Human papillomavirus (HPV) and Epstein Barr virus (EBV) have been found in breast carcinomas (BCs) around the world. In this study, fifty-five BCs from Chile were analyzed for HPV and EBV presence. In addition, HPV-16 viral load/physical status and E6/E7 expressions were determined.The amplification of a housekeeping gene showed that 46/55 samples (84%) had amplifiable DNA. HPV-16 was detected in 4/46 BCs (8.7%) and EBV was detected in 3/46 (6.5%) BCs. The analysis of HPV-16 physical status showed that this virus was integrated in all of the tumors with a relatively low viral load (range: 0.14 to 33.8 copies/cell). E6 and E7 transcripts, however, were not detected in any HPV-16 positive specimens. Using a Cox-regression model, we found a statistically significant association between EBV presence and poor survival (p = 0.013).The findings in this study suggest that it is unlikely that HPV and/or EBV play a direct role in the etiology of BC. Breast cancer (BC) is a leading cause of death in women around the world. In Chile, BC is the second cause of death by cancer with a mortality rate of 11.0/100.000 habitants . Thus, eIn this study, HPV and EBV sequences were searched in BCs from patients living in Santiago of Chile. Fifty-five formalin-fixed paraffin-embedded BCs from different public and private hospitals were collected and the extracted DNAs analyzed for integrity by amplifying a 110 bp fragment of the betaglobin gene. Forty-six samples out of 55 were betaglobin positive (84%). Using conventional PCR for a 65 bp fragment of L1 region, HPV was detected in 4/46 (8.7%) BCs. Using qRT-PCR for EBV EBNA-1, EBV was present in 3/46 (6.5%) BCs. Co-infection with both viruses was only detected in 1/46 2.1%) cases. In addition, ISH for EBER-1 showed that all of the BCs were EBV negative. A non-statistically significant association between HPV presence and age, tumor size, histology and differentiation grade was found  BCs [Other authors have analyzed HPV in BC specimens, suggesting a causal association. Hennig et al. proposed that HPV can be transported by circulation (bloodstream/lymphatic system) from cervix to the breast . In addi.6%) BCs . On the .6%) BCs .INK4a by a negative feedback mechanism [The loss of E2 gene function by HPV integration is known to be one of the major genetic events facilitating transformation and transition to malignancy . The E2 echanism . High-riechanism . The anaechanism . So, wheEven though, HPV-16 was present and integrated in BCs, we were unable to detect E6 and E7 transcript expressions in the specimens. The successful amplification of a fragment of β2-microglobulin was used as control of cDNA preparation and amplification from paraffin-embedded tissues. It has been reported that in general HPV load in extragenital tumors is heterogeneous and lower that in cervical cancer ,24,25 soOn the other hand, there is a relative consensus that for detection of EBV latent infection, the "gold standard" method is ISH for EBER-1 . InteresIn conclusion, we reported here that HPV and EBV prevalence in breast cancer from Chile is relatively low, thus a possible direct etiological role of these virus is unlikely. However, additional studies are warranted to elucidate the function of HPV/EBV in a subset of breast carcinomas from Chile.Fifty-five BCs from patients of different Hospitals in Santiago of Chile were considered for this study. The Hospitals were Las Condes Clinic; San Juan de Dios Hospital; Instituto Nacional del Cáncer; San Borja Arriaran Hospital; José Joaquín Aguirre Hospital; Arturo Lopez Perez Foundation and Santa María Clinic. The average age of the patients was 58.7 years (range: 48.1-69.3). The histological types of BC were: 48 ductal, 6 lobulillar and 1 mucinous. In the present study, histological classification was made using the guidelines of the Japan Cancer Society , which f-1 of proteinase K  and incubated overnight at 56°C. The solution was heated at 100°C for 10 min and subsequently the DNA was purified by phenol-chloroform extraction and precipitated with cold ethanol. DNA quality was tested by PCR for betaglobin using PCO3 5'-ACACAACTGTGTTCACTAGC-3' and PCO4 5'-CAACTTCATCCACGTTCACC-3' primers under the following PCR conditions: denaturation at 95°C for 15 min, 40 cycles with the cycling profile of 95°C for 1 min, 52°C for 1 min, 72°C for 1 min and final extension at 72°C for 5 min. To detect HPV genomes, broad-spectrum SPF10-biotinylated primers were used. The PCR products, 65 bp of the L1 gene, were characterized on a 4% agarose gel and visualized under UV radiation with ethidium bromide staining. HPV genotyping was performed using the Inno-Lipa HPV Genotyping V2 test  [Sections of 10 μm thickness were cut and collected in sterile tubes. For DNA extraction, each sample was treated with 1 mL of xylene and subsequently washed with 1 mL of ethanol. The sediment was resuspended in 50 mM Tris-Cl pH 8.0, 1 mM EDTA, pH 8.0, and 0.5% Tween 20 containing 200 μgmLBelgium) . As negaTo determine the presence, physical status and viral load of HPV-16, real-time PCR was performed with the ABI Prism 7700 Sequence Detection System  and 2 × QuantiTect SYBR Green PCR kit . The HPV-16 primers for E6 ORF amplification were as follows: E6F: 5'-GAGAAACTGCAATGTTTCAGGACC-3' and E6R: 5'-TGTATAGTTGTTTGCAGCTCTGTGC-3'. The HPV-16 primers for E2 ORF amplification were as follows: E2F: 5'-AACGAAGTATCCTCTCCTGAAATTATTAG-3' and E2R: 5'-CCAAGGCGACGGCTTTG-3'. The PCR conditions were 2 min at 50°C, 15 min at 95°C, and a two-step cycle of 95°C for 15 s, and 60°C for 60 s for a total of 40 cycles . The siz2. The conditions of amplification were: denaturation at 95°C for 10 min, 45 cycles of 95°C for 15 s, 55°C for 20 s, 72°C for 20 s and a final extension at 72°C for 20 s. The sequence of primers was: E6F: 5'-CAACAAACCGTTGTGTGAT-3'; E6R: 5'-CGTGTTCTTGATGATCTGC-3'. A melting curve analysis between 62°C to 95°C with a variation of 0.5°C was made. The melting temperature (Tm) of the amplification product from a positive control was 79.85°C. For E7, the reaction mixture was composed by 1X SensiMix, 0.8 μM primers and 3 mM MgCl2. The conditions of amplification were initial denaturation at 95°C for 10 min followed by 45 cycles consisting of denaturation at 95°C for 15 s, annealing at 57°C for 20 s and extension at 72°C for 20 s. The sequence of primers was E7F: ATGCATGGAGATACACCTAC; E7R: 5'-CATTAACAGGTCTTCCAAAG-3'. The amplification of β2-microbulin gene was carrying out as follow: 0.8 μM Primer F; 0.8 μM primer R, 5 mM MgCl2, 1X Sensimix and 50 ng cDNA. The amplification program was initial denaturation at 95°C for 10 min, followed by 45 cycles consisting of denaturation at 95°C for 15 s, annealing at 55°C for 20 s and extension at 72°C for 20 s. The sequence of primers was: B2MF: 5'-TGTAAGCAGCATCATGGA-3' and B2MR: 5'-AGTGTAAGTGTATAAGCATATCAA-3'. The melting point of the amplified product was 81.15°C. All the amplifications were made using Rotor Gene 6000 (Corbett Research) real-time PCR equipment.The RNA purification was carrying out using the High Pure RNA paraffin kit (Roche), according to instructions of the manufacturer. The obtained RNA was resuspended in 50 μL of TE  and stored at -80°C until use. The cDNA preparation was made as follows: 100 ng of purified RNA were transformed to cDNA using RNAsin 1 U/μL ; 1× buffer TR ; 10 μg/μL random primers ; 20 U/μL MMLV  and 2 mM dNTPs in a final volume of 20 μL. The reaction mixture was incubated at 37°C for 1 h, 70°C for 15 min and stored at -20°C. Ten microliters of cDNA were used for qRT-PCR. The amplification reaction was carrying out separately for HPV-16 E6 and E7 transcripts using the SensimixSYBR (Bioline) kit in a final volume of 25 μL. For E6, the reaction mixture was composed by 1X SensiMix, 0.8 μM primers and 5 mM MgClFifty-seven specimens of BC were used to construct a tissue array as previously described . The tumIn situ hybridization (ISH) with a complementary digoxigenin-labeled 30-base probe was used to detect EBER-1 expression according to the procedure previously published . BrieflyFisher's exact test was used for determine differences between groups. P-value < 0.05 was considered statistically significant. For survival analysis, a Cox-regression model was used.The authors declare that they have no competing interests.FA, SAk, AC and CK conceived of the study, analyzed the data and participated in the redaction of the manuscript. CG and NK made the analysis of the clinical specimens and analyzed the data. AC and LS got specimens for analysis, gave clinical information, and gave clinicopathologic support. OP gave statistical support and analysis. YE, CK and SAm gave analytical support for analysis. All authors read and approved the final manuscript.


Interestingly, OEOA also enhances erythroid differentiation in K562 cells through suppressing the expression of Bcr-Abl and phosphorylation of Erk1/2. These findings identify a novel chemical entity for further development as therapeutics against leukemia.Oleanolic acid (3β-hydroxy-olea-12-en-28-oic acid) is a natural pentacyclic triterpenoic acid found in many fruits, herbs and medicinal plants. In the past decade, increasing evidence has suggested that oleanolic acid exhibits inhibitory activities against different types of cancer including skin cancer and colon cancer, but not leukemia. We report here that a derivative of oleanolic acid, olean-12-eno  Leukemia is a malignant disease which broadly covers a number of cancers of the blood, bone marrow, and lymphoid systems. Based on how the disease develops (acute or chronic) and the blood cells affected (lymphocytes or myelocytes), they are categorized into four main types, i.e. acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelocytic leukemia (AML), and chronic myelocytic leukemia (CML) While leukemia affects both adults and children of both genders with most cases (>90%) diagnosed in adults, leukemia is the most common form of cancer in children and adolescents, accounting for about one third of cancers in individuals aged under 20. The United States National Cancer Institute estimates that the number of new cases is 47,150 in 2012 and about 23,540 people will die from leukemia this year Despite substantial progress in current and emerging treatment strategies, short remission duration has been reported Oleanolic acid  is a natural pentacyclic triterpenoic acid 1H-NMR, 13C-NMR and mass spectrometry as illustrated in Oleanolic acid (OA) was purchased from Sigma-Aldrich . OEOA was synthesized from OA as previously described 2 in humidified air. Human neonatal keratinocytes (HEKneo) were purchased from Invitrogen. HEKneo cells were cultured using Epilife with 1% HKGS in a T75 flask . The cultures were maintained at 37°C in a humidified incubator with 5% CO2, following the manufacturer’s protocol.Human erytholeukemia cell lines, K562 and HEL and a T-cell leukemia cell line, Jurket  were maintained in RPMI 1640 medium  supplemented with 10% fetal bovine serum . HepG2 (hepatocellular liver carcinoma), MCF-7 (breast adenocarcinoma), and HeLa  cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS. The cell cultures were incubated at 37°C with 5% CO4 per well and then incubated overnight at 37°C with 5% CO2. Following treatment of cells with OEOA or DMSO (both diluted in culture medium) for 48 h, MTT  assay was performed to measure the cell viability . To examine the cytotoxic effect of OEOA, K562 and HEL cells were seeded onto a 6-well plate at an initial cell density of 1×105 per well. Following incubation at 37°C with 5% CO2 overnight, cells were then treated with OEOA (1 µM) or DMSO for 6 days. Viable cells were counted daily after being stained with 0.4% trypan blue (Sigma Aldrich).Cells were seeded on a 96-well plate at the density of 3×105 per well in a 6-well plate) were treated with OEOA for 24 h. Cells were harvested and washed twice with ice cold PBS with 1% calf serum (Invitrogen). The cell pellets were then resuspended in 100 µl of PBS with 1% calf serum, fixed with 80% ethanol at 4°C for 1 h, resuspended and incubated in 500 µl of Tris-EDTA solution containing 40 µg/ml propidium iodide (PI) and 40 µg/ml of RNaseA at 37°C for 30 min K562 cells (1×10http://rsbweb.nih.gov/ij/).Following treatment with OEOA, cells were harvested and lysed with RIPA buffer containing 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 0.5% deoxycholic acid, 2 µg/ml aprotinin, 1 mM PMSF, 5 mM benzamidine, 1 mM sodium orthovanadate and 10 µg/ml soybean trypsin inhibitor in 50 mM Tris buffer, pH 7.4. Protein concentration was determined by Bio-Rad protein assay . Equal amounts of proteins were resolved by SDS electrophoresis, and then transferred to nitrocellulose membranes for immunoblotting. Primary antibodies against phospho Ser807/811-retinoblastoma protein (p-Rb), phospho-Erk1/2 (p-Erk1/2), Rb, Erk1/2, secondary antibodies  were purchased from Cell Signaling Technology , and an antibody against actin was from Sigma-Aldrich. Antibodies specific for p27, Cyclin D1, Cyclin E, Cdk4, Cdk6 and c-Abl were obtained from Santa Cruz Biotechnology . The polyclonal primary antibody against retinoic acid-regulated nuclear matrix-associated protein (RAMP) was generated as described previously γ-globin forward primer: 5′-TGGCAAGAAGGTGCTGACTTC-3′; γ-globin reverse primer: 5′-TCACTCAGCTGGGCAAAGG-3′cd41b forward primer 5′-GCTGCAGATGGACGCAGCCA-3′; cd41b reverse primer 5′-GCATGTAGTGGGCGCCCTGG-3′. In each experiment, human hypoxanthine phosphoribosyltransferase 1 . Five microgram total RNA of each sample was reverse-transcribed using oligo (dT) primers and SuperScript II reverse transcriptase (Invitrogen) in 20-µl volume. Quantification of the target genes was performed with Power SYBR Green PCR master mix kit in 7500 Fast-real time PCR system, according to the manufacturer’s instructions . The specificity of the SYBR Green PCR signal was confirmed by melting curve analysis. Following primer sequences were used: 5 in a T25 flask) were treated with 1 µM of OA or OEOA, and cell pellets were collected by centrifugation. The culture medium (3 ml) was aspirated and extracted with equal volume of ethyl acetate for two times (30 min each). After combining the ethyl acetate extracts, the samples were vacuum-dried and dissolved in methanol for detection. Cell pellets were washed with cold phosphate-buffered saline and lysed in RIPA buffer. The cell lysates were dried in vacuum and dissolved in methanol for detection. Area under peak (AUC) was obtained and percentage of compounds in cell lysates was calculated as: % of the compound (cell lysate) = AUC (cell lysate)/AUC (medium+cell lysate) x 100%. The remaining samples were then loaded onto the preparative HPLC and the corresponding fractions of OA or OEOA were collected and dried. They were dissolved with methanol-d6 in NMR tube, and then analyzed in the Varian 300 NMR to confirm the identity of compound.To study the cellular permeability of OEOA and OA in K562 cells, culture medium and cell lysates were collected at different time intervals after incubation. K562 cells  were obtained from Animal Care Facility in the Hong Kong University of Science and Technology (HKUST). OA and OEOA were dissolved in a solution of 3% dimethylacetamide/10% Tween-80 in water (3∶10∶87 by vol.) and administered intraperitoneally to the mice at 20 µmol/kg (injection volume 10 ml/kg). The procedures in this study were approved by HKUST Animal Ethics Committee and conducted in accordance with the Code of Practice and Use of Animals for Experimental Purpose. Terminal blood  was collected at indicated time points after OA and OEOA administration. Plasma was obtained by centrifuging the blood at 3,800×g for 10 min at 4°C followed by extraction with ethyl acetate twice and pooled extracts were evaporated to dryness. The sample was then dissolved with 0.1 ml methanol, vortexed for 30 s followed by ultrasonication for 5 min and centrifuged at 17,900×g for 5 min. The supernatant was transferred into vial for high performance liquid chromatography tandem mass spectrometry analysis (HPLC-MS/MS).To examine the concentrations of OEOA and OA OA and OEOA were analyzed by an Agilent 1200 series HPLC system coupled to an AB SCIEX 4000+ triple quadrupole system equipped with a turbo V source with an electrospray ionization (ESI) probe. The compounds in methanol were separated on a C18 column  with the mobile phase consisted of (A) 0.1% formic acid in water and (B) acetonitrile. Five microliters of sample was injected and eluted by the following program at the flow rate of 0.5 ml/min: 0–7 min, 70∼100% B; 7–10 min, 100% B; 10–10.1 min, 100∼70% B; 10.1–15 min, 80% B. Detection of OA and OEOA was carried out in negative ionization mode. The ESI conditions were as follows: declustering potential −50V, entrance potential −10V, collision cell exit potential −15V, collision energies −40 V, curtain gas 20 (arbitrary units), collision gas 5 (arbitrary units), ion spray voltage −4500 kV, source temperature 350°C, ion source gas 1∶40 (arbitrary units), ion source gas 2∶10 (arbitrary units). Multiple-reaction-monitoring was used to measure OEOA and OA in samples. Standard solutions of OA and OEOA (0.2 to 20 µM) were prepared in methanol and added to mouse plasma.t-test. Results were considered statistically significant with p-value <0.05.Data are expressed as mean ± SEM of three independent experiments and analyzed by Student’s 50 = 0.78±0.037 µM) and HEL cells (IC50 = 0.21±0.041 µM) in a dose-dependent manner, whereas OA showed minimal inhibitory effect on the cell growth of these two cell lines (50 = 0.29±0.025 µM). Importantly, OEOA did not suppress the growth of primary human neonatal keratinocytes (HEKneo) at a concentration up to 10 µM  using HPLC-MS/MS analysis. Quantitative analysis indicated that both OA and OEOA were readily detected in plasma at 30 min after injection (0.5t was 4.25±1.277 and 5.51±0.938 µM, respectively. While the concentration of OA at 1t and 4t was 0.92±0.416 µM and 0.02±0.004 µM, OEOA remained at high level, 3.10±0.646 µM and 0.65±0.099 µM, respectively. At 8 h after administration, while the concentration of OA was below detection limit, the concentration of OEOA was 0.29±0.001 µM, suggesting that OEOA is retained in plasma for a longer period than OA.We next investigated whether the structural modification in OEOA altered the pharmacokinetic profile of the compound when compared with OA. First, we have developed the detection parameters of OEOA using HPLC-MS/MS analysis. Under the collision energy −70 V, the ion mass spectrum of OEOA was measured at nd 307.1 . Similar/z 455.3 . To inve/z 455.3 . Interes/z 455.3 . OEOA annjection . The conOA and its derivatives have been shown to suppress the initiation and growth, as well as to induce differentiation and apoptosis of various tumors In the present study, we provide molecular evidence to show that treatment of K562 cells with OEOA triggers cell cycle arrest in G1 phase. Mechanistically, we demonstrate that OEOA treatment significantly attenuated the expression levels of Cyclin D1, Cdk4 and Cdk6 in K562 cells, resulting in reduced phosphorylation of Rb and Cyclin E and accumulation of p27. In early G1 phase, Cyclin D binds to Cdk4 and/or Cdk6 to form cyclin-Cdk complexes, resulting in the activation of Cdks. Phosphorylation of Rb protein by the Cyclin D-Cdk4/6 complex in turn allows the expression of other cell cycle genes, such as Cyclin E. Association between Cyclin E and Cdk2 then leads to the phosphorylation and degradation of p27, allowing the transition from G1 to S phase Given that K562 cells are of CML origin, we also evaluated the potential effects of OEOA on CML by examining the expression of Bcr-Abl in K562 cells. The chimeric Bcr-Abl oncoprotein possesses a constitutive tyrosine kinase activity, which drives CML pathogenesis γ-globin and inhibiting Bcr-Abl expression and Erk1/2 activation in K562 cells. The beneficial role of OEOA on erythroid differentiation, especially to CML patients, may shed light on future leukemia drug development exploiting the differentiation approach.Differentiation therapy has become one of the new strategies for treating leukemia, which induces maturation and eventual senescence of cancer stem cells, instead of killing them through cytotoxicity In conclusion, our findings provide the first demonstration that a derivative of oleanolic acid, OEOA acts through unique mechanisms to inhibit leukemia cell proliferation by inducing G1 arrest and erythroid differentiation. Although further studies are required to determine the underlying target for its action, our results suggest OEOA may be a promising lead compound for leukemia treatment.


Powder X-ray diffraction patterns indicate that the LnxLn′xSbx2−2Se3 Ln: Lu3+/Yb3+ and Lu3+/Er3+ crystals (x = 0.00 − 0.04) are isostructural with Sb2Se3. The cell parameters were increased for compounds upon increasing the dopant content (x). Scanning electron microscopy and transmission electron microscopy images show that co-doping of Lu3+/Yb3+ ions in the lattice of Sb2Se3 produces nanorods, while that in Lu3+/Er3+ produces nanoparticles, respectively. The electrical conductivity of co-doped Sb2Se3 is higher than that of the pure Sb2Se3 and increases with temperature. By increasing the concentration of Ln3+ions, the absorption spectrum of Sb2Se3 shows red shifts and some intensity changes. In addition to the characteristic red emission peaks of Sb2Se3, emission spectra of co-doped materials show other emission bands originating from f-f transitions of the Yb3+ ions.Lu V2VI3 compounds, is a layer-structured semiconductor of orthorhombic crystal structure and exhibits good photovoltaic properties and high thermoelectric power, which allows possible applications for optical and thermoelectronic cooling devices [2Se3 is interesting both for basic and applied research. Doping of some transition metal and lanthanide to the lattice of metal chalcogenides has been investigated [3+/Yb3+ and Lu3+/Er3+ co-doped antimony selenide via co-reduction method at hydrothermal condition.Nanosized semiconductor materials have drawn much research attention because their physical and chemical properties, due to size quantization effect, dramatically change and, in most case, are improved as compared with their bulk counterparts -3. Rare  devices -11. The stigated -20. The 3  and Ln2O3   based on the molecular formula LnxLn′xSbx2−2Se3 (0 ≤ x ≤ 0.04) were added, and the mixture was transferred to a 100-mL Teflon-lined autoclave. The autoclave was sealed, maintained at 180°C for 48 h, and then cooled to room temperature. The optimum conditions for this reaction are pH = 12, temperature = 180°C, and reaction time = 48 h. The black precipitate obtained was filtered and washed with ethanol and water. It was dried at room temperature. Yields for the products were 75% to 85%. Phase identification was performed by powder X-ray diffraction  with Cu Kα radiation. Cell parameters were calculated using the Celref program  from powder XRD patterns, and reflections have been determined and fitted using a profile fitting procedure with the WinXPOW program . The reflections observed in 2θ = 4° to 70° were used for the lattice parameter determination. The morphology of materials was examined by scanning electron microscopy . A linked ISIS-300 Oxford EDS detector  was used for elemental analyses. The high-resolution transmission electron microscopy (HRTEM) image and selected area electron diffraction (SAED) pattern were recorded by a Cs-corrected HRTEM  operated at 200 kV. Photoluminescence measurements were carried out using a Spex FluoroMax3 spectrometer  after dispersing a trace amount of sample via ultrasound in distilled water. Four-point probe method was used for the measurement of electrical and thermoelectrical resistivity of samples. A small oven was needed for the variation of temperature of the samples from the room temperature to about 200°C (maximum). A small chip with 1-mm thickness and 7-mm length was used for this analysis.All chemicals were of analytical grade and were used without further purification. Gray selenium (1 mmol) and NaOH (5 mmol) were added to distilled water (60 mL) and stirred well for 10 min at room temperature. Afterwards, hydrazinium hydroxide , SbClxYbxSbx2−2Se3 samples indicate that the Lu3+/Yb3+ co-doped antimony selenide has the same orthorhombic structure as Sb2Se3 and that single-phase Sb2Se3 is retained at lower doping concentrations of Lu3+/Yb3+. All the peaks in Figure 2Se3 with Pbnm space group and lattice parameters a = 11.62 Å, b = 11.76 Å, and c = 3.95 Å (JCPDS card file 72–1184). For doping levels higher than x = 0.04 for Lu3+ and Yb3+, additional unknown phases were observed , the lattice parameters were increased for these materials, as shown in Figure 2Se3 compounds [The cell parameters of the synthesized materials were calculated from the XRD patterns. With increasing dopant content  luminescence. One can be observed only upon direct excitation of the dopant. The other type is obtained if energy transfer from host to dopant occurs. Binary compounds such as Sbgeration . The fou2Se3 was of the order of 0.2 Ω·m; in the case of Lu0.04Yb0.04Sb1.92Se3, the minimum value of electrical resistivity is 0.009 Ω·m, and for Lu0.04Er0.04Sb1.92Se3, it is 0.032 Ω·m. With the increase in lanthanide concentration, the electrical resistivity of synthesized nanomaterials decreased obviously  are important reasons for decreasing electrical resistivity. The obtained data shows higher electrical resistivity for co-doped samples in comparison with doped samples in the case of Lu3+, Yb3+ and Er3+ doped Sb2Se3[3+/Yb3+-doped Sb2Se3 has the higher electrical conductivity.The temperature dependence of the electrical resistivity for co-doped Sbed Sb2Se3,17. The ed Sb2Se3-20. Comp0.04Yb0.04Sb1.92Se3 are shown in Figure 2Se3 and Lu3+ ions (in the visible domain) and Yb3+ ions in the near-IR domain. By increasing the concentration of Ln3+ ions, the absorption spectrum of Sb2Se3 shows red shifts and some intensity changes  of the Yb3+ ions [UV–vis spectra of Luf Sb2Se3.,22, and b3+ ions .0.04Er0.04Sb1.92Se3, the transition of the Er3+ ions is not observed because of instrument limitation. The peaks between 500 and 620 nm can then be assigned to the lattice of Sb2Se3  are expected around 1.54 μm. These could, however, not be determined due to equipment limitations [2Se3 -based nanomaterials were synthesized by a simple hydrothermal method. The cell parameters were increased for compounds upon increasing the dopant content (x). According to the SEM and TEM images, different morphologies were seen in co-doped Sb2Se3. The HRTEM image and SAED pattern show similar growth [3+/Yb3+ co-doped like Sb2Se3 nanorods. Lanthanide doping promotes the electrical conductivity of Sb2Se3 as well as thermoelectrical conductivity. UV–vis absorption and emission spectroscopy reveals mainly the electronic transitions of the Ln3+ ions in the case of Yb3+-doped nanomaterials.New thermoelectric Lnr growth  directioThe authors declare that they have no competing interests.YH carried out the experiments and drafted the manuscript. SWJ directed the study and provided the analyses. BM carried out the experimental analysis. All authors read and approved the final manuscript.XRD patterns of LuxErxSb2−2xSe3, TEM, HRTEM images, SAED pattern of Sb2Se3 nanorods, absorption spectra of Lu0.02Yb0.02Sb1.96Se3, Lu0.01Yb0.01Sb1.98Se3, and Lu0.02Er0.02Sb1.96Se3 are provided. Figure S1. Powder X-ray diffraction pattern of LuxErxSbx2−Se3 (x = 0.02). Figure S2. Powder X-ray diffraction pattern of LuxErxSbx2−Se3 (x = 0.04). Figure S3. Powder X-ray diffraction pattern of unknown LuxErxSbx2−Se3 phase. Figure S4. TEM image of Sb2Se3 nanorods. Figure S5. HRTEM image of the Sb2Se3 nanorods. Figure S6. SAED Pattern of the Sb2Se3 nanorods. The SAED zone axis is [Figure S7. Absorption spectra of Lu0.02Yb0.02Sb1.96Se3 nanorods at room temperature. Figure S8. Absorption spectra of Lu0.01Yb0.01Sb1.98Se3 nanorods at room temperature. Figure S9. Absorption spectra of Lu0.02Er0.02Sb1.96Se3 nanoparticles at room temperature. (DOC 3322 kb)Click here for file


DNA repair is a cellular defence mechanism responding to DNA damage caused in large part by oxidative stress. There is a controversy with regard to the effect of red blood cells on DNA damage and cellular response.2O2-induced DNA damage and repair in human peripheral blood mononuclear cells.To investigate the effect of red blood cells on H2O2 in the absence or presence of red blood cells, red blood cells hemolysate or hemoglobin. DNA repair was measured by 3H-thymidine uptake, % double-stranded DNA was measured by fluorometric assay of DNA unwinding. DNA damage was measured by the comet assay and by the detection of histone H2AX phosphorylation.DNA breaks were induced in peripheral blood mononuclear cells by HRed blood cells and red blood cells hemolysate reduced DNA repair in a dose-dependent manner. Red blood cells hemolysate reduced % double-stranded DNA, DNA damage and phosphorylation of histone H2AX. Hemoglobin had the same effect as red blood cells hemolysate on % double-stranded DNA.Red blood cells, via red blood cells hemolysate and hemoglobin, reduced the effect of oxidative stress on peripheral blood mononuclear cell DNA damage and phosphorylation of histone H2AX. Consequently, recruitment of DNA repair proteins diminished with reduction of DNA repair. This suggests that anemia predisposes to increased oxidative stress induced DNA damage, while a higher hemoglobin level provides protection against oxidative-stress-induced DNA damage. H2O2 induces double strand breaks (DSBs), which are followed by DNA repair 2O2-induced DNA repair measured in peripheral blood mononuclear cells (PBMC) has been previously used in our laboratory The DNA repair system is a cellular defence mechanism responding to DNA damage caused in large part by oxidative stress. The DNA damaging agents are either external, such as pollution and exposure to sunshine or other types of irradiation, or internal such as a consequence of replication or metabolic pathways that produce reactive oxygen species (ROS). One of the ROS which causes DNA damage is H2O2It has been observed that red blood cells (RBC) may affect the DNA damage and repair system. The active substance may be either the whole hemoglobin molecule or the ferrum component or other metabolites, such as adenosine phosphate, which exist in RBC. The published effects of whole RBC or RBC hemolysate are contradictive: some investigators did not find any association between DNA damage and hemoglobin 2O2; (2) to determine the effect of RBC hemolysate and hemoglobin on % double-stranded DNA as a measure for DNA damage by H2O2. A higher level of % double-stranded DNA indicates less DNA breakage; (3) to assess the effect of RBC hemolysate on H2O2-induced phosphorylation of histone H2AX in PBMC; (4) to evaluate the effect of RBC hemolysate on H2O2-induced DNA breaks in PBMC by the comet assay.The objectives of our study were: (1) to measure the effect of autologous RBC and RBC hemolysate on DNA repair ability of PBMC stimulated in vitro by HThe study was approved by the Rabin Medical Center Institutional Review Board. The whole study was conducted in our center in Petah Tikva, Israel. We did not study humans, but used blood cells, which were purchased from the local blood bank. Hence the Review Board waived the need for informed consent.Buffycoats of fresh blood from apparently healthy donors were obtained from the local blood bank. The buffycoat, which was enriched with white blood cells (WBC) but contained also RBC, was divided into two portions.Cells were separated from one portion by histopaque gradient centrifugation. In order to eliminate RBC contamination, the PBMC underwent a procedure of RBC hemolysation as follows: cell pellet was thoroughly mixed with 10.4 ml 0.24% NaCl hypotonic solution for 20 seconds, followed by 0.88 ml 8.7% NaCl hypertonic solution and diluted to 50 ml with PBS. After centrifugation this procedure was repeated once more. PBMC were suspended in RPMI-1640 complete medium and counted in a Neubauer chamber. DNA repair ability was measured at once. Cells for ds-DNA measurements, phosphorylation of histone h2AX and the comet assay were kept overnight at 4°C in complete RPMI medium and were processed the next morning.v/v per reaction. As the hemolysate was always prepared exactly by the same methods, the range of the hemoglobin level in the hemolysate was small. Thus, 2% v/v RBC hemolysate contained 2.00±0.07 mg hemoglobin per ml reaction medium.The other portion of the buffycoat unit was diluted with PBS and centrifuged at room temperature for 10 minutes at 3000 rpm. Supernatant and the upper layer of white blood cells were removed by suction and the washing procedure of the RBC pellet was repeated twice more until 5 ml of packed red cells were obtained. Five ml of 0.24% NaCl hypotonic solution were added to the packed cells, thoroughly mixed for 1 min and centrifuged for 20 minutes at 3000 rpm. Three ml of clear hemolysate were carefully collected from the upper layer and a sample of it was hematologically analysed. The RBC hemolysate added to the various experiments contained only traces of WBC. Osmolarity of the hemolysate was also measured and crystalline NaCl was dissolved in the hemolysate to reach iso-osmolarity. RBC hemolysate was added to the DNA repair, which was measured the same day, or kept overnight at 4°C and added to ds-DNA measurement, H2AX phosphorylation and the comet assay the next day according to protocols. The RBC hemolysate was added to the reaction mixture at various volumes. The highest concentration of hemolysate was 2% 2O2-stimulated PBMC by the method of 3H-thymidine incorporation, as previously described 6 PBMC per tube in 1.25 ml complete RPMI-1640 medium supplemented with 1% glutamine, 1% antibiotic solution , 1 mmol/L CaCl2, 0.1% bovine serum albumin and 10 mmol/L hydroxyurea (which inhibits scheduled DNA replication). Blank tubes contained no cells. Tubes were kept in an ice bath and then either packed RBCs (3% or 8% v/v) or RBC hemolysate were added to produce a dose-response concentration of 0%, 0.3%, 0.6%, 1% and 2% v/v hemolysate in the reaction mixture. Uniformity of the volume in the reaction tubes was achieved by the addition of saline. Tubes with neither packed RBCs nor RBC hemolysate (0%) were referred to as control. Ten µl of H2O2  were added to enable determination of H2O2-induced DNA repair. The tubes were transferred to a shaking water bath at 37°C for 60 seconds and then to an incubator at 37°C with a humid atmosphere of 5% CO2 for 30 minutes. The tubes were cooled on ice, 10 µl of 1 µCi/ml 3H-thymidine final concentration was added to all the tubes and incubation at 37°C continued for 120 minutes. The tubes were cooled, diluted with cold saline, washed several times with cold saline to remove surplus of radioactivity from the supernatant, and then nuclei-incorporated radioactivity was measured after lysis with NaOH, sedimentation with TCA, filtration through glass fibre filters and washing with plenty of 96% Ethanol. cpm were counted in scintillation fluid. H2O2-induced DNA repairwas expressed as cpm of nuclei-incorporated 3H-thymidine per 2×106 cells, after subtracting cpm of parallel blanks without H2O2.We assessed the effect of both RBC and RBC hemolysate on DNA repair. DNA repair ability was measured in H6 cells/1.06 ml per tube). PBMC were incubated in the presence of increasing concentration of H2O2 as follows, 10, 20, 50, 100 and 200 µmol/L, and ds-DNA was determined. Based on the dose-effect in all the following experiments, we used 200 µmol/L. Ten µl of H2O2 (200 µmol/L end concentration) were added to the reaction mixture, except for tubes in which basal %ds-DNA was measured. ds-DNA was determined in PBMC in the presence of RBC hemolysate, at the following concentrations: 0%, 0.3%, 0.6%, 1% and 2% v/v RBC hemolysate in the reaction, which lasted 40 minutes in a shaking water bath at 37°C, prior to ds-DNA measurement. In some experiments human hemoglobin was added instead of hemolysate at a concentration of 1.8 mg/ml reaction buffer, which is similar to its concentration in 2% v/v hemolysate. After the reaction, FADU was performed as follows: for each hemolysate concentration three conditions were performed in quadruplicates of 0.2 ml of the reaction mixture: total ds-DNA (T), blanks (B) and partial ds-DNA (P), in which T retained natural DNA, in B double strands of DNA were decomposed by sonication and in P a partial unwinding was achieved by the alkaline unwinding condition 2-B2/T2-B2)/(P1-B1/T1-B1), where P2, B2 and T2 are fluorescence counts of P,B and T of the experimental tubes with RBC hemolysate, while P1, B1 and T1 are the fluorescence counts of the control tubes without RBC hemolysate. The value of P1-B1/T1-B1 was constant in every experiment. In all the experiments, the calculated control ds-DNA = 1.00 and the experimental results are expressed as ratios of the control, whereas in the original paper ds-DNA was determined by fluorometric assay of DNA unwinding (FADU) method (12). PBMC which were kept overnight in complete medium were centrifuged at 4°C, suspended in cold myoinositol buffer pH 7.2, and divided between reaction tubes, so that all the tubes contained an equal number of cells  according to the manufacturer’s protocol Following incubation of PBMC with HDNA damage was determined by the comet assay (single-cell gel electrophoresis) using the comet assay kit  according to the manufacturer’s protocol. Electrophoresis was run in an alkaline buffer 3H-thymidine incorporation, 0.5 N HCl was used for neutralisation after hemolysis and 10% TCA solution was used for DNA sedimentation. For ds-DNA analysis human hemoglobin was freshly dissolved before incubation. Myoinositol buffer, 9 M urea, DNA disruption solution, equilibrated NaOH solutions for exact pH control and ethidium bromide solution for fluorescence spectrometry were prepared as instructed in Methods in Enzymology For DNA repair analysis 0.5 N NaOH was used for hemolysis after 3H–thymidine, specific activity 40–60 Ci/mmol, was purchased from Perkin Elmer, Boston, USA. Medium ingredients were purchased from Biological Industries, Beit Ha’emek, Israel. Human hemoglobin was purchased from Sigma, Rehovot, Israel. Scintillation liquid Ultima Gold was purchased from Perkin Elmer, Waltham MA, USA. Other chemicals were purchased from Sigma, Rehovot, Israel.Radioactivity was counted in a LKB 1217 Rackbeta liquid scintillation counter, Wallac, Finland. Fluorescence was measured in disposable cuvettes in a Ratio Master Fluorescence spectrometer, Photon Technology International, USA.Data are presented as mean ± SE. Student’s t test for paired data was used for comparisons, unless otherwise specified. Two tailed p<0.05 was considered significant.2O2 on ds-DNA in PBMC was measured. As illustrated in 2O2 rose from10 µmol/L to 200 µmol/L. Compared to control, 100 and 200 µmol/L of H2O2 reduced %ds-DNA significantly . We did not use a concentration higher than 200 µmol/L, since we had previously observed that a higher concentration affected cell viability.A dose effect of H2O2-induced DNA repair without addition of RBC was 998.9±18.1 (N = 4) and was defined 100%. The addition of 40 µl packed RBC reduced DNA repair by 57.3% to 426.8±39.5 cpm/2×106 .With the addition of 100 µl, RBC DNA repair further decreased by 74.2% to 258.1±24.3 cpm/2×106 cells .H2O2-induced DNA repair in a dose-dependent manner. Tubes with H2O2 but without hemolysate served as a control group and its DNA repair ability was defined 100% (N = 8). Without hemolysate DNA repair ability was 100±8.03%.Addition of various volumes of hemolysate into the reaction medium reduced DNA repair ability as follows: 0.3%, 0.6%, 1% and 2% v/v produced DNA repair ability of 84.6±9.00% (p = NS ), 77.5±5.43% (p = NS), 55.4±5.95% (p<0.005) and 50.9%±6,26% (p<0.005), respectively  between H2O2-induced DNA repair (% of control) and mg hemoglobin in the reaction mixture (mean±SE = 1.90±0.114 mg). In the experiments which contained 1%, 0.6% or 0.3% v/v RBC hemolysate, the correlation coefficient did not reach significance.As hemoglobin concentration was measured in every RBC lysate sample, a correlation between H2O2-induced DNA repair by RBC hemolysate can be attributed to two mechanisms: either hemolysate inhibits the DNA repair reaction or it protects the DNA from damage induced by H2O2 and consequently reduces the need for DNA repair. In order to investigate which of the two possibilities existed in our reactions, the effect of RBC hemolysate on ds-DNA was measured. %ds-DNA was 48.0%±2.40% in PBMC excited by 200 µmol/L H2O2 without hemolysate. In the absence of H2O2, %ds-DNA was 72.1%±3.18%. This difference proved that H2O2 caused DNA damage in our reaction conditions. Without RBC hemolysate, ds-DNA ratio in H2O2-excited cells was 1.00±0.05. With the addition of 0.3%, 0.6%, 1% and 2% v/v RBC hemolysate to the medium, the calculated ds-DNA ratio was 1.40±0.04, 1.55±0.03; 1.62±0.03 and 1.76±0.02, respectively, p<0.001 for all concentrations . Regression line was y = −0.131x +2.432 . This hi2O2. The addition of RBC hemolysate to the reaction with H2O2 prevented the rise in phosphorylation of histone H2AX, which was 1.03±0.27%. The Friedman two way analysis of variance showed a significant difference between all three groups .As illustrated in 2O2 on DNA, we used the comet assay. 2O2 caused DNA breaks, as expected (middle), while the addition of RBC hemolysate prevented the damage to the DNA (right).To ascertain the effect of H2O2-induced DNA breaks. The effect of the RBC hemolysate was dose-dependent. Furthermore, we observed a significant negative correlation between hemoglobin and DNA repair at the concentration of 2% v/v hemolysate. No correlation was seen with lower concentrations of hemolysate.In the present study we found that RBC, RBC hemolysate and hemoglobin reduced DNA repair ability in PBMC following H2O2-induced DNA damage with the appropriate DNA repair response. Indeed, we have previously seen that the rise in DNA damage due to oxidative stress during dialysis was accompanied by a rise in DNA repair 2O2-induced PBMC the ds-DNA increased from a ratio of 1.00 without RBC hemolysate  to a ratio of 1.76±0.02, when 2% v/v RBC hemolysate was present, suggesting that RBC hemolysate inhibited DNA damage by H2O2. Furthermore, there was a significant negative correlation (r = −0.925) between DNA repair and %ds-DNA collectively. These results suggest that the reduction in DNA repair is in large part due to the decrease in DNA breaks in the presence of the RBC hemolysate. These findings were supported by the results of the comet assay and the analysis of the histone H2AX phosphorylation. Reduction in DNA breaks induced by H2O2 with the addition of RBC hemolysate to the reaction was observed by both methods. The phosphorylation of histone H2AX is an early step in recruiting and localising DNA repair proteins to the DSBs sites 2O2 and found a similar effect. Thus, most of the hemolysate effect is probably carried out by the hemoglobin.The reduction in DNA repair by RBC, RBC hemolysate and hemoglobin could be due either to their direct effect on the DNA repair reaction or to a defensive effect against HOur results are in accordance with the studies which found a negative correlation between hemoglobin level and DNA damage DNA damage may lead to cell aging and carcinogenesis


Our results showed that T6FA significantly inhibited auto- and acetylcholinesterase (AChE)-induced aggregation of Aβ1–40in vitro and blocked the cell death induced by Aβ1–40 in PC12 cells. In an AD mouse model by the intracerebroventricular injection of Aβ1–40, T6FA significantly improved the cognitive ability along with increasing choline acetyltransferase and superoxide dismutase activity, decreasing AChE activity and malondialdehyde level. Based on our findings, we conclude that T6FA may be a promising multifunctional drug candidate for AD.We have previously synthesized a series of hybrid compounds by linking ferulic acid to tacrine as multifunctional agents based on the hypotheses that Alzheimer's disease (AD) generates cholinergic deficiency and oxidative stress. Interestingly, we found that they may have potential pharmacological activities for treating AD. Here we report for the first time that tacrine-6-ferulic acid (T6FA), one of these compounds, can prevent amyloid-β peptide (Aβ)-induced AD-associated pathological changes  Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder with progressive and devastating memory impairment 1, 2, in vitro and in vivoTacrine  and 3--2,5- diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (agency in China). 2,7-dichlorofluoroescin diacetate (H2DCF-DA) were purchased from Molecular Probes Co. (agency in China). Tacrine-6-ferulic acid (purity >98%) was synthesized by our laboratory 2DCF-DA was prepared in ethanol. Fresh T6FA (1 mg/mL) was prepared in phosphate-buffered saline. The cells were pre-incubated with 2–50 µM T6FA for 30 min before the addition of 20 µM of Aβ1–40. The commercial kits for the assay of AChE , choline acetytransferase , superoxide dismutase  and malondialdehyde  were purchased from Nanjing Jiancheng Biotech Company, China (www.njjcbio.com).Aβ1–40, lyophilised from 2 mg/mL hexafluoroisopropanol solution and dissolved in DMSO, were incubated for 48 h at room temperature in 0.215 M sodium phosphate buffer (pH 8.0) at a final concentration of 230 µM. For co-incubation experiments aliquots (16 µL) of HuAChE  and HuAChE in the presence of 2 µL of the tested inhibitors in 0.215 M sodium phosphate buffer pH 8.0 solution  were added. Blanks containing Aβ, HuAChE, and Aβ plus inhibitors at various concentrations in 0.215 M sodium phosphate buffer (pH 8.0) were prepared. The final volume of each vial was 20 µL. Each assay was run in duplicate. To quantify amyloid fibril formation, the thioflavin T fluorescence method was then applied. After incubated at 37°C for 24 h, the solutions containing Aβ, or Aβ plus AChE, or Aβ plus AChE in the presence of inhibitors were added to 50 mM glycine–NaOH buffer (pH 8.5) containing 1.5 µM thioflavin T in a final volume of 2.0 mL. Fluorescence was monitored with excitation at 446 nm and emission at 490 nm soon after the solution was mixed. The fluorescence intensities were compared and the percent inhibition due to the presence of test compounds was calculated. The percent inhibition of the HuAChE-induced aggregation due to the presence of increasing test compound concentration was calculated by the following expression: 100−(IFi/IFo×100) where IFi and IFo are the fluorescence intensities obtained for Aβ plus HuAChE in the presence or absence of inhibitor, respectively. Inhibition curves were obtained for each compound by plotting the percentage inhibition versus the logarithm of inhibitor concentration in the assay sample. The linear regression parameters were determined and the IC50 extrapolated, when possible (GraphPad Prism 3.0 GraphPad Software Inc.).Aliquots of 2 µL Aβ1–40 fibril formation using TEM images as previously described 1–40 in the presence or absence of T6FA or FA at different concentrations. A mixture of freshly prepared Aβ1–40 solution (10 µL of 50 µM in 10 mM sodium phosphate at pH 7.4) was incubated for 72 h at 37°C. The TEM samples were prepared by placing 5 µL of the pre-incubated solution on a carbon-coated grid. The samples were stained with 1% uranyl acetate and were placed on a clean paper for removing excess staining solution. The grids were thoroughly examined using a Phillips CM-30 electron microscope. Images were recorded at 52,000× magnification in a Philips/FEI CM120 electron microscope with a Gatan GIF100 imaging filter, equipped with a cooled slow scan CCD camera.Assay of Aβ2 incubator. Cells were pretreated with test compound and incubated for 30 min. After that, Aβ1–40 solution  was added to culture medium and incubated for 24 h. Controls were only treated with the vehicle or with the reverse peptide Aβ40–1 . The reverse sequence Aβ40–1 was prepared in the same way of that of Aβ1–40.PC12 cells originally obtained from American Tissue Type Cell Collection (ATCC) were grown in RPMI 1640 medium supplemented with 10% horse serum, 5% fetal bovine serum, and 1% antibiotics (penicillin/streptomycin) at 37°C in a humidified 95% air/5% CO1–40 or Aβ40–1 treatment as previously described Assays for cell viability were performed after 24 h of Aβ2DCF-DA 2DCF-DA  for 30 min at 37°C after 24 h of Aβ1–40 treatment in the presence or absence of T6FA. The cells were then rinsed with PBS solution. Intracellular esterases convert DCF diacetate to anionic DCFH which is trapped in the cells. The fluorescence of DCF, formed by the reaction of DCFH with ROS was recorded  using a PerkinElmer LS-5B spectrofluorometer.Intracellular ROS formation was measured by fluorescence using HThe animal experiments were performed according to internationally followed ethical standards and approved by the research ethics committee of Sun Yat-sen University (No 20081102). The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . Specific pathogen free SPF C57 BL/6J mice  were supplied by the Experimental Animal Center of Sun Yat-sen University  and housed in separate cages in standard conditions. Animals were housed in standard laboratory conditions: air-conditioned room (20–25°C), 12 h light/dark illumination cycle, free access to food and water.1–40 was prepared as stock solution at a concentration 0.6 µg/µL in sterile 0.1 M phosphate-buffered saline (PBS) (pH 7.4), and aliquots were stored at −20°C. Aβ1–40 was aggregated by incubation in sterile distilled water at 37°C for 4 d before use as described previously 1–40 (400 pmol/mouse) or PBS was administered by icv route using a microsyringe  that was inserted perpendicularly 3 mm deep through the skull. Briefly, the C57 BL/6J mice were anesthetized with 10% chloral hydrate (3.5 ml/kg body weight) dissolved in saline and then 3 µL of Aβ or sterile PBS was injected directly into the lateral ventricle, at the following coordinates from bregma taken from the atlas of mouse n = 10) to receive an intragastric administration of vehicle (Sham and Model), T6FA (2 mg/kg and 20 mg/kg), Donepezil (5 mg/kg) for consecutively 21 d. The protocol was outlined in Aβ1–40 administration, five groups were subjected to the MWM as described. Prior to first trial of each day, mice were placed on platform for 30 s for spatial orientation. Then mice were placed in a random start site, facing tank wall, initially mice were trained for 4 d for hidden platform trial, at 4th day were trained for probe trial after hidden platform trial. The escape latency is recorded as a parameter for hidden platform trial, and the time spent in goal area is recorded as a parameter for probe trial.The testing procedure was carried out as described previously 1–40 administration. The brains were removed quickly and the hippocampi were dissected on ice. Samples were weighed and homogenized to 10% homogenate  for AChE. Aliquote of the homogenate were further diluted with saline to 5% for ChAT assay. The supernatant was used to determine AChE and ChAT activity respectively. Protein concentration was determined by the method of BCA. The activities of ChAT and AChE were determined spectrophotometrically using the assay kit from Nanjing Jiancheng Bioengineering Institute . The absorbance was read at 324 nm, and ChAT activity was expressed as unite per g protein. For the assay of AChE activity, a reaction mixture that contained sodium phosphate  470 µl, 2% DTNB 167 µl and 33 µl of homogenate was incubated for 5 min at 37°C. Then, acetylcholine iodide (2 mM) 280 µl was added to the reaction mixture. After incubation for 3 min at 37°C, the reaction was terminated by adding 50 µl of neostigmine (4 mM). The absorbance was measured at 412 nm at room temperature. AChE activities were expressed as µmol per µg of protein.Following MWM test, animals of each group were decapitated under anesthesia at d22 after AβMDA level and SOD activity were detected using commercial kits  in tissue homogenates diluted to 10% and prepared in accordance with the manufacturer's instructions. The 10% homogenate made as described before was also used here for MDA assay and diluted with saline for the determination of SOD activity. Activity of SOD in hippocampus was measured by the method reported using nicotinamide adenine denucleotide reduced form as a substrate P value of <0.05 was accepted to determine the significance.The means and standard errors of means (SEM) were calculated for all experiments. The data were subjected to one-way analysis of variance (ANOVA) followed by Duncan's multiple-range test to determine whether means were significantly different from the control or model. In all cases, a 1–40 aggregation by 50.27% and 20.23% at 100 µM and 50 µM, respectively, while the effects of FA and tacrine on Aβ aggregation were lower or not detected at such concentrations.Accumulating evidence demonstrates that AChE has secondary non-cholinergic functions including the processing and deposition of Aβ It was well documented that antioxidants, including FA, can inhibit Aβ auto-aggregation 1–40, PC12 were pretreated with T6FA (2–50 µM) for 30 min and then exposed to 20 µM Aβ1–40 for 24 h before cell viability was assayed. Phase contrast images showed that the cells were fewer in number, less viable with shrinked cell body, many fragments, and less adhered after the addition of Aβ1–40. T6FA (10 µM) treatment blocked these effects of Aβ1–40  increased the intracellular ROS accumulation by about 2.0-folds after 24 h in the PC12 cells. Treatment of T6FA (10 µM) profoundly reduced the Aβ1–40-induced production of ROS to basal levels without affecting basal levels of ROS . The increased escape latencies could be significantly attenuated by Donepezil (5 mg/kg) at d1, d2 and d4 (P<0.05). T6FA (20 mg/kg) could markedly shorten the increased escape latencies and such effects were mostly significant from d2 (P<0.01). T6FA (2 mg/kg) showed the same effect at the last two days. In the probe trial, swimming times within the target quadrant of mice in the model group were obviously less than those in the sham-operated group  on spatial learning was evaluated by MWM test in an AD model in mice. As shown in ed group . Howevered group .P<0.05), whereas the T6FA and donepezil treatments dramatically reversed this change. In contrast, the AChE activity was significantly elevated in hippocampus in the model mice, which was reversed to normal levels of mice in sham group.ChAT activity and AChE activity were measured after the learning and memory tests. As shown in P<0.05), while the SOD activity was significantly increased (P<0.05) by T6FA (2 mg/kg) (from 127 to 161 U/mg protein). Interestingly, the effects of T6FA (20 mg/kg) was milder . All the treatments markedly reversed the increased MDA level  prevents the neurotoxicity induced by Aβle in AD . Free-raat 10 µM .in vitro. However, the protective effects of T6FA in vivo were still unknown. Our previous research found that T6FA can improve the scopolamine-induced cognitive dysfunction in mice 1–40, which had been evaluated as a validated animal model for anti-AD drugs development 1–40 analogous to peptides found in neuritic plaques of AD patients, cognitive deficits emerge in mice 1–40 administration.Clearly, T6FA has protective effects against cholinergic deficiency, oxidative stress and Aβ toxicity in vivo (We also measured the inhibitory effects of T6FA on cholinergic systems and anti-oxidative activity in AD mice. Our results showed that T6FA could significantly increased ChAT and SOD activity, decreased AChE activity and MDA level, indicating it also has ability of antioxidation and inhibiting cholinergic deficiency in vivo . Meanwhiin vitro independent of their AChE inhibitory. It should be very interesting whether T6FA can also modulate the level of amyloid protein or not. Future studies are needed to be done to discover the question in vitro and in vivo.In addition, increasing evidences indicated that AChE inhibitors, such as tacrine 1–40 induced cell death and ROS in vitro and chronically oral administration of T6FA protected mice against Aβ1–40-induced cognitive impairment in vivo strongly suggest that T6FA is a novel “one-compound-multi-targets” agent and might be useful as preventive and therapeutic medicines for AD. Additional in vitro and in vivo studies in other models to uncover the anti-AD effects of the novel compound are currently underway in our laboratory.Taken together, the present study demonstrated that, T6FA, a new tacrine-ferulic acid heterodimer, potently inhibit auto- and AChE-induced aggregation. Further, the findings that T6FA blocks or prevents Aβ


HMGA2) gene silencing in retinoblastoma (RB) cells.To explore the molecular mechanisms deregulated by high mobility group protein A2 (HMGA2 short interfering RNA (siRNA) was used to silence the HMGA2 gene in cultured Y79 RB cells that were subjected to whole genome microarray analysis. The expression of differentially regulated key genes was confirmed with quantitative reverse-transcriptase polymerase chain reaction (qRT–PCR) in post-silenced RB cell lines (Y79 and WERI Rb1). These deregulated genes were compared for their constitutive expression in primary RB tumors (n=10). Zymographic determination of matrix metalloproteinase (MMP) activity was performed in RB cells. A cell cycle assay and a proliferation assay were performed in post-transfected RB cells.Synthetic anti-HMGA2 gene silencing in cultured RB cells results in reduced cell proliferation and transition in the G1/S phase. The whole genome microarray analysis of HMGA2 silenced Y79 cells revealed overall upregulation of 1,132 genes (≥1.0 fold) and downregulation of 1,562 genes (≤ −1.0 fold). Specific quantitative pathway analysis of the deregulated genes (using Biointerpreter) revealed 150 upregulated genes and 77 downregulated genes (≥1.0 fold) involved in vital pathways, namely, mitogen-activated protein kinase, Janus kinase/signal transducers and activators of transcription, Ras pathway, Ras-induced extracellular signal-regulated protein kinases 1 and 2, and tumor protein p53. The differential expression of genes obtained from microarray analysis  [CDH1], Homo sapiens snail homolog 1 (Drosophila) [SNAI1], Homo sapiens matrix metallopeptidase 2 [MMP2], and Homo sapiens matrix metallopeptidase 9 [MMP9]) was confirmed with quantitative reverse-transcriptase polymerase chain reaction in post-silenced RB cells. Zymographic analysis revealed that the increase in MMP mRNA expression in the post-silenced RB cells did not correlate with corresponding enzyme activity.HMGA2 silencing in RB cancer cells, offering mechanistic insights into the anticancer potential. HMGA2 may be considered a promising candidate for gene silencing therapy in RB.Our study revealed molecular regulatory changes induced by  Retinoblastoma (RB), a common primary intraocular tumor, occurs in infants and children with a relative incidence of 3% of all pediatric tumors worldwide . Newer mThe HMGA family consists of HMGA1a, HMGA1b, HMGA1c, and HMGA2 ,11. HMGAHMGA2 gene is not detectable in adult human tissues where it is probably completely silenced ..35].HMGA2 gene. Human HMGA2 siRNA.1 , HMGA2 siRNA.2 , and scrambled siRNA  HMGA2 siRNA.3 , were used in this study. The HMGA2-specific siRNAs and the scrambled siRNA were purchased from Qiagen .The transfection protocol was performed using three different short interfering RNA (siRNA) sequences targeting the HMGA2-specific siRNAs (Qiagen) plus 5 µl of Lipofectamine transfection reagent  for 24 h, 48 h, and 72 h. The cells were harvested and processed for quantitative reverse-transcriptase polymerase chain reaction (qRT–PCR) and western blot analysis. The same protocol was applied using 200 nM non-target scrambled siRNA (Qiagen) for the cell proliferation assay.About 1×50,000 cells were plated per well (12 well plates), and allowed to grow for 24–36 h (until they were 40%– 60% confluent) and incubated with 0.5 ml of antibiotic-free media, 0.5 ml of complete media containing 200 nM HMGA2 RNA expression in untreated and siRNA-treated RB  cells, tumor tissues, the total RNA was extracted with the guanidine isothiocyanate and chloroform method . Cells were harvested from cultures and collected in RNase-free vials. To the pellet, 1 ml of TRIzol reagent (TRI Reagent) was added, vortexed vigorously for 2 min, and incubated at room temperature for 5 min. Later, 0.5 ml of chloroform was added to the solution and mixed well for 15 s and centrifuged. The aqueous layer that contains RNA was transferred to new vials. Then 0.5 ml of isopropanol was added and incubated at room temperature for 10 min. After centrifugation, the supernatant was discarded, and 0.5 ml of 75% ethanol was added, mixed well, and centrifuged. Then the supernatant was discarded. The pellet was air dried at room temperature for 2 min and reconstituted in 25 µl of RNase-free water. All centrifugations in the RNA extraction were performed at 15,000 × g for 10 min at 4 °C. All RNA samples were treated with TURBO DNase . For all samples, 1 μg of total RNA was used to synthesize first-strand cDNA with reverse transcriptase  and random primers. The cDNA synthesis was performed at 37 °C for 60 min followed by heat inactivation at 95 °C for 10 min. Gene expression assays for HMGA2 (Hs00171569_m1) and endogenous control, glyceraldehyde-3-phosphate dehydrogenase , were obtained from Applied Biosystems .To quantify the HMGA2 gene expression was normalized with GAPDH expression, which was measured using predeveloped assay reagents (Applied Biosystems). The final volume for each PCR was 20 μl including 1 μl (100 ng) of the investigated sample 1× Universal PCR Master Mix  for HMGA2 gene expression according to the manufacturer’s instructions. Gene expression in each sample was analyzed in triplicate. The PCR for HMGA2 gene expression using TaqMan probes was performed as follows: 2 min at 50 °C, 10 min at 95 °C, and 40 cycles of 15 s at 95 °C, plus 1 min at 60 °C. PCR for the other genes was performed as follows: commercial software  was used to calculate ΔΔCt [GAPDH endogenous control.ate ΔΔCt  relativeg for 15 min at 4 °C and washed with acetone at 4 °C. The dried proteins were dissolved directly in sample buffer . The protein was resolved by using 18% acrylamide gel. The separated proteins were electrophoretically transferred to the nitrocellulose membrane at 100 V for 1 h. The blots were incubated with human HMGA2 primary antibody  and human histone H1  overnight at 4 °C followed by anti-rabbit horseradish peroxidase-conjugated secondary antibody  incubation for 2 h. To determine the p53, p21, and β-actin proteins, the total protein cell lysate of the post-silenced Y79 and WERI Rb1 cells was extracted using lysis buffer containing 50 mM Tris-HCl (pH 7.6), 5 mM EDTA, 150 mM sodium chloride, 0.1% phenylmethanesulfonyl fluoride (PMSF), and 250 ml of 1 mg/ml protease inhibitor cocktail on ice. A total protein of 25 μg was resolved on by using 12% sodium dodecyl sulfate–PAGE. The separated proteins were electrophoretically transferred to the nitrocellulose membrane at 100 V for 1 h. The blots were incubated with human p53 primary antibody , human p21 , and β-actin  overnight at 4 °C followed by anti-mouse horseradish peroxidase-conjugated secondary antibody  incubation for 2 h. After intermittent washes with Tween Tris-buffered saline, the membranes were subjected to the chemiluminescence detection method . To derive the HMGA2 concentration in the individual samples, the intensity of the bands was measured using Quantity One, version 4.7 software in GS-800 calibrated Densitometer  and normalized with the respective histone expression. A similar protocol was followed to determine the concentration of p53 and p21, and normalized with the respective β-actin expression.The post-silenced Y79 and WERI Rb1 cells were tested for HMGA2 protein extraction. Cells were treated with 5% perchloric acid. The proteins were precipitated with equal volumes of cold acetone at 20 °C overnight. The precipitate was collected and centrifuged at 20,000 × 2HPO4, 0.24 g KH2PO4, pH 7.4). Mouse monoclonal primary antibody against caspase 3  was used and incubated for 2 h at 4 °C. Following incubation, cells were washed three times with ice-cold PBS. Fluorescein isothiocyanate–conjugated anti-mouse secondary antibody  was used and incubated for 1 h at 4 °C in the dark. Later, the cells were washed three times with ice-cold PBS. Cells were analyzed using a FACSCalibur flow cytometer , with the CellQuest software program (BD Biosciences).The post-silenced Y79 and WERI Rb1 cells were tested for caspase 3 expression. Cells were harvested, washed, and resuspended in ice-cold phosphate buffered saline  were harvested after transfection with siRNA for 48 h. Cells were fixed for 30 min with 70% cold ethanol, washed twice with cold PBS, and then incubated in PBS buffer 100 μg/ml RNase A for 30 min. Propidium iodide 5 μg/ml was added, incubated for 10 min, and cells detected with a flow cytometer .HMGA2 specific siRNA.1  plus 0.5 µl of Lipofectamine transfection reagent (Invitrogen) for 24 h. This was repeated on days 2 and 3. On the days from 1 to 3, serum-free RPMI medium containing 10 µl of 5 mg/ml 3-2,5diphenyltetrazolium bromide (MTT) was added to the wells, and the cells were incubated at 37 °C for 4 h. Then 100 µl of MTT solubilization solution dimethyl sulfoxide  was added, and the cells were incubated at 37 °C for 10 min. Colorimetric measurements were made using a spectrophotometer  at 562 nm, and the background was subtracted at 650 nm. The assay was performed in triplicate with and without scrambled siRNA as controls.Transfected Y79 and WERI Rb1 cells  were plated in 96 well plates on day 0. On day 1, cells were incubated with 200 nM Total RNA used for the microarray analysis was isolated from siRNA-treated and -untreated Y79 cells using TRIzol Reagent  and treated with TURBO DNase  to remove the DNA. The RNA samples (10 μg each) in a 50-μl reaction were treated with 1 μl of TURBO DNase (2 U) in 1× TURBO DNase buffer at 37 °C for 30 min. After the incubation, the RNA sample was extracted with phenol/chloroform to inactivate TURBO DNase. The Low RNA Input Linear Amplification Kit PLUS  was used to generate fluorescent complementary RNA (cRNA). T7 RNA polymerase was used in this method, which simultaneously amplifies target material and incorporates Cy3-labeled cytidine tri-phosphate. Qiagen’s RNeasy mini spin columns were used to purify the amplified cRNA samples, and the samples were then hybridized to the Human Whole Genome 44 K oligo Microarray for 17 h at 65 °C, as recommended by the manufacturer (Agilent Technologies). Data analysis was done using GeneSpring GX version 10 (Agilent Technologies). Agilent Feature Extraction software  was used to analyze the microarray data.Following the microarray analysis, a panel of genes involved in cell signaling, apoptosis, and cell adhesion mechanisms was selected for further confirmation with qRT–PCR. The differential expression of these genes was investigated in post-silenced Y79 and WERI Rb1 cells, and compared with control primary RB tissues.2 Real Time TM SyBr Green/ROX PCR Master Mix  used according to the manufacturer’s instructions. The primer sequences used in the gene expression study are provided in The extraction of total RNA and the cDNA conversion was performed as described above. The final volume for each PCR was 20 μl, including 1 μl (100 ng) of the investigated sample 1× Universal RTg for 5 min. The supernatant was collected, and the proteins were estimated with the Lowry method. Ten-percent sodium dodecyl sulfate–PAGE gels incorporated with 1 mg gelatin  was prepared. About 50 μg of each sample with equal volume of Native Loading Buffer  were loaded and run at 150 V for 90 min. The gels were renatured in Tris-HCl (pH 6.8) and washed thrice in 5.5 ml milli Q water for 15 min, left overnight in low salt collagenase buffer (LSCB) buffer  for three changes of 30 min each in 2.5% Triton X-100, and then washed with milli Q water, 0.02% Brij 35, and 0.02% sodium azide (pH 7.6) at 37 °C. The gels were then stained with 0.5% Coomassie Blue (Sigma Aldrich) for 90 min and destained in 10% acetic acid to reveal zones of digestion.The transfected cells (Y79 and WERI Rb1) were collected and washed thrice with phosphate buffer. When the protein lysate was prepared, 200 μl of 10 mg/ml phenylmethanesulfonyl fluoride  and 10 μl of 1 mg/ml protease inhibitor cocktail (Sigma Aldrich) were added, and the samples were sonicated  three times for 10 s each, on ice. The samples were then incubated in 4 °C for 15 min and centrifuged under cooling conditions  at 2,655 × t test and p value were determined based on a volcano plot using the Benjamini and Hochberg algorithm. A p value ≤0.05 was considered significant for change in gene expression. Log2 transformed values of gene expression changes showing ≥1.0 fold were considered upregulation, while a ≤1.0 fold change was considered downregulation in gene expression. A minimum of three replicates was performed for all cell culture experiments to derive the standard deviation (SD).For microarray analysis, the statistical HMGA2 gene silencing protocol was optimized in cultured RB cells. Using qRT–PCR analyses, we found that over a period of 48 h, transfection with the siRNA.1 (Hs_HMGA2_6) sequence led to a −4.65 log2 ratio decrease, while the other sequences, siRNA.2 (Hs_HMGA2_7) and siRNA.3 sequence, led to a log2 ratio of −2.0 and 1.76 decrease, respectively, only when compared with and without scrambled siRNA as controls in RB cells  sequence showed decreased expression of the log2 ratio of −4.65 and −3.17, respectively. The protein expression of HMGA2 was confirmed with western blot analysis  cells treated with the anti-HMGA2 siRNA, scrambled siRNA (a control for non-specific effects of siRNA treatment on cell growth), and the untreated Y79 cells at the end of 24 h, 48 h, and 72 h resulted in a significant decrease in cell proliferation to 81.7%, 67.5%, and 45.5% in Y79 cells and 75.4%, 69.4%, and 49.9% in WERI Rb1 cells, respectively  and 77 downregulated (33.9%). Significantly, dysregulated pathways were identified using several databases such as Biologic Pathway Exchange (BioPAX) pathways from Biocarta, Human Protein Reference Database (HPRD), Reactome, KEGG, and NCI-cGAP, by querying the differentially expressed gene list against all the genes annotated with pathway information in the microarray. A total of 100 upregulated and downregulated genes of interest are shown (GEO) [GEO Series accession number GSE31687.The genome-wide expression of anti-re shown . Gene dewn (GEO)  and are HMGA2 gene resulted in upregulating gene transcripts involved in the cellular functions, namely, the apoptosis genes—lactalbumin, alpha- (LALBA), phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), insulin-like growth factor binding protein 1 (IGFBP1), IKK interacting protein (IKIP), tumor necrosis factor receptor superfamily, member 10b (TNFRSF10B), Homo sapiens receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1), Homo sapiens damage-regulated autophagy modulator (DRAM), ataxin 3 (ATXN3), mitogen-activated protein kinase 13 (MAPK13), Homo sapiens activating transcription factor 3 (ATF3), Homo sapiens nuclear protein 1 (NUPR1), Homo sapiens cyclin-dependent kinase inhibitor 1A , lectin, galactoside-binding, soluble, 7 ; cell cycle and differentiation genes—Homo sapiens 5-hydroxytryptamine (serotonin) receptor 5A (HTR5A), tachykinin 3 , Homo sapiens activating transcription factor 3 (ATF3); anti-proliferation—Homo sapiens maternally expressed 3 (MEG3); and cell adhesion—Homo sapiens cadherin 11, type 2, OB-cadherin , Homo sapiens cadherin 1, type 1, E-cadherin , Homo sapiens integrin, alpha 1 (ITGA1), Homo sapiens integrin, alpha 2 (ITGA2), Homo sapiens laminin, alpha 3 (LAMA3), Homo sapiens laminin, beta 3 (LAMB3), Homo sapiens laminin, gamma 2 (LAMC2), Homo sapiens matrix metallopeptidase 2 (MMP2), Homo sapiens matrix metallopeptidase 9 (MMP9), and Homo sapiens collagen, type IV, alpha 3 (COL4A3).In the Y79 cells, silencing of the HMGA2 gene resulted in downregulating gene transcripts such as the oncogenes— Homo sapiens v-abl Abelson murine leukemia viral oncogene homolog 1 (ABL1), Homo sapiens v-raf murine sarcoma 3611 viral oncogene homolog (ARAF), Homo sapiens v-crk sarcoma virus CT10 oncogene homolog , Homo sapiens ELK1, member of ETS oncogene family (ELK1), Homo sapiens v-yes-1 Yamaguchi sarcoma viral related oncogene homolog (LYN), Homo sapiens v-myb myeloblastosis viral oncogene homolog (avian)-like 2 (MYBL2), Homo sapiens v-myc myelocytomatosis viral oncogene homolog ; cell cycle, proliferation, and differentiation genes—Homo sapiens transcription factor 4 (TCF4), Homo sapiens cyclin-dependent kinase 6 (CDK6), Homo sapiens cell division cycle 25 homolog A(CDC25A), Homo sapiens E2F transcription factor 4, p107/p130-binding (E2F4), Homo sapiens cyclin-dependent, Homo sapiens antigen identified by monoclonal antibody Ki-67 (MKI67), Homo sapiens polo-like kinase 1 —Homo sapiens CNDP dipeptidase 2 , pre-B-cell leukemia transcription factor 1 (Homeobox protein PBX1), Homo sapiens snail homolog 1 ; apoptosis genes—Homo sapiens cDNA FLJ13706 fis, clone, Homo sapiens T-box 3 , transcript variant 2, Homo sapiens topoisomerase (DNA) II alpha; and angiogenesis genes—Homo sapiens fascin homolog 1, actin-bundling protein , Antiapoptosis gene: Homo sapiens G-2 and S-phase expressed 1 (GTSE1).In the Y79 cells, silencing of the CRK, ELK1, MYC, CDC25B, and GRB2. The downregulated genes involved in the Ras pathway are Rac1, RALGDS, and ELK1. The downregulated genes involved in the Jak/STAT pathway are SPREAD2, PIAS3, CCND2, and CNTFR. The increased levels of ATM, PUMA /BB3, PTEN, and DRAM, the downstream molecules of p53-mediated apoptosis, reveal the modulation of cell apoptosis. The role of p53-mediated apoptosis was confirmed by the overexpression of p53 and p21 proteins as well as the caspase 3 protein in HMGA2-silenced Y79 and WERI Rb1 cells  kinase, Ras, Janus kinase/signal transducers and activators of transcription (Jak/STAT), and p53 signaling pathways. The downregulated genes involved in the MAP kinase pathway are b1 cells .ELK1, CDK6, E2F4, GTSE1, DRAM, CDH1, SNAI1, MMP2, and MMP9) in the microarray analysis was consistent with the qRT–PCR findings in the transfected Y79 cells. Although most of the genes were consistent in the expression obtained with the microarray and qRT–PCR analyses, a few genes in the post-transfected WERI Rb1 cells differed in levels of expression with respect to microarray findings. These genes include ELK1, CDK6, and E2F4, which were not downregulated, unlike in the Y79 cells. The SNAI1 gene that was significantly downregulated in Y79 cells was not downregulated to the same extent in the HMGA2-silenced WERI Rb1 cells  was compared for their relative expression in non-transfected primary RB tumors. We observed an inverse correlation of gene expression between the untransfected tumors and the HMGA2-silenced RB cells (ELK1 (9.21), GTSE1 (6.23), CDK6 (10.76), E2F4 (10.51), DRAM (−4.79), CDH1 (−0.430), and SNAI1 (3.60), The RB cells . For theMMP2 at the mRNA level, activity staining with zymography did not reveal a substantial difference between the pre- and post-transfected cells .Chau et al.  reportedromoters . In the 1/S phase with an increase in dead cell percentage. This also correlates with the significant upregulation of p21/CDKN1A (log transformed ratio=2.93), which is a direct target of miR-106b as it plays a key role in miR-106b-induced cell cycle growth [The cell cycle assay showed a marked transition in the Ge growth .HMGA2 gene in the RB cell lines  revealed deregulation of many functional genes. Their possible molecular mechanisms are discussed as follows.HMGA2, as DNA binding proteins often referred to as architectural transcriptional factors, specifically interact with several transcription factors  and participate in forming stereospecific multiprotein enhanceosome complexes . SilenciGTSE1 (G2 and S phase-expressed-1), a microtubule localized protein [HMGA2 siRNA treated RB cells (Y79 and WERI Rb1) and its upregulation in primary RB tissues were validated. The GTSE-1 protein negatively regulates p53 transactivation and p53-dependent apoptosis. The inhibition of HMGA2 gene expression in RB cells inhibited cell proliferation, which corroborates with earlier studies in the nude mice model of RB [DRAM, damage-regulated autophagy modulator, a critical effector of p53- induced autophagy [HMGA2 silenced RB cells (Y79 and WERI Rb1). The constitutive expression of DRAM was downregulated in primary RB tumors. The upregulation of other p53-regulated genes involved in initiating apoptosis such as ATM, PUMA/BB3, NOXA, FOXO4, and PTEN  is indicative of p53-dependent apoptosis [ATF and CREB families, which are increased in the post-HMGA2 silenced RB cells [HMGA2-silenced RB cells.The downregulation of  protein , in the utophagy  in post-poptosis . This waRB cells . Taken tE2F4 and CDK6 in HMGA2 post-silenced Y79 cells. Cyclin-dependent kinases (CDKs) are important regulators of cell cycle progression. CDK6, which first appears in the mid-G1 phase, is important for G1 phase progression and G1/S transition. Coupled with CDK4, they negatively regulate the activity of the RB tumor suppressor protein (The PubMatrix database). Exit from the G1 phase of cell cycle division is regulated by phosphorylation of pRb by cyclin D/CDK4 and cyclin D/CDK6 complexes. This results in the suppression of the cell cycle. The constitutive expression of E2F4 and CDK6 genes was increased in our study cohort of RB tumor tissues analyzed here . This is possibly because of the differential activation and dominance of specific apoptotic and cell cycle pathways that may relate to tumor aggressiveness. WERI Rb1 represents a non-metastatic model of RB while Y79 represents more aggressive and metastatic characteristic of RB [The study also shows the downregulation of various transcription factors and cyclin-dependent kinases involved in cell cycle regulation, namely, zed here . Anotheric of RB .SNAI1  along with simultaneous upregulation of E-cadherin in the post-anti-HMGA2 silenced RB cells. HMGA2 silencing induced transcriptional derepression of E-cadherin with decreased Snail has been reported in a pancreatic cancer cell line by Sugiko et al. [SNAI1 and CDH1 in RB tumor tissues revealed the inverse correlation between them  with no significant change in the expression of their inhibitors (TIMPs) was observed in post-HMGA2 silenced RB cells. The microarray analysis did not reveal significant changes in the levels of thrombospondin-2 (activator of MMP2) and thrombospondin-1 (activator of MMP9) [The other gene modulations in the current study include the significant downregulation of o et al. . The coneen them . This hatachment . The preilencing ,59. Matrilencing –64. In tof MMP9) ,52. ThusHMGA2 silencing in RB cancer cells resulted in the deregulation of genes responsible for apoptotic, cell cycle, and cell adhesion mechanisms, thereby explaining the mechanisms by which cancer cell progression is suppressed in HMGA2-silenced RB cells. These findings are further substantiated by the inverse correlations between the deregulated gene expression in the HMGA2-silenced RB cells and in the primary RB tumor tissues. The HMGA2 gene silencing approach is thus suggested to be a promising strategy in RB therapy.In conclusion,


Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination.Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os  Seed germination is a complex event, which commence with the uptake of water by the quiescent dry seed and terminate with the elongation of the embryonic axis In mammalian cells, there are three types of PI3K with different substrate specificities phox to the cell membrane, which affects the assembly of NADPH oxidase complex and eventually the activity of NADPH oxidase phox and p47phox, which influences their abilities to translocate p67phox and finally alters the activity of NADPH oxidase NADPH oxidase, which plays a significant role in generating ROS, has been implicated in seed germination In this study, we revealed that PI3K altered the quantity of ROS and played an essential role in rice seed germination, through the regulation of the expression and activity of NADPH oxidase. It was also characterized how PI3K regulated the activity of NADPH oxidase. Finally, we proposed a novel hypothetical model that PI3K regulated the translocation of Rac-1 from cytoplasm to plasma membrane, resulting in activated assembly of NADPH oxidase in rice seed germination.PI3K in rice seed germination, the total RNA was extracted from the rice seed embryo after imbibition. The result of RT-PCR analysis suggested that PI3K might involve in the regulation of seed germination. Meanwhile, it was found that the expression of PI3K was increased with the prolongation of imbibition time (2+ (below 10 mM) could further increase the expression of PI3K in rice seeds after germinated for 12 h. However, when the concentration of Ca2+ was up to 20 mM, the PI3K expression began to decrease slightly compared with the treatment with 10 mM CaCl2  rice seed germination 2O2) could partly rescue the inhibitory effects of PI3K inhibitors on rice seed germination  or Wortmannin (20 µM). To further investigate this relationship between PI3K and ROS, ROS probe H2O2 (20 mM) could partly rescue the decrease in seed germination caused not only by KI and ASA but also by DPI , gibberellins (GAs) and ethylene 2O2 abated the inhibition of PI3K inhibitors during the rice seed germination  3 in animal cells. Moreover, in the early seed germination, a temporary structural perturbation, particularly to membranes, was occurred following the influx of water into the cells of dry seeds The analysis of western-blot was shown by using Rac-1 antibody. As expected, under control conditions, the percentage of Rac-1, which was transmitted to membrane, was more than that of inhibitor treatment. The translocation of Rac-1 to the membrane was suppressed, but total Rac-1 in the homogenate was not changed obviously under the treatment of LY294002. In addition, compared with that of LY294002, another PI3K inhibitor Wortmannin seemed to alter the amount of total Rac1 while it reduced the amount of membrane Rac1 severely . The difTogether with the above work, the data presented here provided a comprehensive view of the mechanism of PI3K-dependent seed germination with ROS as key signaling elements in rice seed germination. A scheme showing this mechanism was presented in Oryza sativa L. Nipponbare) seeds were kindly provided by Professor L.X. Zeng . H2DCFDA was obtained from Molecular Probes . DPI, nitroblue tetrazolium (NBT) and XTT were purchased from Sigma-Aldrich . LY294002 and Wortmannin were obtained from BiYunTian . Rac-1 antibody was bought from Cell Signalling Technology (USA).Rice  (100 mM) and KI (10 mM) were used to treat the seeds. In addition, DPI (100 µM) was also used to test if NADPH oxidase was one of the major ROS sources. The average germination rate of the triplicate of 100 seeds was calculated every 24 h over a period of 5 days.UBQ was used as a control in these reaction as described in the previous study rboh reaction was initiated at 94°C for 5 min followed by 30 cycles at 94°C for 15 s, 64.5°C for 1 min and 72°C for 1 min. The UBQ reaction was initiated at 94°C for 5 min followed by 30 cycles at 94°C for 15 s, 60.5°C for 1 min and 72°C for 1 min. The PI3K reaction was initiated at 94°C for 5 min followed by 30 cycles at 94°C for 15 s, 64.5°C for 1 min and 72°C for 1 min. The primers that were used in PCR were as follows: PI3K , UBQ , Os rboh1 , Os rboh2 , Os rboh3 , Os rboh4 , Os rboh5 , Os rboh6 , Os rboh7 , Os rboh8 , Os rboh9 .Embryos after being isolated from dry seeds (kept in 4°C) and germinating seeds  were frozen in lipid nitrogen and stored at −80°C until used. For each extract, 100 embryos were grounds to a fine powder in lipid nitrogen, and total RNA was extracted using Trizol RNA isolation reagent  following the manufacturer's instructions. Before synthesizing the cDNA, the isolated RNA were added DNase I  and incubated for 30 min at 27°C for digesting DNA impurities. DNase I was then inactivated by heat treatment for 2 min at 80°C 2DCFDA), as described previously 2DCFDA at a final concentration of 5 µM for 20 min at 30°C on a shaker. And then 200 µl of the solution was taken and its fluorescence  was measured within a few minutes using a microtiter plate reader  ROS release from embryos was determined by the fluorescence of DCF, which was the product of oxidation of dichlorofluorescin diacetate  containing 500 µM XTT in darkness at 25°C on a shaker for 3 h, and XTT reduction was determined at 470 nm. Blanks without plant material were run in parallel and used for subtracting spontaneous fluorescence changes −1 fresh weight) containing 0.25 M sorbitol, 1 mM EGTA, 20 µM phenylmethanesulfonyl fluoride, 1% (w/v) PVP, 2 mM dithiothreitol, and 50 mM Tris-acetate, pH 7.5, and filtered through four layers of gauze. The resulting filtrate was centrifuged at 15,000×g for 20 min. The recovered supernatant was centrifuged at 100,000×g for a further 30 min and the resulting membrane pellet  was gently resuspended in 5 mM potassium phosphate buffer (pH 7.8) including 330 mM Suc, 5 mM KCl, 1 mM DTT, 0.1 mM EDTA and protease inhibitors. The suspension was then fractionated by the aqueous two-phase partitioning method according to the batch procedure g for 5 min. The upper phase, enriched in plasma membrane (PM) vesicles, was repartitioned twice with fresh lower phase without PEG and, after dilution with 0.25 mM sorbitol, 1 mM EGTA, 2 mM MgCl2, 2 mM dithiothreitol, and 20 mM Tris-acetate, pH 7.5, and then the PM vesicles were collected by centrifuging at 100,000×g for 45 min. The pellets were then resuspended in Tris–HCl dilution buffer and stored at −80°C for further analysis. All procedures were carried out at 4°C.Plasma membranes were isolated by an aqueous two-phase partitioning system according to the method reported previously with minor modifications 2 or 10 mM EGTA. Protein concentrations were determined by the approach of Bradford The NOX activity of PM vesicles was determined by an assay based on reduction XTT by superoxide anion 2, and 1 mM CaCl2, pH 7.4) for 20 min, and then with 0.5 mM NADPH until the appearance of blue formazan bands was observed. The reaction was stopped by immersion of the gel in distilled water.The NADPH-dependent superoxide anion coming from the isolated membrane fractions were assayed in native gels by a modified NBT reduction method To obtain the optimal anti-body, amino acid sequences of Rac-1 in different species was compared using DNAman .Membrane proteins and total proteins were separated by 12% SDS-PAGE and transferred onto PVDF membranes (American Pharmacia Biotech) and subjected to immunodetection with Rac-1/Cdc42 polyclonal antibody, which was the result of Rac-1 sequence comparison from different species. The Rac-1/Cdc42 antibody could recognize endogenous levels of total rac-1 and cdc42 protein. The antigen-antibody complex was visualized with anti-rabbit secondary antibody and enhanced chemiluminescence. The Coomassie Brilliant blue-stained gel was used to show that an equal amount of proteins Figure S12O2 abate the inhibition of PI3K inhibitor on the rice seed germination.H Germination of rice seed embryos came from the same seed lot at 27°C in the dark. Rice seed treated with 10 mM KI, 1 mM ASA, 100 µM DPI, 20 µM Wortmannin, 60 µM LY294002, KI and H2O2 combined (10 mM KI+10 mM H2O2), ASA and H2O2 combined (1 mM ASA+10 mM H2O2), DPI and H2O2 combined (100 µM KI+10 mM H2O2), Wortmannin and H2O2 combined (20 µM Wortmannin+10 mM H2O2), LY294002 and H2O2 combined (60 µM LY294002+10 mM H2O2), respectively. The germination of rice seed after 5 days imbibition was counted. Data are means of three replicates ± SD. * indicates the values that are significantly different from control (P<0.05).(TIF)Click here for additional data file.Figure S2Treatment with PI3K inhibitors decreases intracellular ROS level based on the treatment of DPI. Rice seed embryos was pretreated with 100 µM DPI, DPI and Wortmannin combined (100 µM DPI+20 µM Wortmannin), DPI and LY294002 combined (100 µM DPI+60 µM LY294002), respectively. ROS was determinated by H2DCFDA. DCF fluorescence was measured using a microtiter plate reader as described in Materials and Methods. Data are means of three replicates ± SD.(TIF)Click here for additional data file.Figure S3Comparison of amino acid sequences of Rac-1 in different species. To obtain the optimal anti-body, amino acid was compared using DNAman, and the protein sequences used correspond to the following GenBank ID (from top to bottom): AAA36537, CAA40545, AAC49851, CAD42726, AAA96980, BAA84492.(TIF)Click here for additional data file.Table S1rboh gene was searched from the rice genome database and appraised nine genes. Compared with vps34 gene in Arabidopsis, pi3k gene was found from rice genome database. ubq gene was used as a control as described in the previous study.List of Primers used in semiquantitative reverse transcriptase -polymerase chain reaction (RT-PCR). (DOC)Click here for additional data file.