has always been critical for comprehensive and in-depth glycoproteomics analysis, but the lack of materials with superior capture capacity and high specificity still makes it a challenge. In this work, we introduce a way first to synthesize a novel boronic-acid-functionalized magnetic graphene@phenolic-formaldehyde resin multilayer composites via a facile process. The as-prepared composites gathered excellent characters of large specific surface area and strong magnetic responsiveness of magnetic graphene, biocompatibility of resin, and enhanced affinity properties of boronic acid. Furthermore, the functional graphene composites were shown to have low detection limit (1 fmol) and good selectivity, even when the background nonglycopeptides has a concentration 100 fold higher. Additionally, enrichment efficiency of the composites was still retained after being used repeatedly (at least three times). Better yet, the practical applicability of this approach was evaluated by the enrichment of human serum with a low sample volume of 1 μL. All the results have illustrated that the magG@PF@APB has a great potential in glycoproteome analysis of complex biological samples

HPV16 E6 suppresses the ING4 mediated p53 transcriptional activity and apoptosis.

<p>(A ) Saos-2 (p53^−/−) cells were cotransfected with a wild-type p21WAF1/CIP1 promoter construct, different combinations of plasmids expressing Myc-p53, PCDNA-ING4, flag-tagged HPV16 E6 or its mutant L50G. At 36 h posttransfection, cells were harvested and lysed in reporter lysis buffer. The bars plot the means of the results of two independent experiments. Error bars represent standard deviations (SD). The results showed that HPV16 E6 attenuates ING4-triggered p53 transcriptional activity. The expression levels of each target proteins were detected by western blotting and shown at the bottom panels. (B) Saos2 cells were transfected with expression plasmids for myc-p53, PCDNA-ING4, in combinations with flag-E6 or its mutant L50G. After a 2-week selection, cells were fixed on the plates with 4% formaldehyde and stained with 0.1% crystal violet. A representative of colony formation was shown. The area of colonies (pixels) in each dish was calculated by Li-Cor Odyssey. The number represents the averages of data from two independent experiments.</p

Additional file 1 of Laser therapy decreases oral leukoplakia recurrence and boosts patient comfort: a network meta-analysis and systematic review

Supplementary Material 1

Anti-Tumor Effect of a Novel Soluble Recombinant Human Endostatin: Administered as a Single Agent or in Combination with Chemotherapy Agents in Mouse Tumor Models

<div><p>Background</p><p>Angiogenesis has become an attractive target in cancer treatment. Endostatin is one of the potent anti-angiogenesis agents. Its recombinant form expressed in the yeast system is currently under clinical trials. Endostatin suppresses tumor formation through the inhibition of blood vessel growth. It is anticipated that combined therapy using endostatin and cytotoxic compounds may exert an additive effect. In the present study, we expressed and purified recombinant human endostatin (rhEndostatin) that contained 3 additional amino acid residues (arginine, glycine, and serine) at the amino-terminus and 6 histidine residues in its carboxyl terminus. The recombinant protein was expressed in <i>E. Coli</i> and refolded into a soluble form in a large scale purification process. The protein exhibited a potent anti-tumor activity in bioassays. Furthermore, rhEndostatin showed an additive effect with chemotherapy agents including cyclophosphamide (CTX) and cisplatin (DDP).</p><p>Methods</p><p>rhEndostatin cDNA was cloned into PQE vector and expressed in <i>E. Coli</i>. The protein was refolded through dialysis with an optimized protocol. To establish tumor models, nude mice were subcutaneously injected with human cancer cells (lung carcinoma A549, hepatocellular carcinoma QGY-7703, or breast cancer Bcap37). rhEndostatin and/or DDP was administered peritumorally to evaluate the rate of growth inhibition of A549 tumors. For the tumor metastasis model, mice were injected intravenously with mouse melanoma B16 cells. One day after tumor cell injection, a single dose of rhEndostatin, or in combination with CTX, was administered intravenously or at a site close to the tumor.</p><p>Results</p><p>rhEndostatin reduced the growth of A549, QGY-7703, and Bcap37 xenograft tumors in a dose dependent manner. When it was administered peritumorally, rhEndostatin exhibited a more potent inhibitory activity. Furthermore, rhEndostatin displayed an additive effect with CTX or DDP on the inhibition of metastasis of B16 tumors or growth of A549 tumors.</p><p>Conclusion</p><p>Soluble rhEndostatin exhibits a potent anti-tumor activity in mouse xenograft models and it also has an additive effect with CTX and DDP, implying possible applications in clinical settings.</p></div

HPV16 E6 forms complex with ING4 independent of p53.

<p>(A) Lysates from HPV negative cervical carcinoma cell line C33A and two HPV16 positive cell lines CaSki and SiHa were subjected to IP with HPV16 E6 specific antibody C1P5 and detected by Western blotting (WB) for the indicated proteins. (B) Saos2 cells were co-transfected with Flag-tagged E6 and PCDNA-ING4, balanced with empty vector. The cell lydates were subjected to IP with ING4 specific antibody and detected by WB. (C) and (D) Either GST control, GST-ING4 full length or truncates beads were incubated with HPV16 E6 in vitro translated protein with ³⁵S-radiolabeled. 5% of in vitro translated protein input was used as a comparison. Precipitated proteins were resolved by SDS-PAGE, exposed to phosphorimager screen and scanned by Typhoon 9410 imaging system. Coomassie blue staining of SDS-PAGE-resolved purified GST and GST-ING4 proteins was shown under the panel. (E) Colocalization of ectopically expressed ING4 and HPV16 E6. Saos2 cells plated on coverslips were transfected with Flag-E6 and PCDNA-ING4 using Lipofectamine 2000. (F) Colocalization of endogenous HPV16 E6 and ING4 in CaSki cells. All panels are representative pictures from approximately 50 cells of five different fields of three independent experiments.</p

HPV16 E6 suppresses the ING4 mediated p53 transcriptional activity and apoptosis.

<p>(C) U2OS cells were respectively transfected with siRNA Luciferase or siRNA E6AP, with ING4 or ING4+E6 using the Lipofectamin 2000. Cells were collected at 36 h posttransfection after a 12-h serum starvation and fixed. Levels of cells undergoing apoptosis (sub-G1 phase) in individual PI-stained samples were analyzed by flow cytometry, and the data were analyzed by FlowJo software. The bar diagram represents the mean of three independent experiments. Western blot showing the protein level of E6AP in the lentivirus-mediated E6AP of control knockdown cell lines. GAPDH was used as the loading control.</p

HPV16 E6 binds to ING4 and hinders its association with p53.

<p>(A) HPV16 E6 does not affect ING4 stability. p53^−/−Mdm2^−/−MEF cells were transfected with ING4 and an increasing amount of either Flag-HPV16 E6 or control vector. The levels of ING4, HPV16 E6 and GAPDH were examined by Western blot. (B) HPV16 E6, ING4 and p53 bind each other. Flag-E6, PCDNA-ING4, and Myc-p53 were transfected into p53^−/−Mdm2^−/−MEF cells. Cell lysates were immunoprecipitated with EzviewTM Red anti-Flag M2 Affinity Gel (lanes 3 and 4), Flag-E6 and the associated proteins were eluted with 3XFlag peptide. Twenty percent of the eluent was subject to Western analysis using indicated antibodies. The remaining eluent was used for secondary immunoprecipitation with anti-myc antibody (lanes 5and 6). (C) Saos2 cells were transfected with PCDNA-ING4, Myc-p53, and either increasing amounts of HPV16 E6 or the vector control. Transfected cells were treated with MG132 for 6 h. The association of PCDNA-ING4 and Myc-p53 was analyzed by immunoprecipitation assay with anti-p53 antibody. (D) Knockdown of HPV16 E6 increases the ING4-p53 interaction. CaSki and SiHa cells with lentivirus-delivered GFP labeled shRNA against HPV16 E6 (shE6) or scramble control (shC) were shown on the left panel. On the other side, the top panel showed the whole cell lysates were immunoprecipitated with anti-p53 antibody and checked ING4 expression by Western blot. Input equivalent to 10% of the whole cell lysates used for immunoprecipitation was subjected to Western blot using the indicated antibody. (E) ING4 mediated p53 acetylation was attenuated by HPV16 E6. Saos2 cells were transfected with PCDNA-ING4, Myc-p53, in the present of either HPV16 E6 or its mutant L50G. At 24 hr post transfection, trichostatin A were added for additional 6 hours, immunoprecipitation and western blot showed that HPV16 E6L50G attenuated binding and acetylation induced by ING4 on p53 without p53 degradation mediated by E6AP. (F) HPV16 E6 attenuates the binding affinity between ING4 and p53 in vitro. Myc-tagged p53 was incubated with bacterially-expressed GST or GST-ING4 in the presence of Flag-tagged HPV16 E6 or control vector for GST-pull down assay. Concentration of each fusion protein used in GST-pull down assays was kept the same. The level of myc-p53 pulled down by GST-ING4 was less in the presence of HPV16 E6.</p

Inhibition of human tumor growth by administrating rhEndostatin intravenously.

<p>Hepatocellular carcinoma QGY-7703(A), A549 lung carcinoma(B), or Bcap37 breast cancer cells(C), were implanted to nude mice at the dosage of 6×10⁶ cells each, subcutaneously. Tumor-bearing mice were treated with either DDP (2 mg/kg/day) for successively 7 days(•), or rhEndostatin 10 mg/kg/day(▴), rhEndostatin 5 mg/kg/day(▾), and rhEndostatin 2.5 mg/kg/day(□) for successively 15 days. PBS(▪) was injected as the vehicle control. Results were presented as mean±SD (n = 10, each). Mice administered with rhEndostatin i.v. exhibited a significant reduction in tumor volume, compared with that in vehicle control group.</p

Additive effect of rhEndostatin with CTX on the metastasis of B16 melanoma.

<p>Nude mice (n = 10, each) were inoculated with 2.5×10⁵ tumor cells intravenously to establish a metastasis model. Mice were either administered i.v. with rhEndostatin alone at 10 mg/kg/day, 5 mg/kg/day, or 2.5 mg/kg/day for 15 days successively, or combined treated with CTX at the dosage of 15 mg/kg/d for successively 7 days, then intravenously administered with rhEndostatin at 10 mg/kg/day, 5 mg/kg/day, or 2.5 mg/kg/day for successively 15 days. The mice were sacrificed to count the metastasis colony in the lung on day 25 post inoculation. The formula, [(mean colony of PBS control- mean colony of treated mice)/mean colony of PBS control]×100, was used to calculate the inhibition rate of metastasis. #: <i>P</i><0.05 vs mice administered with single rhEndostatin treatment at the same dose, respectively; *: <i>P</i><0.05 vs mice treated with CTX alone, at the dosage of 15 mg/kg/day.</p

<i>In vivo</i> inhibition of angiogenesis using chicken embryo CAMs.

<p>Treated with rhEndostatin at 6 µg/disc (<b>A</b>), or treated with saline vehicle control disc (<b>B</b>) the vessels numbers were calculated(n = 8, each) in CAMs assay after 10 days treatment, and data were presented as mean±SD. <b>#</b> indicated <i>P</i><0.05. (<b>C</b>).</p