However, little is known about JNK1/2 and p38 MAPK signaling pathways in enterovirus 71 (EV71)-infected immature dendritic cells (iDCs). Thus, iDCs were induced from peripheral blood mononuclear cells (PBMC) and performed to explore the expressions and phosphorylation of molecules in the two signaling pathways as well as secretions of inflammatory cytokines and interferons during EV71 replication.c-Jun NHWe showed that EV71 infection could activate both JNK1/2 and p38 MAPK in iDCs and phosphorylate their downstream transcription factors c-Fos and c-Jun, which further promoted the production of IL-2, IL-6, IL-10, and TNF-α. Moreover, EV71 infection also increased the release of IFN-β and IL-12 p40. Pretreatment of iDCs with SP600125 and SB203580 (20 μM) could severely impair viral replication and its induced phosphorylation of JNK1/2,p38 MAPK, c-Fos and c-Jun. In addition, treatment of EV71-infected iDCs with SP600125 and SB203580 could inhibit secretions of IL-6, IL-10 and TNF-α.JNK1/2 and p38 MAPK signaling pathways are beneficial to EV71 infection and positively regulate secretions of inflammatory cytokines in iDCs. EV71 is a positive-stranded RNA virus in the genus enterovirus of the family Picornaviridae, usually leading to hand, foot, and mouth diseases (HFMD) and herpangina. MoreoveMitogen-activated protein kinase (MAPK) belongs to a family of serine/threonine protein kinases. It is widely conserved among eukaryotes and involved in many cellular processes such as inflammation, proliferation, differentiation, movement, and death-9. To daDCs are the first line of defense which could not only promote innate immune response but also initiate specific host immune responses by both capturing and processing antigens to MHC-I and II molecules on the cellular surface, regulating naïve T cells and differentiation-21. It hAll the patients provided informed consents, which was approved by the Ethics Committee of the Third Affiliated Hospital of Suzhou University.Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and RPMI 1640 were purchased from Thermo Scientific HyClone . Hybond C membrane and ECL Western blot detection system were from Pierce . Rabbit polyclonal antibodies against JNK, p-JNK, p38 MAPK, p-p38 MAPK, c-Fos, p-c-Fos, c-Jun, p-c-Jun and horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG were purchased from SAB . Antibodies against anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from ProteinTECH Group . Rabbit polyclonal antibody against EV71 VP1 was purchased from Abcam . The JNK1/2 and p38 MAPK specific inhibitor (SP600125 and SB203580) were acquired from LC Laboratories  and freshly prepared using DMSO solution.2 atmosphere, and passaged when reaching 90% confluence. EV71 strain was from China Center for Type Culture Collection (CCTCC)/GDV083 (ATCC VR-784) and propagated in RD cells. Viral titer was determined by CPE and expressed as 50% tissue culture infective dose (TCID50) per ml[RD cells were purchased from Chinese Academy of Sciences Cell Bank of Type Culture Collection (CBTCCCAS), cultured in high glucose DMEM supplemented with 10% fetal bovine serum  at 37°C in a humidified incubator under 5% CO) per ml.Peripheral venous blood obtained from healthy blood donors was kindly provided by Changzhou Blood Center and used to purify mononuclear cells using Ficoll-Hypaque  density gradient centrifugation. Monocytes were isolated from PBMC by adhesion to plastic dishes for more than 2 h at 37°C as previously described. iDCs were generated from monocytes by culturing in RPMI 1640 medium containing 10% FBS, 100 ng/mL of GM-CSF , 50 ng/mL of IL-4 , and antibiotics for 7 days. On Day 7, cells were collected and analyzed by flow cytometry  for CD3, CD11c, CD80, CD83, CD86 and HLA-DR. The induced DCs were assigned in two groups. One group was not infected with EV71 and used as control. The other group was infected with EV71 at a MOI of 5 for 1 h at 37°C. After washed twice with PBS, all cells were cultured in RPMI medium for 24 h and analyzed using flow cytometry. Meanwhile, the supernatants were collected and stored at -80°C.-ΔΔCt.After incubating at 37°C for 1/2 h, 2 h, 8 h and 24 h, both uninfected and infected iDCs were harvested and used to extract total RNA using the SV total RNA isolation system . PCR arrays were performed with customized PCR containing pre-dispensed primers  on the LightCycler 480  using SYBR MasterMix . Each PCR contained 10 ng of synthesized cDNA. The thermocycler parameters were performed with an initial denaturation at 95°C for 5 min followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 15 s and extension at 72°C for 20 s. Relative change in gene expression was calculated using ΔΔCt (threshold cycle) method. The housekeeping genes such as B2M, ACTB, GAPDH, RPL27, HPRT1 and OAZ1 were used to normalize to the amount of RNA. Fold changes in gene expression were calculated using the formula of 2β-mercaptoethanol, 50 mM Tris–HCl (pH 6.8), 1 mM phenylmethylsulfonylfluoride). Cell lysates were obtained by centrifugation at 45,000 × g for 1 h at 4°C. Total protein concentration was determined by the bicinchoninic acid protein assay kit (Pierce). Equal amount of proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto PVDF membranes (Millipore). The membranes were blocked for 2 h with 5% nonfat dry milk solution in Tris-buffered saline containing 0.1% Tween-20 and then incubated with specific primary antibodies. After washed with PBS, the membranes were incubated with HRP conjugated secondary antibodies and washed with PBS. The immunoreactive bands were detected by ECL reagents , visualized on Super RX film (Fujifilm) and quantitated by densitometric analysis . The level of phosphoproteins was normalized to its respective control at 0 h, which was arbitrarily set to 1.iDCs were pre-incubated for 1 h with SP600125 and SB203580 (20 μM), and then infected with EV71 at a MOI of 5 in the presence of SP600125 and SB203580 for 24 h. Cells were harvested by centrifugation, washed and lysed with a lysis buffer  using luminex fluorescent technique according to the manufacturers’ instruction. The fluorescence data in each standard, quality control and samples were detected with the FLEXMAP3D  and subsequently analyzed using the MILLIPLEX™ Analyst V5.1 . The standard curves were generated for each cytokine with Bio-plex manager software and used to calculate cytokine concentrations in supernatants using stepwise five-fold dilution of protein standards.All data were presented as the mean ± SE and statistically analyzed using GraphPad Prism software . P values less than 0.05 were considered statistically significant.iDCs were prepared from monocytes purified from peripheral blood by induction with GM-CSF and IL-4. Flow cytometric analysis indicated that 90.8% and 92.9% of DCs were positive for CD80 and CD11c, respectively, and only 3.5% and 6.8% of cells were positive for CD3 and CD83, respectively, confirming that they were indeed iDCs. At 1/2 h, 2 h, 8 h and 24 h p.i., iDCs were collected and the expressions of molecules in JNK1/2 and p38 MAPK signaling pathways were examined by PCR arrays. The results showed that the mRNA levels of MEK3/6, MEK4/7, JNK1, JNK2, JNK3, and p38 MAPK(α/β) were upregulated by 2.02 - 3.08 - fold at different times of EV71 p.i. in different time, while c-Jun and c-Fos were increased by 3.03 to 9.17 - fold. In addition, the mRNA levels of IL-2, IL-6, IL-12, TNF-α, and IFN-β were upregulated by 2.24 - 4.32 - fold at different times of EV71 p.i.  for 1 h and infected with EV71 at a MOI of 5 for 24 h and the repilcation of EV71 was measured by TCID50. The results showed that the two inhibitors markedly inhibited EV71 replication  is a heterodimeric transcription factor composed of proteins in the subfamilies of c-Jun, c-Fos, Maf, and activating transcription factor (ATF). It regulates gene expression in response to a variety of stimuli, including cytokines, growth factors, stress, and bacterial and viral infections,29. The iDCs can secrete several cytokines once they are activated by viral infection. To examine the role of JNK1/2 or p38 MAPK pathways in cytokine secretion in iDC, the culture supernatants of control iDCs, EV71-infected iDCs and iDCs pretreated with inhibitor SP600125 or SB203580 (20 μM) prior to EV71 infection were collected at 24 h p.i. and used to detect the levels of IL-2, IL-6, IL-10, IL-12 p40, IL-12 p70, TNF-α, IFN-α and IFN-β using luminex fluorescent technique. The results showed that EV71 infection (MOI = 5) significantly increased secretions of IL-2, IL-6, IL-10, IL-12 p40, TNF-α and IFN-β in iDCs, and pretreatment with SP600125 or SB203580 only significantly inhibited the production of IL-6, IL-10 and TNF-α, but not that of IL-2, IL-12 p40, IL-12 p70, IFN-α and IFN-β, indicating that production of the formers, but not the latters, were mediated by JNK1/2 or p38 MAPK pathways , and they are strongly activated in response to cytokines, UV irradiation, growth factor deprivation, DNA damaging agents, growth factors,and viral infection,37. JNK1Four isoforms of p38 MAPK have been identified and named as p38 MAPK α/β/γ/δ. Like alThe transcription factor activator protein 1 (AP-1) is a major downstream target of JNK1/2 and p38 MAPK. It is a dimeric complex composed of members of the c-Jun, c-Fos, Maf, and ATF protein subfamilies. After activation in the cytoplasm, JNK1/2 and p38 MAPK translocate to the nucleus, where they phosphorylate Ser and Thr residues of specific AP-1 subunits to augment AP-1 transcriptional activity. Both JNK1/2 and p38 MAPK target to ATF2 (ATF subfamily), while JNK1/2 also targets to c-Jun and JunD. Our resProinflammatory cytokines such as IL-6, TNF-α, and IFN-β are usually induced by oxidant stress, cytokines, and virus infection, which play important roles in host cell damages, chronic inflammation, and other immunoresponses-49. EV71To our knowledge, this study is the first report showing that EV71 infection activates JNK1/2 and p38 MAPK pathways in iDCs and leads to increased viral yield and proinflammatory cytokine secretions. Moreover, inhibition of JNK1/2 and p38 MAPK pathways could effectively reduces viral replication and cytokine release, supporting the idea that the activation of these two pathways are important for EV71 infection. We speculate that JNK1/2 and p38 MAPK regulate viral replication by acting at certain specific steps of viral replication cycle, including attachment, entry, gene transcription, protein expressions, and assembly, as well as viral pathogenesis. However, the underlying mechanisms need to be further studied in vitro or in vivo to highlight JNK1/2 or p38 MAPK as a potential broad antiviral molecular target for treatment of EV71 infection.The authors declare that they have no competing interest.WS conceived and designed the experiments, participated in the data analysis and manuscript preparation. HP, LZ and LY performed cell culture, Western blot and flow cytometry. MS and YJ participated in the data analysis and manuscript preparation. JS and LZ performed PCR-fluorescence probing assay and analyzed the data. XW and XX detected cytokine levels. XZ and YM analyzed PCR array. All authors have read and approved the final manuscript.


Studies with JNK specific inhibitor, SP600125, showed that Huaier extract induced S phase arrest and decreased β-catenin and cyclin D1 expression via JNK signaling pathway. In conclusion, we verify that Huaier extract causes cell apoptosis and induces hepatocellular carcinoma cells arrest in S phase via JNK pathway, which advances our understanding on the molecular mechanisms of Huaier extract in hepatocarcinoma management.Huaier aqueous extract, the main active constituent of Huaier proteoglycan, has antihepatocarcinoma activity in experimental and clinical settings. However, the potential and associated antihepatoma mechanisms of Huaier extract are not yet fully understood. Therefore, in this study, we aimed to elucidate the inhibitory proliferation effect of Huaier extract on apoptosis and cycle of HepG2 and Bel-7402 cells. Our data demonstrated that incubation with Huaier extract resulted in a marked decrease in cell viability dose-dependently. Flow cytometric analysis showed that a 48 h treatment of Huaier extract caused cell apoptosis. Typical apoptotic nucleus alterations were observed with fluorescence microscope after Hoechst staining. Immunoblot analysis further demonstrated that Huaier extract activated caspase 3 and PARP. Additionally, Huaier extract inhibited the activity of p-ERK, p-p38, and p-JNK in terms of MAPK. Furthermore, Huaier extract induced HCC cells arrest in S phase and decreased the cycle related protein expression of  Hepatocellular carcinoma (HCC) is the fifth most frequent malignancy and the second leading fatal disease worldwide . There a Trametes robiniophila Murr. (Huaier) has been applied in traditional Chinese medicine for approximately 1600 years  × 100%.Cell proliferation was measured using the CellTiter 96 Aqueous One Solution cell proliferation assay . Cells were seeded in 96-well plates at a concentration of 105 cells were seeded into a 6-well plate. After 48 h exposure to different concentrations of Huaier aqueous extract as above, all the adherent cells were collected with 0.25% trypsin without EDTA, including the floating cells in the medium. Annexin V-FITC and propidium iodide (PI) were used for staining according to the manufacturer's instructions. The double-stained cells were subsequently analyzed by a FACSCanto flow cytometer . At least 10,000 cells were counted each time.Cell apoptosis was determined using the Annexin V-FITC apoptosis detection kit . Briefly, 2 × 10μL Hoechst 33258  for 15 min at room temperature in the dark and then washed with PBS. Afterwards, the cells were mounted and examined under fluorescence microscopy . Apoptotic cells were identified by the condensation and fragmentation of their nucleus. The apoptotic ratio was obtained by the following calculation: The Apoptotic Ratio = apoptotic cell number/seeded cell number × 100%.Following treatment with Huaier extract at various concentrations for up to 48 h in a 6-well plate, cells were washed twice with PBS and fixed in 1 mL of 4% paraformaldehyde for 10 min at 4°C. After washing twice with PBS, cells were stained with 500 5 cells were seeded into a 6-well plate and starved in serum-free medium on the second day. After 12 h starvation, the cells were treated with gradient concentrations of Huaier solution for 48 h. The cells were then trypsinized, washed with cold PBS, and fixed overnight with 70% cold ethanol at 4°C. The next day, the fixed cells were centrifuged at 1200 g for 5 min and washed once with PBS. After that, the cells were suspended in PI/RNase staining buffer for 30 min in the dark according to the manufacturer's instructions of Cell Cycle Detection Kit . Then, the DNA contents of the cells were analyzed in a FACScan flow cytometer . At least 10,000 cells were collected for each measurement.Briefly, 2 × 10μg/lane) were resolved onto SDS-PAGE and transferred onto a PVDF membrane in a wet transfer system  at 70 V at 4°C. For immunoblotting, the PVDF membrane was incubated with Tris-buffered saline plus Tween-20 (TBS-T) containing 5% nonfat milk for 1.5 h and then incubated with a specific primary antibody overnight at 4°C. Horseradish peroxidase- (HRP-) conjugated IgG was used as the secondary antibody and incubated for 2 h. Afterwards, reactive protein was detected using an enhanced chemiluminescence (ECl) commercial kit . The results were recorded using the MicroChemi Bio-Imaging Systems  and Quantity One version 4.5.0 software .The cells treated with Huaier extract or SP600125  for 48 h were washed twice with ice-cold PBS and lysed in ice-cold protein lysis buffer supplemented with 1% (v/v) protease inhibitor cocktail and PMSF. The lysates were centrifuged at 12,000 rpm for 10 min at 4°C. The suspension protein concentrations were determined using a BCA Protein Assay kit  and were then denatured by boiling. Total proteins , mouse anti-cyclin D1 monoclonal antibody , mouse anti-GAPDH monoclonal antibody , rabbit anti-ERK polyclonal antibody , rabbit anti-p38MAPK polyclonal antibody , rabbit anti-JNK polyclonal antibody , mouse anti-pERK monoclonal antibody , mouse anti- pp38MAPK monoclonal antibody , mouse anti-pJNK monoclonal antibody , and mouse anti-tubulin monoclonal antibody . The secondary antibodies included goat anti-rabbit IgG serum  and goat anti-mouse IgG serum .The primary antibodies used in this study were as follows: rabbit anti-pro-caspase 3 polyclonal antibody , rabbit anti-cleaved-caspase 3 polyclonal antibody , rabbit anti-P < 0.05, P < 0.01, and P < 0.001.All experiments were performed three times. Data were presented as means ± standard deviations (SD). The differences were analyzed using one-way ANOVA followed by the Student-Newman-Keuls test and all statistical analyses were performed using GraphPad Prism 5 software. Statistical differences are presented at probability levels of 50 value of 7.6 and 10.6 mg/mL, respectively, after 48 h. But the IC50 value in the case of THLE-3 was 13.8 mg/mL, which means that the Huaier extract is less toxic to the normal liver cells than to HCC cells.To evaluate the proliferative effect of Huaier extract on HepG2 and Bel-7402 cells, we measured cell proliferative viability using the MTS assay after the cells were dose-dependently treated with Huaier extract for 48 h. As shown in To demonstrate the apoptosis effect of Huaier extract, we used FCM analysis with Annexin V-FITC and PI double staining. After treatment with different doses of Huaier extract for 48 h, early apoptotic cells and late apoptotic cells were differentiated from viable or necrotic ones. In the control group, there were almost normal cells, rarely apoptotic cells, while in Huaier extract groups, the rates of apoptotic cells gradually increased along with increasing concentrations of Huaier extract. The rates of apoptosis in different Huaier extract  groups were 5.50 ± 1.04%, 13.57 ± 0.58%, 29.40 ± 3.00%, 49.53 ± 8.50%, and 96.22 ± 3.06%, respectively, in HepG2 cells, and 1.5 ± 0.5%, 6.1 ± 2.1%, 16.6 ± 2%, 43 ± 1.5%, and 72.4 ± 1.6% respectively, in Bel-7402 cells .In addition, we verified the apoptotic effect of Huaier extract in HepG2 cells by morphological changes. After treatment with different doses of Huaier extract for 48 h, HepG2 cells were stained with Hoechst 33258. The normal cells in morphology are round and homogenous, while the morphological changes of cell apoptosis include cell shrinkage, nuclear condensation, and fragmentation. Fluorescence dye stains condense chromatin of apoptotic cells more brightly than chromatin of normal cells. The number of HepG2 cells adhering to the culture plates in Huaier extract treatment was greatly reduced compared to control group. The apoptotic morphological changes were observed in the Huaier extract-treated groups, whereas few apoptotic cells were found in the control group. The percentage of apoptotic cells in different Huaier extract  groups was 4.27 ± 1.80%, 14.27 ± 1.20%, 27.60 ± 2.00%, 33.17 ± 1.90%, and 62.67 ± 2.40%, respectively, in HepG2 cells .To further confirm the apoptotic mechanisms of Huaier extract on HCC cells, we tested the expression of procaspase 3, cleaved caspase 3, and cleaved PARP with Western blot. Huaier extract activated the caspase 3, resulting in increased expression of cleaved caspase 3, cleaved PARP, and decreased expression of procaspase 3 .To further investigate the effect of Huaier extract on the cell cycle, the cell cycle profiles of HepG2 and Bel-7402 cells were analyzed using flow cytometry. The cells were treated with Huaier extract at concentrations of 0, 2, 4, 8, and 16 mg/mL for 48 h and stained with PI. Huaier extract treatment resulted in a significant increase in the percentage of cells in the S phase and a significant decrease in the percentage of cells in the G0/G1 phase. The percentage of cells accumulated in the S phase were 25.31 ± 1.53%, 28.43 ± 1.25%, 30.48 ± 0.76%, 33.55 ± 0.81%, and 47.57 ± 0.87%, respectively, in HepG2 cells, and 20.25 ± 2.06%, 25.87 ± 2.06%, 32.43 ± 2.02, 37.11 ± 2.05, and 43.61 ± 2.33, respectively, in Bel-7402 cells. The accumulation of G0/G1 phase cells was maximal in the control group and declined with increasing concentrations of Huaier extract. The decrease in the number of G0/G1 phase cells was 60.36 ± 0.71%, 57.43 ± 0.95%, 56.13 ± 0.96%, 55.76 ± 0.54%, and 40.38 ± 0.88%, respectively, in HepG2 cells, and 67.4 ± 2.07, 63.62 ± 2.30, 57.5 ± 1.84, 55.66 ± 2.41, and 51.23 ± 2.10, respectively, in Bel-7402 cells . These rβ-catenin and cyclin D1 expression by Western blotting and found that Huaier extract treatment significantly decreased the expression of β-catenin and cyclin D1 in a dose-dependent manner  for 1 h and then incubated with Huaier extract (8 mg/mL) for 48 h. Using Western blotting assay, it was found that treatment with either Huaier extract (8 mg/mL) or SP600125 (10 μM) reduced β-catenin protein levels by 19% and 28.4% and cyclin D1 protein levels by 41% and 51.7%, respectively, and that combination treatment reduced β-catenin and cyclin D1 protein levels by 54.7% and 60.7%. These data suggest that Huaier suppresses the expression of β-catenin and cyclin D1 through the downregulation of JNK signaling in HepG2 cells. Moreover, in a functional assay of cycle inhibitory properties, SP600125 also increased the percentage of HepG2 cells in the S phase, decreased the percentage of cells in the G0/G1 phase, and facilitated the Huaier extract induced S phase arrest in HepG2 cells (To further investigate whether JNK plays a role in reducing the expression of G2 cells . All theHuaier electuary ointment, of which the active ingredient is extracted from fungi of Huaier, has been used in clinic for the treatment of hepatocellular carcinoma with satisfactory results . It has In the current study, the results of MTS assay demonstrated that Huaier extract significantly attenuated the proliferation of HCC cells in a dose-dependent manner. To our knowledge, the inhibition of cell proliferation was involved in apoptosis and block of cell cycle progression. It has been reported that Huaier extract induced melanoma cells and breast cancer cells apoptosis via the increased expression of P53 and the modulation of Bcl-2/BAX protein expression , 18. MorEukaryotic cell proliferation is primarily regulated by the cell cycle, which consists of four phases: the G1 phase, the S phase, the G2 phase, and the M phase . It is wβ-catenin were analyzed following the treatment of HepG2 and Bel-7402 cells with Huaier extract and the results were consistent with previous observations that S phase arrest is accompanied by the decreased expression of cyclin D1 and β-catenin [In the current study, the expression of the important cycle regulatory protein, cyclin D1, and its upstream effector -catenin , 31. Theβ-catenin. Noticeably, treatment with SP600125 obviously decreased expression of cyclin D1 and β-catenin and also caused a significant increase in the percentage of cells in S phase and decrease in the percentage of cells in the G0/G1 phase, which was consistent with the treatment of Huaier extract on cells. All these results demonstrate that Huaier extract induces hepatocellular carcinoma cells arrest in S phase via JNK signaling pathway.Activated MAPK pathways play a central role in HCC proliferation and cell cycle development , 33. HowOverall, our findings verify that Huaier extract causes HCC cell apoptosis and induces hepatocellular carcinoma cells arrest in S phase via JNK signaling pathway, which advances our understanding on the molecular mechanisms of Huaier extract in hepatocarcinoma management. The clinical treatment for liver cancer is difficult due to its malignant biological characteristics such as invasion, metastasis, and malignant proliferation. In this study, we explored the antiproliferative mechanisms of Huaier extract on HCC cells, providing a novel prospect for liver cancer treatment. Huaier extract serves as a promising therapeutic drug for liver cancer, not only for a potent apoptosis inducing, antiangiogenic, and anti-invasive agent, but also for the block role in cell cycle progression.


The objective of this study was to evaluate the radiation dose and response in terms of local-regional progression-free survival (LRPFS) and overall survival (OS) of patients with stage IV non-small cell lung cancer (NSCLC) undergoing concurrent chemotherapy and thoracic three-dimensional radiotherapy.In all, we enrolled 201 patients with stage IV NSCLC in this study and analyzed OS in 159 patients and LRPFS in 120.P = 0.000). The 1-, 2-, 3-, and 5-year LRPFS rates of patients undergoing four to five cycles with doses ≥63 Gy and <63 Gy were 77.4% and 32.6%, 36.2% and 21.7%, 27.2% and 0, and 15.9% and 0, respectively (P = 0.002). According to multivariate analyses, four to five cycles of chemotherapy, gross tumor volume <175.00 cm3 and post-treatment Karnofsky Performance Status score stable or increased by at least 10 units were independent prognostic factors for better OS . Radiation dose to the primary tumor ≥63 Gy resulted in better OS (P = 0.057) and LRPFS (P = 0.051), both findings being of borderline significance.The 1-, 2-, 3-, and 5-year OS rates were 46.2%, 19.5%, 11.7%, and 5.8%, respectively, the median survival time being 12 months. The median survival times in differential treatment response of primary tumors were 19 of complete response, 13 of partial response, 8 of stable disease, and 6 months of progressive disease, respectively (3 and post-treatment Karnofsky Performance Status score not lower than pretreatment values.Treatment of IV NSCLC with joint administration of four to five cycles of chemotherapy and three-dimensional radiotherapy may prolong survival, particularly in patients receiving ≥63 Gy radiotherapy, with gross tumor volume <175.00 cm Systemic chemotherapy is the standard treatment option for these patients,2. NSCLCz et al.,14. The 9/L, platelet count 80 × 109/L, hemoglobin concentration ≥80 g/L, aspartate aminotransferase and alanine aminotransferase concentrations ≤2× the upper limit of the institutional normal range, total bilirubin ≤1.25× the upper limit of the institutional normal range, and creatinine concentration ≤120 μmol/L). The exclusion criteria were as follows: 1) previous thoracic surgery, radiotherapy, or chemotherapy; 2) malignant pleural or pericardial effusion; 3) pregnancy or lactation; and 4) previous malignancy or other concomitant malignant disease.This prospective study was performed with the approval of the Institutional Review Board of the Affiliated Hospital of Guiyang Medical College, and Guizhou Cancer Hospital China and informed consent was obtained from all patients. The inclusion criteria were as follows: 1) histologically or cytologically confirmed NSCLC; 2) newly diagnosed stage IV disease according to the staging system of the 2002 American Joint Committee on Cancer; 3) age 18–80 years with a Karnofsky Performance Status (KPS) score ≥70; 4) limited metastatic disease (≤five sites); 5) no contraindications to radiotherapy and chemotherapy; 6) underwent at least two cycles of chemotherapy and received radiation at doses ≥36 Gy; and 7) adequate bone marrow, liver, and renal function  scanning of the chest and abdominal region, plain or contrast-enhanced magnetic resonance imaging (MRI) of the head, and bone scintigraphy. Additional investigations were performed if indicated. All the same assessments, except for fiberoptic bronchoscopy and bone scintigraphy, were also performed after therapy.3 (16.55–892.00 cm3). The median number of cycles of chemotherapy was 4 (2–5 cycles), and the median dose to the planning target volume was 63 Gy (36–72 Gy). Post-treatment progression of the primary tumor occurred in 11 patients. Twenty-eight patients participated in post-treatment follow-up via telephone but refused re-examination by imaging; thus, 120 were assessed for local-regional progression-free survival (LRPFS). These patients’ clinical characteristics are listed in Table In all, 201 patients diagnosed with stage IV NSCLC between January 2003 and July 2010 were enrolled in this study. Nineteen patients underwent only one cycle of chemotherapy, 10 received <36 Gy and 13 cases refused post-treatment evaluation by imaging. These 42 cases were omitted from the analyses. Combination chemotherapy and radiation were administered to intrathoracic primary tumors in the remaining 159 patients, 119 of whom were male and 40 female. The patients were aged 30–80 years (median age 59 years). The median gross tumor volume (GTV) was 175.00 cm2 cisplatin (C) or carboplatin  intravenously on day 2, 140 mg/m2 paclitaxel (P) or 75 mg/m2 docetaxel (D) on day 1, and 25 mg/m2 vinorelbine (V) on days 1 and 8 every 21–28 days during thoracic radiotherapy. Concurrent thoracic radiation was given within 1 week of starting chemotherapy. After completion of thoracic radiotherapy, patients with a response or stable disease continued chemotherapy for up to four to six cycles, whereas second-line therapy was substituted in patients with progressive disease or unacceptable toxicity. PC or PCb combination chemotherapy was administered to 40.3% of patients (64/159), DC or DCb to 53.4% (85/159), and VC to 6.3% (10/159). Thirty-one, 41, 83, and four patients underwent two, three, four, and five cycles of chemotherapy, respectively. In all, 537 cycles were administered.Platinum-based doublets chemotherapy was administered according to the following regimens: 80 mg/m3 radiation treatment planning software (version 7.4f). The GTV encompassed both the primary lung lesion and any hilar and mediastinal lymph nodes that were visible on the treatment planning CT scan. The GTV plus a margin of 15 mm was defined as the planning target volume, the area covered by at least a 90% isodose surface. The V20 , maximum point dose to the spinal cord and mean esophageal dose were required to be ≤32%, 50 Gy and ≤35 Gy, respectively, in each individual treatment plan. Patients received late-course accelerated hyperfractionated radiotherapy to the thoracic primary site using 3D-conformal or intensity-modulated radiation therapy. The first course of radiotherapy was given in two Gy fractions for 5 days a week to a total dose of 36–40 Gy, whereas late-course accelerated hyperfractionated radiotherapy was delivered in two fractions of 1.5 Gy each with an interval of 6–8 h each day. The protocol required delivery of a prescription dose of 60–70 Gy to patients; individuals who did not tolerate this were to receive ≥36 Gy. Provided radiation doses to normal tissue were acceptable, the radiation dose to the primary thoracic tumor could be escalated to 72 Gy. Sixty-seven patients received <63 Gy and 92 ≥ 63 Gy (70 received 63 Gy and 22 received 63–72 Gy). Metastatic lesions were treated with 3D-conformal radiation therapy or 2D hypofractionated radiotherapy. In all, 98 patients received radiotherapy concurrently or sequentially with chemotherapy for metastatic lesions in 3–10 Gy daily fractions to a total dose of 20–60 Gy.The treatment was planned using ADAC PinnacleThe responses of the primary tumors, including complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD), were evaluated according to the World Health Organization response criteria. LRPFS was defined as the length of time between the start of treatment and either progression of the primary tumor or death. A comprehensive evaluation of both the primary tumor and distant metastases was performed 1 month after completion of chemoradiotherapy treatment. For primary tumors and metastatic lesions other than those in bone, response rate was defined as CR or PR. However, for patients with bone metastases, SD (i.e. no evidence of PD) of bone metastases was also regarded as a response. Nonresponse of primary tumors was defined as larger and/or stable lesions and of metastatic tumors as SD, PD, or development of new metastatic lesions.χ2 test. Kaplan–Meier analyses were performed to estimate LRPFS and OS and the log-rank test to compare the survival curves. The Cox proportional hazards model was used to perform multivariate analyses to assess the LRPFS and OS. Results were considered statistically significant when the two-tailed P value was <0.05.After completion of treatment, the patients underwent contrast-enhanced CT scanning of the chest and abdominal region and MRI of the head every 3 months for 2 years, and every 6 months thereafter. Bone scintigraphy was performed every 6 months for 2 years, and every 12 months thereafter. All statistical analyses were performed using the Statistical Package for Social Sciences version 13.0 . The significance of differences in proportions was assessed with the χ2 = 24.330, P = 0.000). The 1-, 2-, 3-, and 5-year OS rates of patients with CR/PR and SD/PD were 54.4% and 23.8%, 22.3% and 11.9%, 13.4% and 7.1%, and 5.4% and 4.8%, respectively, their MSTs being 14  and 7  months, respectively .Follow-up ended on 1 March 2013. Four patients were lost to follow-up; the remaining 155 patients (97.5%) underwent follow-up for 2–80 months (median 12 months). One hundred and forty-five patients had died by the study end-point, 34 (23.5%) from local progression, 26 (17.9%) from local progression and distant metastases, and 85 (58.6%) from distant metastases alone. Ten patients were still alive, their survival times being 34–80 months. The 1-, 2-, 3-, and 5-year OS rates of the 159 study patients were 46.2%, 19.5%, 11.7%, and 5.8%, respectively; their median survival time (MST) was 12  months. Twelve/159 patients (7.5%) achieved CR, 66.0% (105/159) PR, 19.5% (31/159) SD, and 6.9% (11/159) PD, the MSTs being 19 , 13 , 8 , and 6  months, respectively . The 1-, 2-, 3-, and 5-year LRPFS rates were 57.5%, 30.5%, 21.0%, and 10.2%, respectively. The LRPFS of patients in CR seemed to be longer than of those in PR and SD; however, these differences were not significant   and 10  months, respectively   was not significant.The 1-, 2-, 3-, and 5-year LRPFS rates of patients undergoing four to five cycles with doses ≥63 Gy versus <63 Gy were 77.4% and 32.6%, 36.2% and 21.7%, 27.2% and 0, and 15.9% and 0, respectively, their MSTs being 20  and 9  months, respectively . The 1-, 2-, 3-, and 5-year LRPFS rates of patients who received four to five cycles with doses ≥63 Gy and two to three cycles with doses ≥63 Gy were 77.4% and 43.5%, 36.2% and 26.1%, 27.2% and 19.6%, and 15.9% and 0, respectively, their MSTs being 20  and 9  months, respectively . In addition, the 1-, 2-, 3-, and 5-year LRPFS rates of patients who received four to five cycles with doses ≥63 Gy compared with other treatment regimens  were 77.4% and 40.5%, 36.2% and 25.4%, 27.2% and 15.8%, and 15.9% and 0, respectively, their MSTs being 20  and 10  months, respectively   and 7  months, respectively (P = 0.035), GTV <175.00 cm3  and post-treatment KPS score stable or increased by at least 10 units  were independent predictors of better OS. Both N stage  and the primary tumor radiation dose  were of borderline significance with respect to OS. GTV <175.00 cm3  and post-treatment KPS score  were independent predictors of LRPFS. The primary tumor radiation dose was of only borderline significance with respect to LRPFS  [10 BED) palliative thoracic radiotherapy reportedly improves the total symptom score and 1-year overall survival compared with lower dose radiotherapy. These data indicate that some patients benefit from receiving local radiotherapy[In patients with stage IV NSCLC, four to six cycles of platinum-based combination chemotherapy typically results in 1-year survival rates of approximately 30%–45%, median time to progression of 3–5 months, and median survival time of 8–10 months,15-17. Het al. performeet al.,21. Seve= 0.000), indicat= 0.000),18. In t= 0.000). A systeotherapy. A systeotherapy. Thus, fotherapy. In our otherapy. In the otherapy. The mosThe prognosis of NSCLC patients is closely associated with both pre-treatment and post3 and whose post-treatment KPS scores do not decrease.Four to five cycles of chemotherapy given concurrently with 3D radiotherapy for intrathoracic tumors may prolong survival, particularly in patients who receive radiotherapy at doses ≥63 Gy, GTV < 175.00 cmNSCLC: Non-small cell lung cancer; LRPFS: Local-regional progression-free survival; OS: Overall survival; PFS: Progression-free survival; MRI: Magnetic resonance imaging; CT: Computed tomography; 2D: Two-dimensional; 3D: Three-dimensional; KPS: Karnofsky Performance Status; GTV: Gross tumor volume; C: Cisplatin; Cb: Carboplatin; P: Paclitaxel; D: Docetaxel; V: Vinorelbine; MST: Median survival time.The authors declare that they have no competing interests.BL designed the study. W-WO, S-FS, Y-XH, ZM, Q-SLi, H-QLi, and Y-CG collected the data. W-WO, S-FS, Y-XH, and BL undertook the data analyses and interpretation, performed the statistical analyses and wrote the report. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/491/prepub


The calibration curve was linear in the concentration range of 0.0525–21.0 μg/mL (r = 0.9998). The accuracy was higher than 90.92%, while the intra- and interday precision were less than 6.26%. The extraction procedure provides recovery ranged from 89.44% to 92.32%, for both analyte and IS. Finally, the method was successfully applied to a bioequivalence study of a single 500 mg dose of cefuroxime axetil in 22 healthy Chinese male subjects under fasting condition. Bioequivalence was determined by calculating 90% Cls for the ratios of Cmax, AUCt0−, and AUC∞0− values for the test and reference products, using logarithmic transformed data. The 90% Cls for the ratios of Cmax (91.4%~104.2%), AUCt0− (97.4%~110.9%), and AUC∞0− (97.6%~111.1%) values were within the predetermined range. It was concluded that the two formulations  analyzed were bioequivalent in terms of rate and extent of absorption and the method met the principle of quick and easy clinical analysis. A simple, robust LC-MS/MS assay for quantifying cefuroxime in human plasma was developed. Cefuroxime and tazobactam, as internal standard (IS), were extracted from human plasma by methanol to precipitate protein. Separation was achieved on a Zorbax SB-Aq (4.6 × 250 mm, 5  Cefuroxime is a second-generation cephalosporin used against a variety of infections. Due to its low oral bioavailability, cefuroxime is administered orally as a prodrug in the form of cefuroxime axetil . Upon adSeveral methods, including HPLC-DAD, LC-MS/MS, and UPLC-MS/MS, had been reported for the determination of cefuroxime in human plasma. However, they all need a complicated and expensive sample pretreatment method, or solid-phase extraction –6, or prIn this study, we designed a sensitive and robust LC-MS/MS method following simple protein precipitation extraction with tazobactam as IS for determination of cefuroxime in human plasma. This method was accurate, sensitive, robust, and simple and was successfully applied to a bioequivalence study of a single 500 mg dose of cefuroxime axetil formulations (test and reference) in 22 healthy Chinese male subjects under fasting condition.Batch No. 130493-200704, purity 91.4%) and tazobactam (IS)  were supplied by the National Pharmaceutical Institute of China. The chemical structures are shown in Cefuroxime . The HPLC system was coupled with an API 4000 triple-quadrupole mass spectrometer  via electrospray ionization interface for mass analysis and detection. Data acquisition was performed with Analyst 1.4.2 software (Applied Biosystems).μm) at a column temperature of 30°C. An isocratic mobile phase consisting of methanol/0.05% formic acid in water  was used at a flow rate of 1 mL/min, with the injection volume of 2 μL. The autosampler was set at 4°C. A primary flow rate of 1 mL/min was split to 500 μL/min using a T-piece. All measurements were carried out with mass spectrometer operated under the negative ESI mode. The multiple reaction monitoring transitions were m/z 423.0 → 317.9 for cefuroxime and m/z 298.9 → 138.0 for IS. Other parameters were as follows: collision gas, curtain gas, ion source gas 1 and ion source gas 2 (nitrogen) 6, 15, 55, and 50 psi, respectively; dwell time 200 ms; ion spray voltage −4500 V; ion source temperature 400°C; declustering potential (DP) −40 V for cefuroxime and −34 V for IS; collision energy −10 V for cefuroxime and −22 V for IS; collision exit potential (CXP) −10 V for cefuroxime and −9 V IS; and entrance potential (EP) −10 V for cefuroxime and IS. Unit resolution was used for both Q1 and Q3 mass detection.Separation was performed on an Agilent Zorbax SB-Aq  were also prepared in the same way. Tazobactam (IS), stock solution 1.86 mg/mL prepared in 50% methanol, was diluted with 50% methanol to give a final concentration of 18.6 μg/mL. Both of these stock solutions were stored at 4°C avoiding light for using.Stock solution (1.05 mg/mL) of cefuroxime was prepared in 50% methanol and was further diluted with 50% methanol to achieve standard working solutions at concentrations of 210, 105, 52.5, 21.0, 10.5, 5.25, 2.10, 1.05, and 0.525 μL) were used to spike blank plasma samples (200 μL). The final concentrations of cefuroxime standard calibration plasma samples were 21.0, 10.5, 5.25, 2.10, 1.05, 0.525, 0.210, 0.105, and 0.0525 μg/mL, respectively. The QC samples were also prepared in the same way by adding 20 μL diluted QC stock solutions to 200 μL blank human plasma. The final concentrations of cefuroxime in the low-, medium-, and high-QC plasma samples were 0.0842 μg/mL, 1.68 μg/mL, and 16.8 μg/mL, respectively.The standard working solutions  and 20 μL IS (18.6 μg/mL) solution. After a thorough vortex mixing for 30 s, mixtures were precipitated with 600 μL methanol, vortex-mixed for 30 s, and centrifuged at 13000 rpm for 5 min. Finally, 2 μL of supernatant was injected into the LC-MS/MS system.After frozen human plasma samples were thawed at ambient temperature and adequately vortexed, a total of 200 The current method was validated prior to the analysis of human plasma samples according to the guidance of bioanalytical method validation . The selμg/mL. The validity of the linear regression equation was indicated by the correlation coefficient (r).To evaluate selectivity, drug-free plasma samples from 6 individuals were analyzed to check for the presence of any interfering peaks at the elution times of both cefuroxime and IS. The calibration curves were constructed using 9 standards ranging in concentration from 0.0525 to 21.0 n = 6): LLOQ (0.0525 μg/mL), low (0.0842 μg/mL), medium (1.68 μg/mL), and high (16.8 μg/mL).The intraday and interday precision and accuracy were evaluated by assessing QC samples at the following concentrations . The study followed a single dose, two-way randomized crossover design with a 1-week washout period between doses. After an overnight fast of at least 10 h, subjects received a single oral 500 mg dose of either the test or reference formulation with 240 mL of water. During both treatment periods, heparinized blood samples were collected at the following times: before (0.0 h) and at 0.33, 0.67, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, 8.0, and 10.0 h after dosing. The blood samples were centrifuged at 4000 rpm for 10 min, and plasma samples were separated and stored at −20°C until analyzed.Cmax and Tmax obtained directly from the measured data, other PK parameters  were calculated by noncompartmental analysis using Drug Statistics (DAS) software 2.1.1 . The relative bioavailability (F%) of the tested formulation was calculated as follows: F% = AUCt0–(test)/AUCt0–(reference) × 100%. Analysis of variance (ANOVA) using DAS 2.1.1 was performed on Cmax, AUCt0–, and AUC∞0– values evaluating treatment, period, sequence, and subject within sequence effects. Their ratios (test versus reference) of log-transformed data were analyzed for relative bioavailability. The 90% Cls served as interval estimates and were determined by two 1-sided t-tests. If the differences in PK parameters between the two formulations were not statistically significant (P > 0.05) and the 90% Cls for the ratios of Cmax, AUCt0–, and AUC∞0– are located within the bioequivalence criteria range (80~125% for AUC and 70~143% for Cmax), then the two formulations were considered to have met the regulatory requirement for bioequivalence.In addition to 18, Zorbax SB-Aq, Hypersil GOLD Aq, and Atlantis T3) were carefully evaluated. Finally, Agilent SB-Aq column was chosen in the present study for its high efficiency and peak symmetry. Different mobile phases  were examined to obtain efficient chromatography and relatively short run time for cefuroxime and IS. It was found that the addition of formic acid could remarkably improve the peak symmetry and ionization of cefuroxime and IS. When methanol was used as the organic phase, the peak of cefuroxime was further improved. Therefore, the mobile phase was selected as methanol-mixture of 0.05% formic acid in water to achieve better separation and less interference from other components in the plasma. The retention time for cefuroxime and IS was 6.8 and 5.9 min, respectively. The total chromatographic run time was 8.0 min  least squares linear regression analysis with a coefficient of determination r = 0.9998. Typical equations for the calibration curve were as follows: Y = (0.186 ± 0.002)X + (0.00024 ± 0.00049) (n = 3), where X represents the plasma concentration of cefuroxime (μg/mL) and Y represents the ratios of cefuroxime peak area to that of IS. LLOQ under the optimized conditions was 0.0525 μg/mL for cefuroxime, which was judged from the fact that the precision and accuracy were less than 20% , and the interday precision was less than 6.26%. The accuracy ranged from 90.92% to 101.8%. The above values were within the acceptable range, which demonstrated the good stability and repeatability of this described method.QC samples at three concentration levels were calculated over three validation runs (once a day). Six replicates of each QC level were determined in each run. μg/mL, respectively. Mean recovery for the IS was 92.32 ± 0.90%. The RSDs for all recoveries were less than 5.21% throughout the entire concentration ranges, indicating assay consistency.The recoveries of the protein precipitation for cefuroxime were 89.44 ± 4.66%, 91.94 ± 0.94%, and 91.39 ± 1.67% at concentrations of 0.0842, 1.68, and 16.8 μg/mL, and IS. The results obtained were well within the acceptable limit [The matrix effect was evaluated to determine the influence of matrix components on analyte quantification. Average matrix effect values obtained were 110.6 ± 5.10%, 109.8 ± 1.58%, 111.4 ± 2.12%, and 108.8 ± 1.52% for QC samples at concentrations of 0.0842, 1.68, and 16.8 μg/mL, except for long-term stability for 0.105, 1.68 and 16.8 μg/mL. The results indicated that cefuroxime was stable in untreated plasma when placed in the short-term (6 h) at room temperature, repeated three freeze/thaw cycles and stored at −20°C for 145 days. In addition, it was found also stable in treated-plasma samples when placed in autosampler at 4°C for 20 h , AUCt0– (97.4%~110.9%), and AUC∞0–(97.6%~111.1%) values for the test and reference products were all located within the bioequivalence criteria range (80~125% for AUC and 70~143% for Cmax), proposed by China Food and Drug Administration [No significant differences in AUCstration . It was A simple and sensitive LC-MS/MS method for the quantification of cefuroxime in human plasma was developed. Method validation has been demonstrated by a variety of tests for selectivity, linearity, sensitivity, precision, recovery, matrix effect, and stability. This method is attractive for the pharmacokinetic and bioequivalence analysis of cefuroxime, because of its high sensitivity and accuracy.


Salinicoccus halodurans H3B36 is a moderately halophilic bacterium isolated from a sediment sample of Qaidam Basin at 3.2 m vertical depth. Strain H3B36 accumulate Nα-acetyl-α-lysine as compatible solute against salinity and heat stresses and may have potential applications in industrial biotechnology. In this study, we sequenced the genome of strain H3B36 using single molecule, real-time sequencing technology on a PacBio RS II instrument. The complete genome of strain H3B36 was 2,778,379 bp and contained 2,853 protein-coding genes, 12 rRNA genes, and 61 tRNA genes with 58 tandem repeats, six minisatellite DNA sequences, 11 genome islands, and no CRISPR repeat region. Further analysis of epigenetic modifications revealed the presence of 11,000 m4C-type modified bases, 7,545 m6A-type modified bases, and 89,064 other modified bases. The data on the genome of this strain may provide an insight into the metabolism of Nα-acetyl-α-lysine. The amount of Nα-acetyl-α-lysine in the cell was increased and could be accumulated to a high level when strain H3B36 was subjected to salt stress or heat stress. Unlike other compatible solutes, Nα-acetyl-α-lysine has only been found to date in Salinibacter ruber to date, and the molecular mechanisms through which this compound is synthesized and stored are unclear [Moderately halophilic bacteria are a group of halophilic microorganisms that grow optimally in media containing between 3 % and 15 % (w/v) NaCl. These bacteria exhibit strong salt tolerance and are widely distributed in different high-salt habitats, such as hypersaline soils and lakes, solar salterns, and salted foods . To copesmolytes , such asectoine) . In stra unclear , 10.Salinicoccus halodurans W24T (= CGMCC 1.6501T = TDSM 19336) [Salinicoccus, which was first described by Ventosa et al. [Staphylococcaceae. To date, 16 validly named species of Salinicoccus have been identified; however, only six genome sequences are available. All species of the genus Salinicoccus are defined as moderately halophilic bacteria. These organisms may have potential applications in various fields, including as additives in the food industry; for production of polymer compounds, enzymes, and stress protectants; and in environmental protection and biodegradation [Based on analysis of the 16S rRNA gene sequence, this strain is most closely related to  19336T) . The gena et al. , 13, belradation –19.Nα-acetyl-α-lysine and explore the genome of the Salinicoccus spp, we performed complete genome sequence analysis and annotation of Salinicoccus halodurans H3B36.To obtain insights into the metabolic pathway of Salinicoccus halodurans W24 T with 99.9 % 16S rRNA gene sequence identity, and as such, strain H3B36 was classified as a strain of Salinicoccus halodurans.Strain H3B36 Table  was isolThe cell morphology of strain H3B36 was determined using scanning electron microscopy Fig. . MicroscSalinicoccus halodurans H3B36 was selected for genome sequencing because we observed the presence of a unique compatible solute for protection and potential industrial applications. The complete genome sequence has been deposited in GenBank under the accession number CP011366. Sequencing, annotation, and analysis were performed at WUHAN Institute of Biotechnology, China. The project information and its association with MIGS version 2.0 are shown in Table Salinicoccus halodurans H3B36 was grown aerobically in GMH medium containing 5 g/L casamino acid, 5 g/L yeast extract, 4 g/L MgSO4 · 7H2O, 2 g/L KCl, 0.036 g/L FeSO4 · 7H2O, 0.36 mg/L MnCl2 · 7H2O, and 60 g/L NaCl, at pH 7.0 (titrated with 1 M NaOH). Genomic DNA from freshly grown cells harvested in the exponential growth phase was extracted using the QIAGEN Genomic DNA Buffer Set and QIAGEN Genomic-tip 100/G according to the manufacturer’s protocols. The prepared DNA was evaluated on a 0.75 % agarose gel to verify the integrity of the molecular weight fragments. Qualification and quantification of the prepared DNA sample was measured with a NanoDrop instrument  and Qubit  to confirm the suitability of the DNA sample for high-throughput next-generation sequencing.Salinicoccus halodurans H3B36 was sequenced using third-generation sequencing technology on a PacBio RS II instrument. The analysis produced a total of 573,153,827 bp, and 54,457 post-filter reads with a mean length of 10,524 bp were obtained. The Hierarchical Genome Assembly Processing pipeline, version 2.2.0, was used to assemble the genome [de novo to one final 2,778,378-bp complete contig with 212 × depth of coverage.The genome of e genome . Long reThe RAST Prokaryotic Genome Annotation Server was used to predict protein-coding open reading frames, tRNAs, and structural RNA genes . The CluSalinicoccus halodurans H3B36 was found to be 2,778,378 bp and had a G + C content of 44.54 %. No plasmids were found. RAST predicted 2,853 coding sequences, 61 tRNA genes, and 16 structural RNA genes. The predicted CDSs represented 88.79 % of the total genome sequence, with an average length of 864.72 bp. Genome analysis showed that the genome of strain H3B36 contained 58 tandem repeats, six minisatellite DNA sequences, and 11 genome islands. Further analysis of epigenetic modifications revealed 11,000 m4C-type modified bases, 7,545 m6A-type modified bases, and 89,064 other modified bases in the genome. Furthermore, several restriction modification genes were found, with eight belonging to the type I system, three belonging to the type II system, and one belonging to the type IV system. The genome statistics and gene distributions into COG functional categories are presented in Tables The complete genome sequence of Salinicoccus halodurans H3B36 contained many genes related to the stress response, such as choline and betaine transporters, glycerol uptake facilitator protein, cold-shock protein, chaperones proteins, and others. These genes allowed the strain to cope with different environmental stresses. Experimentation and additional analysis of these genes may help to elucidate the mechanisms mediating the stress response and facilitate the development of Salinicoccus halodurans H3B36 for use in industry applications. In addition, several genes encoding hydrolases, including amylase (1), protease (19), pullulanase (2), lipase (3), phosphoesterase (5), and glucosidase (4), were identified in the genome. Hydrolases are highly valuable resources for some specific industrial processes, and hydrolases from various extremophiles may have many advantages [Salinicoccus halodurans H3B36 might have the potential for application in industrial biotechnology as a producer of miscellaneous hydrolases.Genome analysis showed that vantages , 19. TheNα-acetyl-α-lysine was found play a key role in protecting Salinicoccus halodurans H3B36 cells under different stresses . Genome annotations showed that lysine may be synthesized through the acetyl-dependent diaminopimelic acid pathway in Salinicoccus halodurans H3B36. One 8-kb gene cluster containing eight genes was predicted to be involved in Nα-acetyl-α-lysine biosynthesis. Six genes in the cluster map to enzymes in the acetyl-dependent diaminopimelic acid pathway, including the genes encoding aspartokinase, aspartate-semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-acetyltransferase and diaminopimelate decarboxylase. Nα-acetyl-α-lysine is a derivative of lysine, so this gene cluster may participate in the synthesis of Nα-acetyl-α-lysine. Further studies are required to verify this assumption and identify the metabolic pathway mediating Nα-acetyl-α-lysine biosynthesis in Salinicoccus halodurans H3B36.Salinicoccus halodurans. The genome size of Salinicoccus halodurans H3B36 (2.78 M) is larger than the other sequenced members of genus Salinicoccus including Salinicoccus sp. SV-16 (2.59 M), Salinicoccus luteusTDSM 17002 (2.55 M), Salinicoccus albusTDSM 19776 (2.64 M), Salinicoccus carnicancri CrmT (2.67 M), and Salinicoccus roseus W12 (2.56 M). Salinicoccus halodurans H3B36 has a G + C content (44.5 %) higher than Salinicoccus albusTDSM 19776 but lower than those of Salinicoccus carnicancri CrmT, Salinicoccus sp. SV-16, Salinicoccus luteusTDSM 17002, and Salinicoccus roseus strain W12 . Further comparative genomic study shows that the Nα-acetyl-α-lysine related gene cluster is also found in other sequenced members of genus Salinicoccus. The gene cluster in Salinicoccus sp. SV-16, Salinicoccus luteusTDSM 17002, Salinicoccus carnicancri CrmT, and Salinicoccus roseus W12 containing eight genes are similar to that in Salinicoccus halodurans H3B36. Salinicoccus albusTDSM 19776 has a slight discrepancy, which lacks aspartokinase in its gene cluster. The genome of Salinicoccus halodurans H3B36 provides important insights into our understanding of the metabolism of Nα-acetyl-α-lysine. Furthermore, the sequence of Salinicoccus halodurans H3B36 provides useful information and may contribute to facilitate applications of genus Salinicoccus in industrial biotechnology.This is the first report describing the genome sequence of


Up to now there are no studies that focus on the epigenetic changes produced as a result of Aβ-42 accumulation and the possible involvement in the different expression of related AD-genes. Under several behavioral and cognition test, we found impairment in memory and psychoemotional changes in female 5XFAD mice in reference to wild type that worsens with age.APP, BACE1), Oxidative Stress  and inflammation ; no changes were found in non-amyloidogenic pathway indicators such as ADAM10.Cognitive changes correlated with alterations on protein level analysis and gene expression of markers related with tau aberrant phosphorylation, amyloidogenic pathway (Dnmt1 increases in younger 5XFAD and Dnmt3a and b high levels in the oldest transgenic mice. Similar pattern was found with histone methyltransferases such as Jarid1a and G9a. Histone deacetylase 2 (Hdac2) or Sirt6., both related with cognition and memory, presented a similar pattern. Taken together, these hallmarks presented by the 5XFAD model prompted its use in assessing different potential therapeutic interventions based on epigenetic targets after earlier amyloid deposition.Epigenetics changes as higher CpG methylation and transcriptional changes in DNA methyltransferases (DNMTs) family were found.  APOE) [APOE exerts this influence remains largely undefined, with indications including a key role in mediating Aβ metabolism and clearance [Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder  and the APOE) -10. The learance . Numeroulearance . Howeverlearance . Reasonslearance . The occlearance ; thus, iIn addition to genetic factors, growing evidences demonstrated that the effects of environmental factors and epigenetic mechanisms also play an important role in the pathogenesis of AD -20. EpigIn addition, Aβ changes in DNA methylation are associated with induction of several genes, causing apoptotic and cell loss. It remains unclear how these changes in DNA methylation began, but it is suggested that various cellular mechanisms, such as OS and Aβ-42, are involved . OS and The detection and identification of mutations in APP, PS1 and PS2 genes associated with Familial AD (FAD) have allowed the generation of various transgenic murine models. These transgenic models have the potential to enable us to understand the pathogenesis of the disease, and to develop and evaluate new and effective therapeutic targets. The 5XFAD mouse was formally established and declared in 2006 by Oakley and the specific characteristic of this model being that it is generated to promote rapid, aggressive and complete development of AD pathological interactions. This novel model of Aβ pathology consists of two APP mutations (APP K67ON/M671L (Swedish), I716V (Florida), and V717 (London) and two PS1 mutations (PS1 M146 and L286V) on B6/SJL and each transgene is regulated by Thy1 promoter .The present work focuses on the study of the possible involvement of Aβ-42 accumulation in the production of OS and in subsequent modifications in the epigenetic machinery in the murine model of AD, 5XFAD. We aimed to discuss the role of global DNA methylation and the histone epigenetic enzymes involved in epigenetic modification acting in the control of gene expression and memory in this AD mouse model.NORT analysis demonstrated significant differences between the 5XFAD and Wt strains at 8, but not at 2 months of age  = 4.5631, p = 0.0416; genotype, F = 4.563, p = 0.001, and interaction age × genotype F = 6,211, p = 0.0189). The DI was significantly decreased in 5XFAD mice at 8 months of age compared with young 5XFAD mice and with age-matched Wt, indicating impairment in hippocampal memory processes Fig. . AlthougBehavioral and emotional tests showed differences in anxiety-like behavior, through EPM test Figs. . Old 5XFMolecular analysis demonstrated that there a significant increase in BACE1 and APP gene expression Figs.  in 8 monAccording to gene expression results, when protein levels for BACE1 were studied significant increases of BACE1 in 5XFAD in comparison with Wt were demonstrated  = 0.5599, n.s.; genotype, F = 15.05, p = 0.0047, and interaction age × genotype F = 11.17, p = 0.0102) Fig. . sAPPβ pTau hyperphosphorylation was also studied with a focus on pTau (Ser396), whose levels exhibited an age dependent increase in pTau both in Wt and in 5XFAD mice  = 69.06, p <0.0001; genotype, F = 8.256, p = 0.0207, and interaction age × genotype F = 2.108, n.s.) Fig. .Aldh2 enzyme transcription levels were demonstrated in older 5XFAD  = 9.098, p =0.0107; genotype, F = 1.789, p = n.s., and interaction age × genotype F = 16.06, p = 0.0017)  = 97.05, p <0.001; genotype, F = 136.4, p < 0.0001, and interaction age × genotype F = 135.0, p < 0.0001) and IL-6  = 17.74, p <0.0119; genotype, F = 51.40, p < 0.04871, and interaction age × genotype F = 17.74, p < 0.0012) were found among animal groups. Both cytokines were significant higher in 8 months-old 5XFAD mice compared with young animals and age mated Wt indicating an age dependent inflammatory process  = 17.9969, p <0.0028; genotype, F = 6.875, p = 0.0306, and interaction age × genotype F = 5.446, p = 0.049), jointly with 4-HNE  = 76.55, p <0.0001; genotype, F = 36.13 p = 0.0003, and interaction age × genotype F = 3.451, n.s.) Figs.  were fou17) Fig.  althoughups Fig. . With ress Figs. .OS leads to gliosis and neuronal loss. In our hands increases in Glial Fibrillary Acidic Protein (GFAP) staining was found in 5XFAD animals compared with Wt, indicating harmful environment Fig. .Quantification of fluorescence intensity demonstrated higher astrogliosis in hippocampus of 5XFAD with regard to Wt and significant worsening with age  = 0.1417, n.s.; genotype, F = 0.1785, n.s., and interaction age × genotype F = 20.68, p = 0.007)  = 12.90, p =0.0037; genotype, F = 63.51, p < 0.0001, and interaction age × genotype F = 16.18, p = 0.0017; Dnmt3b: age, F = 11.06, p =0.0060; genotype, F = 20.76, p < 0.0007, and interaction age × genotype F = 2.668, n.s.)  = 0.4151, n.s.; genotype, F = 0.1934, n.s. and interaction age × genotype F = 25.93, p = 0.003), without changes in Tet2  in 5XFAD mice with respect to Wt which reached significance at the age of 2  = 2.383, n.s.; genotype, F = 29.32, p = 0.0006, and interaction age × genotype F = 2.583, n.s.) Fig. , whereas007) Fig . Dnmt3a .) Figs. . TET oxiet2 Fig. .Jarid1a, a specific H3K4 histone demethylase, increased in older 5XFAD  = 12.90, p =0.0037; genotype, F = 0.0367, n.s., and interaction age × genotype F = 8.272, p = 0.0139)  between non-transgenic and 5XFAD mice. In Wt mice, there was a significant G9a increase with age, that was absent in 5XFAD animals  = 3.604, n.s.; genotype, F = 5.779, p = 0.0333, and interaction age × genotype F = 14.61, p = 0.0024)  = 8.394, p =0.0134; genotype, F = 2.184, n.s., and interaction age × genotype F = 17.60, p = 0.0012)  = 19.88, p =0.0008; genotype, F = 2.378, n.s., and interaction age × genotype F = 6.581, p = 0.0225)  Fig. . In addi24) Fig. . In term12) Fig. . No diffage Fig. . Sirtuint2 Figs. , but an 25) Fig. .Epigenetic modifications such as global DNA methylation/hydroxymethylation, histone demethylation levels and histone acetylation levels play a crucial role in gene regulation . EpigeneAPP and Bace1 expression, higher protein levels of BACE1 and sAPPβ, and higher number of amyloid plaques indicating the development of AD pathology as described elsewhere in aged 5XFAD [5XFAD mice are characterized by higher levels in amyloidogenic pathology . These med 5XFAD . Anothered 5XFAD . In our ed 5XFAD . InteresThese molecular and biochemical findings are phenotypically demonstrated by loss in memory abilities and also by impairment in behavioral and emotional parameters in aged 5XFAD. Therefore, in our hands female 5XFAD initiate the AD characteristics as earlier as 2 months but the disease is completely developed at 8 months of age in a similar way described by Oakley and coworkers (2006).Here we confront the implication of epigenetic changes with AD hallmarks in female 5XFAD (including Aβ deposition and OS processes), which we addressed from a holistic point of view, in order to demonstrate a possible relationship between the processes gated to earlier Aβ deposition that account for, in the 5XFAD brain, the epigenetic response, both to delay or to contribute the evolution of the disease.Dnmt1 and Dnmt3a. Dnmt3b is maintained as increased in older mice. Although no changes in hydroxymethylCytosine (5-hmC) levels were found, Tet1 but not Tet2  increases in 2 month-old transgenic mice, followed by a reduction in older animals. In reference to histone modification by acetylation or deacetylation, again we found differences in transgenic mice, with increases in Hdac2, Jarid1a or G9a.By means of this approach, we demonstrate that a significant increase in global DNA methylation, measured by 5-methylCytosine (5-mC) levels in young animals when Aβ initiate deposition and with concomitant increase of Dnmt1 and Dnmt3b in young 5XFAD lead to higher cytosine methylation, prior to the development of AD hallmark in this mouse, underlying the earlier participation for methylation processed at the onset of the pathology. Some authors reported that in aged mice there occurs a diminution in hippocampal expression of Dnmt3a, and that increasing this expression reverses memory deficits whereas knockdown in young mice impairs memory formation [Dnmt3a and Dnmt3b in transgenic animals could be an attempt to surpass the harmful effect of the Aβ increment as well as the high oxidant environment that occurs in 5XFAD when the disease is completely developed, as depicted in negative correlations found among the results obtained in the present work  and ARID domain, containing histone lysine demethylase 1a (JARID1a) increased histone acetylation by inhibiting histone deacetylase 1 function and modifying gene transcription in a demethylase-independent manner. nescence . Moreovenescence .G9a gene expression in 5XFAD compared with non-transgenic mice is coincident with the loss of memory in this AD mouse model.Different histone methylations are also associated with behavioral disorders. G9a methylates H3K9 and this hypermethylation leads to the downregulation of BDNF . It wellHdac2 increased significantly in 2-month old transgenic mice whereas diminution of Sirt6 was found. No significant changes were found for these and other histone acetylases or deactetylases tested at older age . A very significant negative correlation between amyloid plaques and HDAC2 was found  and tap water and were maintained under standard temperature conditions (22 ± 2°C) and 12h:12h light-dark cycles (300 lux/0 lux).Female Wild type  and 5XFAD  mice at both 2 and 8 months of age, were used. These animals had Studies were performed in accordance with the Institutional Guidelines for the Care and Use of Laboratory Animals established by the Ethical Committee for Animal Experimentation at the University of Barcelona.® ver. 3.0 software .This test is employed to assess anxiety . The bas® ver. 3.0 software, and each trial were recorded for later analysis, utilizing a camera fixed to the ceiling at a height of 2.1 m situated above the apparatus. Mice were placed at the center, or at one of the four corners, of the Open Field and allowed to explore the apparatus for 5 min. After the 5-min test, the mice were returned to their home cages and the Open Field was cleaned with 70% ethyl alcohol and allowed to dry between tests. To assess the animal's habituation process to the novelty of the arena, the mice were exposed to the apparatus for 5 min on 2 consecutive days. The behaviors scored included Line Crossing, Center Entries, Center Stay Duration, Rearing, Defecation, and Urination. Each animal was then given a score for total locomotor activity, which was calculated as the sum of total distance, line crosses and number of rears.Open Field Test (OFT) apparatus was constructed of white plywood (50 × 50 × 25 cm) . Red linThis test was conducted in a 90-degree, two-arm, 25-cm-long, 20-cm-high maze . Light iAn open circular pool  was filled halfway with water  and wate® ver. 3.0 statistical software. The learning phase consisted of 6 days of trials for each mouse. The animals were submitted to five trials each day starting from the positions set (in random order) and without a resting phase between each trial and the subsequent one. At each trial, the mouse was placed gently into the water, facing the wall of the pool, and allowed to swim for 60 sec. If not able to locate the platform in this period of time, the mouse was guided to the platform by the investigator. Animals were left on the platform each time for 30 sec in order to allow spatial orientation.The animal's swimming paths were recorded by a video camera mounted above the center of the pool, and the data were analyzed with SMARTThe parameters measured were latency time in finding the platform, time spent in each quadrant, and distance swum for each trial; the mean was calculated for each trial day. A memory test was performed at the end of the learning days, in which the platform was removed and the time spent by each mouse in each quadrant was measured.Mice were euthanized 3 days after the last trial was conducted, and the brain was quickly removed from the skull. Hippocampus were dissected and frozen in powdered dry ice and maintained at −80°C for further use. Tissue samples were homogenized in lysis buffer containing phosphatase and protease inhibitors , and cytosol and nuclear fractions was obtained as described elsewhere. Protein concentration was determined by the Bradford method. Fifteen μg of protein was separated by Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) (80002013)15%) and transferred onto PolyVinylidene DiFlouride (PVDF) membranes (Millipore). The membranes were blocked in 5% non-fat milk in Tris-Buffered Saline containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature, followed by overnight incubation at 4°C with primary antibodies diluted in TBS-T and 5% Bovine Serum Albumin (BSA) (See details in Table 315%) and™ , nuclear staining was performed with Hoechst 2 μg/mL for 5 min at room temperature. Slides were allowed to dry overnight after mounting and image acquisition was performed with a fluorescence laser microscope . At least four images from four different individuals by group were analyzed with ImageJ/Fiji software available online from the National Institutes of Health, NIH).For immunohistochemical studies, the frozen brains were embedded into OCT Cryostat Embedding Compound , cut into 20-μm-thick sections on a cryostat  at −20°C, and placed on slides. After 3 h of drying time at room temperature, the slices were fixed with acetone at 4°C for 10 min, allowed to dry overnight, and finally stored at (0002212)20°C until their further staining. For the staining procedure, the brain sections were first rehydrated by 5-min incubation in Phosphate-Buffered Solution (PBS). Afterward, the blocking/permeabilization step was performed (20 min in PBS 1% BSA + 1% Triton). Following two, 5-min washings in PBS, the slices were incubated overnight at 4°C with the primary antibodies  for 20 min at room temperature in the dark. Subsequently, these were submitted to washes in 3-min series, specifically with 80% ethanol (two washes), 90% ethanol (one wash), and three washes with PBS. Finally, the slides were mounted using Fluoromount (EMS), allowed to dry overnight at room temperature in the dark, and stored at 4°C. Image acquisition was performed with an epifluorescence microscope . For plaque quantification, similar and comparable histological areas were selected, focusing on adjacent positioning of the hippocampus and the whole cortical area.Aldh2), amyloid beta A4 Precursor (PreAPP), β-secretase 1 (Bace1) and Disintegrin and Metalloproteinase 10 (Adam10), inflammatory genes Interleukin 6 (IL-6) and Tumor necrosis factor alpha (TNF-α), inducible Nitric Oxide Synthase (iNOS), epigenetic enzymes genes DNA (cytosine-5-)-methyltransferase 1 (Dnmt1), DNA (cytosine-5-)-methyltransferase 3 alpha (Dnmt3a), DNA (cytosine-5-)-methyltransferase 3 beta (Dnmt3b), Tet methylcytosine dioxygenase 1 (Tet1), Tet methylcytosine dioxygenase 2 (Tet2), lysine (K)-specific demethylase 5A (Jarid1a), histone-lysine N-methyltransferase 2 (G9a), histone deacetylase 1 (Hdac1), histone deacetylase 2 (Hdac2), sirtuin 1 (Sirt1), sirtuin 2 (Sirt2), sirtuin 6 (Sirt6). Normalization of expression levels was performed with Actin for SYBER Green and TATA-binding protein (Tbp) for TaqMan. The primers were as follows: for Aldh2, forward 5′-GCAGGCGTACACAGAAGTGA-3′ and reverse 5′-TGAGCTTCATCCCCTACCCA-3′, for PreAPP, forward 5′-AGGACTGACCACTCGACCAG-3′ and reverse 5′-CTTCCGAGATCTCTTCCGTCT-3′, for Bace1, forward 5′-AAGCTGCCGTCAAGTCCATC-3′ and reverse 5′-GCGGAAGGACTGATTGGTGA-3′, for Adam10, forward 5′-GGGAAGAAATGCAAGCTGAA-3′ and reverse 5′-CTGTACAGCAGGGTCCTTGAC-3′, for IL-6, forward 5′-ATCCAGTTGCCTTCTTGGGACTGA-3′ and reverse 5′-TAAGCCTCCGACTTGTGAAGTGGT-3′, for TNF-α, forward 5′-TCGGGGTGATCGGTCCCCAA-3′ and reverse 5′-TGGTTTGCTACGACGTGGGCT-3′, for iNOS, forward 5′-GGCAGCCTGTGAGACCTTTG-3′ and reverse 5′-GAAGCGTTTCGGGATCTGAA-3′, for Dnmt1, (Mm01151063_m1), for Dnmt3a, forward 5′-GGGCCACACGGCAGAG-3′ and reverse 5′-CACGGTTCTCCTCCTGTTCC-3′, for Dnmt3b, forward 5′-TGCCAGACCTTGGAAACCTC-3′ and reverse 5′-GCTGGCACCCTCTTCTTCAT-3′, for Tet1, forward 5′-CTGCCAACTACCCCAAACTCA-3′ and reverse 5′-TCGGGGTTTTGTCTTCCGTT-3′, for Tet2, forward 5′-CCATCATGTTGTGGGACGGA-3′ and reverse 5′-ATTCTGAGAACAGCGACGGT-3′, for Jarid1a, forward 5′-TCCGTGTGTCATCAGCCAAA-3′ and reverse 5′-CAAGCCTACGCCAGAGTCAA-3′, for G9a, forward 5′-TTCCTTGTCTCCCCTCCCAG-3′ and reverse 5′-GACGGTGACAGTGACAGAGG-3′, for Hdac1, forward 5′-TCACCGAATCCGCATGACTC-3′ and reverse 5′-TCTGGGCGAATAGAACGCAG-3′, for Hdac2, forward 5′-CTATCCCGCTCTGTGCCCT-3′ and reverse 5′-GAGGCTTCATGGGATGACCC-3′, for Sirt1, forward 5′-AACACACACACAAAATCCAGCA-3′ and reverse 5′-TGCAACCTGCTCCAAGGTAT-3′, for Sirt2, forward 5′-TGCAGGAGGCTCAGG ATTC-3′ and reverse 5′-GTCACTCCTTCGAGGGTCAG-3′, for Sirt6, forward 5′-GTCTCACTGTGTCCCTTGTCC-3′ and reverse 5′-GCGGGTGTGATTGGTAGAGA-3′, for Actin, forward 5′-CAACGAGCGGTTCCGAT-3′ and reverse 5′-GCCACAGGTTCCATACCCA-3′, Tbp, (Mm00446971_m1).Total RNA isolation was carried out by means of Trizol reagent following the manufacturer's instructions. RNA content in the samples was measured at 260 nm, and sample purity was determined by the A260/280 ratio in a NanoDrop™ ND-1000 (Thermo Scientific). Samples were also tested in an Agilent 2100B Bioanalyzer (Agilent Technologies) to determine the RNA integrity number. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was performed as follows: 2 μg of messenger RNA (mRNA) was reverse-transcribed using the High Capacity (complementary DNA) cDNA Reverse Transcription kit (Applied Biosystems). Real-time quantitative PCR (qPCR) was employed to quantify the mRNA expression of Aldehyde dehydrogenase 2  employing SYBR Green PCR Master Mix (Applied Biosystems). Each reaction mixture contained 7.5 μL of cDNA, whose concentration was 2 μg, 0.75 μL of each primer (whose concentration was 100 nM), and 7.5 μL of SYBR Green PCR Master Mix (2X) and for TaqMan gene expression assays (Applied Biosystems), each 20 μL of TaqMan reaction, 9 μL cDNA (18ng) was mixed with the 1 μL 20x probe of TaqMan Gene Expression Assays and 10 μL of 2X TaqMan Universal PCR Master Mix.Data were analyzed utilizing the comparative Cycle threshold (Ct) method (ΔΔCt), where the actin transcript level was utilized to normalize differences in sample loading and preparation. Each sample (n=4(0002013)5) was analyzed in triplicate, and the results represented the n-fold difference of transcript levels among different samples.t test for independent samples. Statistical significance was considered when p values were <0.05. Statistical outliers were performed out with the Grubs test and were removed from analysis.Data are expressed as the mean ± Standard Error of the Mean (SEM) from at least 4(0002013)5 samples. Data analysis was conducted using GraphPad Prism ver. 6 statistical software. Means were compared with two-way Analysis of Variance (ANOVA) and post hoc analysis. Comparisons between groups were performed by the unpaired Student Dnmt1 and Hdac2.In addition, partial correlation controlling for group were calculated using SPSS 19.00, between the following variables: DI, Time in open arms, Locomotor activity, Amyloid plaques, SOD1, 4-HNE, DNMT1 and HDAC2. Partial correlation coefficients were calculated using the data from number of Amyloid plaques obtained with S-Thioflavin staining; behavioral parameters from NORT, EPM and OFT; protein expression for SOD1 and 4-HNE; and gene expression for


Daily dose of rimonabant (10 mg/kg) was administered to HFR and R group by oral gavage for two weeks. Treatment induced a decrease in hepatic malondialdehyde concentration in HFR group compared to HF group (P < 0.01). The concentration of nitrites + nitrates in liver was decreased in HFR group compared to HF group (P < 0.01). Liver content of reduced glutathione was higher in HFR group compared to HF group (P < 0.01). Total liver superoxide dismutase activity in HFR group was decreased in comparison with HF group (P < 0.01). It was found that rimonabant may influence hepatic iron, zinc, copper, and manganese status. Our study indicates potential usefulness of cannabinoid receptor type 1 blockade in the treatment of HFD-induced NAFLD.The present study deals with the effects of rimonabant on oxidative/nitrosative stress in high diet- (HFD-) induced experimental nonalcoholic fatty liver disease (NAFLD). Male mice C57BL/6 were divided into the following groups: control group fed with control diet for 20 weeks (C;  Secondly, oxidative/nitrosative stress, mitochondrial dysfunction, lipotoxicity, and overproduction of proinflammatory cytokines have been involved in progression of liver steatosis to steatohepatitis and fibrosis [Nonalcoholic fatty liver disease (NAFLD) includes a wide spectrum of liver diseases ranging from simple steatosis to steatohepatitis, which can progress to fibrosis, cirrhosis, and, ultimately, hepatocellular carcinoma . NAFLD, fibrosis , 4.Excessive oxidants , reactive nitrogen species (RNS), etc.) and decreased antioxidant defense have been observed in NAFLD patients , 5, 6. T N-arachidonoyl ethanolamine, also known as anandamide /ANA/, and 2-arachidonoyl glycerol /2-AG/), and several enzymes involved in the synthesis and degradation of endocannabinoids [Endocannabinoid system (ECS) is a signaling cascade consisting of cannabinoid receptors (G-protein-coupled cannabinoid receptor type 1 /CB1/ and type 2 /CB2/), endocannabinoids  that were raised on Military Medical Academy in Belgrade. Animals were kept in individual cages under standard laboratory conditions  with free access to tap water and standard chow diet. Daily food intake was measured during the experiment. All experimental procedures were in full compliance with Directive of the European Parliament and of the Council (2010/63EU) and were approved by the Ethical Committee of the University of Belgrade (Permission No 695/2).n = 31) were randomly divided into the following groups: (1) control group fed with control chow diet for 20 weeks ; (2) group fed with high saturated fat diet for 20 weeks ; (3) group fed with standard chow diet and treated with rimonabant after 18 weeks ; (4) group fed with high saturated fat diet and treated with rimonabant after 18 weeks . Composition of high saturated fat diet  is shown in All animals  anesthesia. Liver samples were taken for pathohistology analysis and determination of oxidative/nitrosative stress parameters and oligoelement levels.Liver samples for biochemical analysis were homogenized on ice, in cold buffered 0.25 M sucrose medium , 10 mM phosphate buffer (pH 7.0), and 1 mM ethylenediaminetetraacetic acid . The homogenates were centrifuged at 2000 ×g for 15 minutes at 4°C. Crude sediments were dissolved in a sucrose medium and centrifuged. The supernatants were transferred into the tubes and centrifuged at 3200 ×g for 30 minutes at 4°C. Obtained sediments were dissolved in deionized water. After one hour of incubation, the samples were centrifuged at 3000 ×g for 15 minutes at 4°C, and supernatants were stored at −70°C. Proteins were determined by the Lowry method using bovine serum albumin as the standard .Lipid peroxidation, measured as malondialdehyde (MDA) level, was determined spectrophotometrically in a reaction with thiobarbituric acid as described by Girotti et al. . The resx) as a measure of NO production was determined by using Griess reagent. After reduction of the nitrates, the total nitrites were reacted with sulfanilamide and N-(1-naphthyl) ethylenediamine to produce an azo dye, which was measured spectrophotometrically at 492 nm [The concentration of nitrites + nitrates  in the liver was measured spectrophotometrically, as an inhibition of epinephrine autooxidation at 480 nm. After addition of 10 mM epinephrine , analysis was performed in the sodium carbonate buffer  containing 0.1 mM EDTA . Samples for manganese SOD (MnSOD) were previously treated with 8 mM KCN  and then analysed as previously described . ActivitThe content of reduced glutathione (GSH) was determined spectrophotometrically using 5,5-dithio-bis-2-nitrobenzoic acid (DTNB). DTNB reacts with aliphatic thiol compounds at pH 8.0 forming yellow p-nitrophenol anion whose absorption is measured spectrophotometrically at 412 nm . ResultsA MDS-2000 system microwave digestion lab station was used for digesting the samples of tissue with 10 mL of diluted nitric acid (1 : 1), followed with addition of 10 mL concentrated nitric acid. Samples were treated according to the program of 3 steps. This was done over the period of 20 min with the power of 630 ± 30 W and within the temperature range of 140–160°C. After complete digestion samples were transferred in 50 mL volumetric flask .Zn and Cu liver content was determined by means of flame atomic absorption spectrometry (FAAS), while graphite furnace atomic absorption spectrometry (GF AAS) was applied for determining of Mn and iron (Fe) liver content .Oligoelement liver content is expressed as milligram per kilogram (mg/kg).All chemicals for the digestion of the samples were of the analytical reagent grade and were supplied by J.T. Baker Chemical Co. .μm and then stained with Hematoxylin-Eosin (HE) and prepared for light microscopy analysis. All samples were evaluated by an experienced pathohistologist who was blinded to the experiment. Preparations were analyzed and photographed using a combined photobinocular light microscope Olympus BX51 equipped with Artcore 500MI artery, Co. Ltd. Japan Camera.Liver tissue was incubated in 10% formalin solution at room temperature. After the fixation, the liver samples were processed by the standard method. Tissues were incorporated in paraffin sectioned at 5 P < 0.05. For statistical analysis computer software SPSS 15.0 was used.All results are expressed as means ± SD. As the normal distribution of parameters was confirmed by Kolmogorov-Smirnov test, one-way analysis of variance (ANOVA) with Tukey's post hoc test was used for testing the difference among groups. The difference was considered significant if P = 0.955), as well as in HF group (4.86 ± 0.44%) and HFR group (4.67 ± 0.38%) (P = 0.907).There was no statistically significant difference in liver/body weight ratio between C group (4.65 ± 0.33%) and R group (4.54 ± 0.41%) (P = 0.004). Treatment with rimonabant induced a significant decrease in food intake in HFR group in 19th and 20th weeks (2.66 ± 0.19 g resp.) compared to first the 18 weeks (3.03 ± 0.21 g) (P = 0.033). Similarly, decrease in food intake was observed in R group in 19th and 20th weeks (2.56 ± 0.23 g) compared to the first 18 weeks (4.31 ± 0.34 g) (P = 0.002) (Food intake was significantly higher in group fed with control chow diet (4.30 ± 0.22 g) compared to HFD fed mice (3.06 ± 0.18 g) (= 0.002) .P = 0.023). On the other hand, rimonabant treatment induced a significant decrease in MDA concentration in HFR group (197.04 ± 17.99 nmol/mg prot.) in comparison with HF group (P = 0.001). Significant decrease in MDA concentration was also observed in control chow diet fed mice treated with rimonabant (483.18 ± 26.59 nmol/mg prot.) compared to control (P = 0.001) (MDA concentration was significantly increased in HF group (777.99 ± 70.87 nmol/mg prot.) compared to control (661.09 ± 106.72 nmol/mg prot.) (= 0.001) .x content (18.10 ± 2.94 nmol/mg prot.) compared to control (15.51 ± 2.02 nmol/mg prot.) (P = 0.007). Rimonabant treatment induced a significant decrease in NOx concentration in HFR group (10.31 ± 1.66 nmol/mg prot.) in comparison with HF group (P = 0.001) (HFD caused an increase in NO= 0.001) .P = 0.001). Besides, higher total SOD activity was registered in R group (32.57 ± 3.06 U/mg prot.) compared to control (P = 0.001). On the other hand, total SOD activity in HFR group (22.84 ± 3.27 U/mg prot.) was significantly decreased in comparison with HF group (P = 0.004). Analysis of SOD isoenzymes revealed that expression of MnSOD and Cu/ZnSOD followed the changes of total SOD activity in all groups  compared to C group (14.88 ± 2.86 U/mg prot.) (l groups .P = 0.003). However, GSH level was significantly higher in HFR group (100.09 ± 5.39 nmol/mg prot.) compared to HF group (P = 0.009). Besides, decrease in GSH concentration was observed in R group (92.46 ± 10.19 nmol/mg prot.) when compared to control (P = 0.041) (GSH content was significantly decreased in HF group (62.43 ± 8.86 nmol/mg prot.) in comparison with control (104.59 ± 13.16 nmol/mg prot.) (= 0.041) .P = 0.033). However, Zn content in HFR group (50.1 ± 8.65 mg/kg) was higher compared to HF group (P = 0.001) and also in R group (45.79 ± 6.16 mg/kg) compared to C group (P = 0.009) (It was found a significant decline in Zn content in HF group (29.10 ± 2.38 mg/kg) in comparison with C group (36.69 ± 5.02 mg/kg) (= 0.009) .P = 0.004). In contrast, treatment with rimonabant reduced Fe content in HFR (70.46 ± 10.07 mg/kg) in comparison with HF group (P = 0.003) (It was shown a significant HFD-induced increase in Fe content (110.59 ± 10.07 mg/kg) when compared to control (82.90 ± 4.83 mg/kg) (= 0.003) .P = 0.001). Significant increase in Cu content was also observed in HFR group (5.53 ± 1.38 mg/kg) compared to HF group (P = 0.005), as well as in R group (4.04 ± 0.48 mg/kg) in comparison with C group (P = 0.001) (Cu content in HF group (4.33 ± 0.24 mg/kg) was significantly increased in comparison with control (2.66 ± 0.15 mg/kg) (= 0.001) .P < 0.003). Administration of rimonabant increased Mn content in HFR group (10.01 ± 1.99 mg/kg) in comparison with HF group, too (P < 0.001). In contrast to Cu content, Mn content in R group (0.06 ± 0.01 mg/kg) was decreased when compared to C group (P < 0.001)  compared to control value (1.27 ± 0.01 mg/kg) (< 0.001) .There were no pathohistological changes in the liver in C and R groups . HFD cauResults of our study clearly showed that HFD induced lipid peroxidation and nitrosative stress in the liver that are associated with steatosis and portal inflammation . The rolα) production and inactivation of nuclear factor-kappaB (NF-κB). Similarly, our study clearly suggests that anti-inflammatory effect of rimonabant may be additionally mediated by decline in lipid peroxidation. Besides, this finding is in accordance with higher level of ANA and upregulation of CB1 receptor in HFD-induced NAFLD [Our study suggests that endocannabinoids also aggravate oxidative stress in NAFLD and contribute to the development of nonalcoholic steatohepatitis (NASH) through CB1 receptors. In fact, this is the first study that deals with effects of endocannabinoids and the selective CB1 antagonist, rimonabant, on oxidative/nitrosative stress in the liver. Namely, prooxidative effects of endocannabinoids are suggested by reduction of lipid peroxidation in both standard diet- and HFD-fed animals after rimonabant treatment . Howevered NAFLD .x content compared to control  and MnSOD  . In our In present study higher activity of total SOD and its isoenzymes was registered in liver of rimonabant-treated mice . Given tIn the present study total hepatic SOD activity and its isoenzymes were significantly decreased in HFD fed mice treated with rimonabant in comparison with mice treated with HFD . This deIn the current study, HFD caused an increase in iron liver content when compared to control values . This reOur study has shown that administration of rimonabant to HFD fed mice reduced hepatic iron content in comparison with mice fed with HFD . Since uα (PPAR-α) and hepatocyte nuclear factor-4α (HNF-4α), play important roles in regulation of hepatic lipid metabolism [α and lipid metabolism genes [The result of the present study was found a significant decline in zinc content in mice fed with HFD compared to control values . This detabolism , 74. Howtabolism . In suchtabolism . In additabolism , 76. Bestabolism . Also, rsm genes .A growing body of evidence indicates that zinc offers a protection from acute and chronic liver injury, but its hepatoprotective properties have not been fully identified , 78–80. Results in our study showed that copper and manganese content was significantly higher in HFD fed mice in comparison with control . These fIn our study significant increase in copper and manganese content was also observed in HFD fed mice after rimonabant treatment compared to mice fed on HFD, as well as in rimonabant treated mice in comparison with control group. On the other hand, in contrast to copper content, manganese content in rimonabant treated mice was decreased when compared to control values . JudgingRimonabant was the first drug developed to block CB1 receptors in the brain and in the periphery . The effIn recent years, it becomes clear that G-protein-coupled receptor antagonists may have agonistic effects on other signaling pathways related to their receptors , 89. ThuTo date, no effective therapy has been proposed for patients with NAFLD. To our knowledge, this is the first study that investigates the effect of CB1 receptor blockade on oxidative/nitrosative liver injury in NAFLD. Based on our results, it can be noted the potential usefulness of CB1 blockade in the treatment of HFD-induced NAFLD, particularly due to attenuation of hepatic oxidative/nitrosative stress parameters and improvement of liver histology. However, further investigations regarding precise mechanisms by which HFD stimulate ECS production in the liver are required before moving toward clinical phase of investigation.