N-sulfo carbamoyl toxins C1 and C2, in sea shellfish. The samples were extracted by acetonitrile/water  with 0.1% formic and purified by dispersive solid-phase extraction (dSPE) with C18 silica and acidic alumina. Qualitative and quantitative detection for the target toxins were conducted under the multiple reaction monitoring (MRM) mode by using the positive electrospray ionization (ESI) mode after chromatographic separation on a TSK-gel Amide-80 HILIC column with water and acetonitrile. Matrix-matched calibration was used to compensate for matrix effects. The established method was further validated by determining the linearity (R2 ≥ 0.9900), average recovery (81.52–116.50%), sensitivity (limits of detection (LODs): 0.33–5.52 μg·kg−1; limits of quantitation (LOQs): 1.32–11.29 μg·kg−1) and precision (relative standard deviation (RSD) ≤ 19.10%). The application of this proposed approach to thirty shellfish samples proved its desirable performance and sufficient capability for simultaneous determination of multiclass PSP toxins in sea foods.In this study, a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for simultaneous determination of eight paralytic shellfish poisoning (PSP) toxins, including saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins (GTX1–4) and the  Collision energies of precursor–product ion transitions were optimized to give the maximum intensity of the product ions. The precursor–product ion transition with the highest signal abundance was selected for quantification, while the secondary product ion and the abundance ration of two transitions were used for qualification. The optimized MS/MS parameters for the eight PSP toxins in MRM mode are shown in The MS/MS conditions were optimized for each PSP toxin with direct injection of the mixed stock solution. Precursor ions were observed by Q1 full scan in the positive mode, and the MS/MS conditions in the multiple reaction monitoring (MRM) mode were further optimized. The results showed that abundant [M + H]v/v); (2) acetone/water ; (3) dichloromethane/water ; (4) ethyl ether/water ; (5) ethyl acetate/water ; and (6) acetonitrile/water . Each extraction solvent was tested in blank sample, and each experiment was done in triplicate, and a total of 18 samples were ran in the experiment. The extraction efficiencies of each extraction solvent are averaged and shown in v/v), so it was employed in the subsequent toxin extraction.Extraction solvent was optimized by evaluating recoveries of analytes which was affected by extraction capacities and possibly matrix effects with difference solvents . In thisdSPE adsorbents were applied for purification to minimize the matrix effects. C18, a hydrophobic silica-based sorbent, was used commonly because of its strong affinity with non-polar compounds. Graphitized carbon black (GCB), which has a high affinity with planar molecules, is suitable for purification procedures, especially removal of the pigment and sterols. Ethylenediamine-N-propyl-silane (PSA) with two amino groups can remove pigment and metal ions. Activated carbon (AC) is capable of cleaning up impurities with negative charge by ion exchange. Acidic alumina (Al-A) was selected here as well, purification of PSP toxins by using merely Al-A has been reported . Accordiv/v), methanol-water  containing 10 mmol·L−1 ammonium acetate, methanol-water  containing 0.1% of formic acid and 0.1% formic acid aqueous solution were compared in the pilot test. The results showed that the best peak shapes were obtained with water with 0.1% formic acid, so it was applied as sample solvent in this study.The composition of reconstitution solvent was considered to directly affect the peak shape and separation of the analytes in chromatographic system. In order to optimize the sample solvent, methanol, methanol-water  are a common problem that occurs when using LC-MS/MS. The extent of the signal suppression/enhancement (SSE) was observed differently for the eight PSP toxins. The SSE range between −20% and +20% is typically considered as tolerable . The sigAccording to the EU guideline , method R2 ≥ 0.9900. The LODs and LOQs of this established method were in the range of 1.32–11.29 µg·kg−1 and 0.33–5.52 µg·kg−1. LOQs of STX (2.48 µg·kg−1), NEO (4.14 µg·kg−1), GTX1 (8.28 µg·kg−1), GTX2 (9.03µg·kg−1), GTX3 (4.79 µg·kg−1) and GTX4 (4.05 µg·kg−1) in clam were obviously lower than those of STX (11.3 µg·kg−1), NEO (19.0 µg·kg−1), GTX1 (26.7 µg·kg−1), GTX2 (38.3 µg·kg−1), GTX3 (17.3 µg·kg−1) and GTX4 (5.30 µg·kg−1) previously reported  × 100%. ME of 0% indicated that no matrix effect occurred in the selected concentration ranges. ME above 0% revealed signal enhancement, while ME below 0% indicated signal suppression. The blank matrix was prepared using the analyte-free shellfish by following the sample pretreatment procedures. The mixed stock solutions were diluted with the blank matrix to yield a serial of concentrations , respectively.−1), intermediate  and high concentration  of the mixed standards. Intra- and inter-day precision was investigated by spiking the analyte-free samples with three levels of the mixed standards and performing analysis on five consecutive days with six replicates every day.The parameters evaluated in the validation were linearity, precision (expressed as RSD in terms of intra-day and inter-day precision) and recovery. LODs were determined by measurements with successive dilution of matrix-spiked standard solution until a signal-to-noise ratio (S/N) of 3:1, and LOQs were defined as the concentration of a PSP toxin producing S/N = 10:1. Recovery experiments were performed in the analyte-free shellfish by employing the method of standard addition with low (9.92 for STX, 10.34 for NEO, 12.42 for GTX1, 22.57 for GTX2, 9.58 for GTX3, 4.05 for GTX4, 26.96 for C1 and 8.06 for C2 ng·mL


The in vitro biological assays demonstrated that complex 3 had increased cytotoxicity against lung cancer cells, especially non-small-cell lung cancer (NSCLC) compared to its mono-substituted complex 3a, indicating that the sterically hindered alkyl moieties have significant influences on its antitumor property. However, the mechanism still remains unclear. The further studies revealed that complex 3 could induce ROS overproduction, severe DNA double strands breaks and inhibit the activation of DNA damage repair proteins within nucleus, leading to cell-cycle arrest and cell death. Moreover, complex 3 could induce autophagy via the accumulation of autophagic vacuoles and alterations of autophagic protein expression. Interestingly, the ROS scavengers, N-acetyl-cysteine (NAC) could reverse complex 3-induced DNA double strands breaks and autophagic responses more significantly compared to complex 3a. The results demonstrated that the ROS generation plays an important role in the DNA double strands breaks and autophagic responses in the antitumor effect of complex 3 with N1,N2-diisobutyl moiety. Our study offered a novel therapeutic strategy and put new insights into the anticancer research of the complexes with N1,N2-diisobutyl moiety served as steric hindrance.A series of novel platinum(II) complexes with -N In the past three decades, lung cancer was the most frequently diagnosed cancer and the leading cause of cancer death among males as well as females in the developed countries . The mor1,N2-disubstituted 1,2-DACH derivatives have been used as carrier ligands + or [M+Cl]− peaks in ESI-MS spectroscopy.The newly synthesized platinum(II) complexes were characterized by elemental analysis, IR, 50 values are presented in Table R,2R)-N1-(2-methylpropyl)-1,2-cyclohexanediamine-N,N’] , which is an important proapoptotic index for early apoptosis. We chose a mitochondria-specific and voltage-dependent fluorescent probe, JC-1, to observe whether there was loss in ΔΨm. The results in Figure Since ATP production is a significant consequence attributed to the dysfunction of mitochondrial energy production, we measured intracellular ATP levels after complex 3 treatment. As shown in Figure 2O2 has been recently suggested to act as a central player in signal transduction pathways −= 526(100%).(C16H30N2O4Pt): Yield: 51%, white powder. Elem anal. Calcd for C16H30N2O4Pt: C, 37.72; H, 5.93; N, 5.50. Found: C, 37.53; H, 5.68; N, 5.25. IR : 3860(br), 3147, 2955, 1675, 1462, 1396, 805; 1H NMR : 0.90–1.06 , 1.33–1.97 , 2.11–2.21 , 2.57 , 6.50 ; ESI-MS: m/z [M+Na]+= 532(100%).(C17H32N2O4Pt): Yield: 21%, white powder. Elem anal. Calcd for C17H32N2O4Pt: C, 39.00; H, 6.12; N, 5.35. Found: C, 38.81; H, 6.01; N, 5.15. IR : 3399(br), 1579, 1355, 696; 1H NMR : 0.99–1.02 , 1.12–1.63 , 2.06–2.12 , 2.24–2.33 , 3.19 22CH), 6.12 ; ESI-MS: m/z [M+Na]+= 546(100%).(C20H36N2O4Pt): Yield: 33%, white powder. Elem anal. Calcd for C20H36N2O4Pt: C, 42.63; H, 6.39; N, 4.97. Found: C, 42.75; H, 6.47; N, 4.72. IR : 3122(br), 2951, 2868, 1646, 1463, 1350,1114, 515; 1H NMR : 0.89–1.03 , 1.05–1.70 , 2.07–2.11 , 2.23–2.36 , 2.61–2.67 , 2.70–2.76 , 5.85 ; ESI-MS: m/z [M+Na]+ = 586(100%).  and 1% penicillin/streptomycin , while HCT-116, SGC-7901, A549, NCI-H1299 and L02 cells were cultured at 37 °C in 5% CO2 with RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were passed every 2 days and restarted from frozen stocks upon reaching pass number 20.Human cancer cell lines HepG2, SGC-7901, HCT-116, A549, NCI-H1299 and MCF-7 as well as normal liver cell line L02 were purchased from the Cell Bank of Shanghai Institute of Cell Biology. Among them, MCF-7, HepG2 cells were cultured at 37 °C in 5% COIn vitro cytotoxicity of the platinum compounds against human hepatocellular carcinoma (HepG-2), gastric carcinoma (SGC-7901), non-small-cell lung cancer (A549), and colorectal cancer (HCT-116) cell lines were measured by the MTT assays. Briefly, the cells were seeded in 96-well cultured plates at a density of 5000 cells/well. After overnight incubation (16 h), the cells were treated with the platinum complexes. After 48 h of incubation, 10 μL of a freshly diluted 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) solution (5 mg/mL) were added to each well and the plates were incubated at 37 °C in a humidified 5% CO2 atmosphere for 4 h. At the end of the incubation period the medium was removed and the formazan product was dissolved in 150 μL of DMSO. The cell viability was evaluated by measurement of the absorbance at 490 nm, using an Absorbance Reader (BioRad). IC50 values (compound concentration that produces 50% of cell growth inhibition) were calculated from curves constructed by plotting cell survival inhibitory rate (%) versus drug concentration logarithm. All experiments were repeated in three times. The reading values were converted to the percentage of control . Cytotoxic effects were expressed as IC50 values.4. The DNA concentration was determined by UV-vis spectra at 260 nm with an extinction coefficient of 6600 M−1. The mixture of DNA with platinum complexes were incubated at 37°C in the dark. The fluorescence was measured in 0.4 M NaCl to avoid the second fixation site of EtBr to DNA. EtBr (0.04 mg/mL) was added to the 0.01 mg/mL DNA solution before the fluorescence measurement. Fluorescence spectra were recorded under following conditions: scan speed 2000 nm min−1; excitation slit width was 5 nm and emission slit width was 10 nm. The excitation and emission wavelength was 530 nm and 592 nm, respectively.Herring sperm DNA was dissolved in 10 mM phosphate buffer (pH 7.4) containing 10 mM NaClOThe cellular uptakes of oxaliplatin, complex 3 and complex 3a were measured on A549, NCI-H1299 and L02 cells. The cells were seeded in 6-well plates overnight and then incubated with 10 μM drugs at 37°C in standard culture conditions. After 4 h, the cells were washed with PBS buffer (pH 7.4) for three times, and harvested by trypsinization. After re-suspension in PBS, the pellet was washed with PBS and collected per centrifugation  at 500 g for 5 min at 4°C. The organelles were then isolated via differential centrifugation. All cellular compartments  were isolated from A549 and NCI-H1299 cancer cells for direct comparative purposes. The supernatant phases discarded during the isolation of nuclei, lysosomes and mitochondria procedures were collected and formed the “residual” fraction. An aliquot of crude lysate after homogenization, nuclear, mitochondrial (pellet lysed via freeze and thaw cycles followed by 20 min incubation in ultrasonic bath), lysosomal and residual fraction was each used for protein quantification using the Bradford method . The har2 and 140 mM NaCl), incubated with Annexin V-FITC and then PI for 10 min in the dark at room temperature, the processed cells were analyzed by flow cytometry  and a computer station running Cell-Quest software. .Annexin V-FITC apoptosis detection kit  assay was performed according to the manufacturer's protocol. Briefly, the A549 and NCI-H1299 cancer cells were treated with oxaliplatin, complex 3, complex 3a of 10 μM respectively for 24 h and washed with PBS. Then the cells were collected, resuspended in binding buffer  FITC Staining Kit (ab65613)) for 1 h at 37°C under 5% CO2. Subsequently, centrifugation of the cells was performed to collect cell pellets and washed 2 times with washing buffer. Each group of cells was resuspended with 300 mL of the washing buffer and transferred 100 mL per well in a microtiter plate reader. The quantification of caspase-3 was evaluated as Relative Fluorescence Unit(RFU) using Fluorophotometer  at an emission wavelength of 535 nm after excitation at 485 nm. The statistical significance was evaluated with one-way ANOVA test using Graph Pad Prism 4 software.For the quantification of activation of caspase-3 in cancer cells, 5 × 10Cells were trypsinized and plated at a density of 500 perplate. Fourteen days later, 50 μM oxaliplatin, complex 3 and complex 3a in cell culture medium were added to the plates, and cells were incubated for a further 7 days. Cells were then fixed with 3% paraformaldehyde, stained with crystal violet and imaged with a light microscope. The experiment was performed in triplicate. The number of colonies, defined as containing > 50 cells, was counted.Cell cycle was analyzed by flow cytometry as described previously . Data we6 cells/ml, then the cells were permeabilized with 0.3% Triton X-100, washed with ice-cold PBS, incubated with 10 μM JC-1 for 15 min at 37°C in the dark and observed under a fluorescence microscope . Red fluorescence is attributable to a potential-dependent aggregation in the mitochondria. Green fluorescence, reflecting the monomeric form of JC-1, appeared in the cytosol after mitochondrial membrane depolarization. Relative fluorescence intensities were monitored using the flow cytometry , and analyzed by the software Modfit and Cell Quest  with settings of FL1 (green) at 530 nm and FL2 (red) at 585 nm.The electrical potential difference across inner mitochondrial membrane (ΔΨm) was monitored using the ΔΨm-specific fluorescent probe JC-1 , a sensitive fluorescent dye. In brief, the A549 and NCI-H1299 cancer cells treated with 10 μM of oxaliplatin, complex 3 and complex 3a for 24 h were harvested with ice-cold PBS and resuspended in RPMI-1640 medium at a density of 0.5 ×10The ATP production assay kit  was used to measure intracellular ATP level according to the manufacturer's instructions. In brief, cells were treated with 10 μΜ oxaliplatin, complex 3 and complex 3a for 12 h, then incubated with 100 μL Nuclear Releasing Reagent for 5 min at 37°C with gentle shaking, followed by further incubation with 1 μL ATP monitoring enzyme. Detection was performed using the luminometer Orion II .2DCFDA; Molecular Probes, Invitrogen, Darmstadt, Germany) as described [2DCFDA diffuses into the cell, where it is enzymatically converted by intracellular esterases and oxidized into the high fluorescence compound DCF, which allows the determination of H2O2, peroxynitrite anions and peroxyl radicals. Approximately 4×103 A549 and NCI-H1299 cancer cells per well were plated on white bottom 96-well plates in extracellular fluid . After 24 h incubation, cells were treated with 10 μM oxaliplatin, complex 3 and complex 3a for 6 h at 37°C. Then, cells were loaded with 10 mM H2DCFDA for 30 min at 37°C and 5% CO2. Then, cells DCF generation was measured over time using a fluorometer  at 492 nm excitation and 520 nm emission.The generation of ROS induced by complex 3 was determined with the cell permeant fluorogenic probe 2′,7′-dichlorodihydrofluorescein diacetate . In the presence of horseradish peroxidase (HPR), the Amplex® Red reagent reacts in a 1:1 stoichiometry to produce the red-fluorescent oxidation product, resorufin. Consequently, resorufin generation allows the detection of the H2O2 released from biological compounds. Approximately 4 × 103 A549 and NCI-H1299 cancer cells were plated on black bottom 96-well plates in 120 ml of working solution containing 40 mM Amplex® Red reagent and 0.1 U/ml HPR, and incubated in the presence of 10 μM oxaliplatin, complex 3 and complex 3a for 2 h at 37°C. Resorufin generation was measured over time using a fluorometer (Fluostar BMG Labtech) at 544 nm excitation and 590 nm emission.The production of HA549 and NCI-H1299 cancer cells in 6 cm culture plates were treated as follows: 10 μM oxaliplatin, complex 3 and complex 3a. After 4 h, medium was removed and dishes were rinsed three times with cold PBS. The cells were harvested and cell numbers were counted. Then, intracellular GSH levels were measured using a GSH test kit (KeyGen KGT006). A549 and NCI-H1299 cancer cells without treatment were used as control groups.DNA cleavage produced by oxaliplatin, complex 3 and complex 3a was investigated by agarose gel electrophoresis. pET28a plasmid DNA (50 ng/mL) was used as the target. Appropriate dilutions of tested complexes were made, and the required volumes of solutions were added to achieve a set of concentrations in the range of 0–100 mm; pET22b DNA  was added to each tube, and the mixtures of platinum complexes and pET22b plasmid DNA were then incubated at 37°C for 6 or 24 h. Afterward, the agarose gel (made up to 1% w/v) containing ethidium bromide was prepared with TA buffer . The mixtures with loading buffer (1 mL) underwent electrophores is in agarose gel in TA buffer at 100 V for 60 min. Bands were imaged using a Molecular Imager  under UV light.5 cells) were combined with molten LMAgarose (Trevigen) at a ratio of 1:10 (v/v) and were immediately pipetted onto CometSlide (Trevigen). Slides were incubated at 4°C in the dark for 10 minutes, then immersed in prechilled Lysis Buffer and incubated at 4°C for 30 min. Slides were immersed in Alkaline Unwinding Solution, pH > 13  for 20 min at room temperature in the dark. Electrophoresis was done at 21 V for 30 minutes using Alkaline Electrophoresis solution . The slides were washed twice in water for 5 minutes and once in 70% EtOH for 5 min, then dried overnight and visualized by microscopy. Under these conditions the formation of “comet tail” is indicative of SSBs, DBSs, and/or active excision repair of DNA cross-links.After the same treatment of oxaliplatin, complex 3 and complex 3a at 10 μM for 24 h, A549 cells (1×102HPO4 and 1 mM KH2PO4). The numbers of SSBs were determined by an alkaline elution assay as previously described [SSB were detected by exposing cancer cells to 10 μM oxaliplatin, complex 3 and complex 3a, respectively, for 15 min, 30 min and 60 min in RPMI 1640 medium at 37°C. Then the cells were washed three times with PBSCMF  at 55 rpm on a rotating shaker for 15 min. Non-specific binding was blocked by incubation with 5% FCS/PBS/0.1% Triton X-100 for 1 h at RT. Immunostaining for MDC1 (1:400), and XRCC1 (1:1000) was followed by detection with species specific secondary AlexaFluor conjugates . Numbers of γH2AX foci were counted in 50–60 nuclei and plotted over time as previously described [For assessment of DSBs repair kinetics we detected the total nuclear volume for an average of 50–60 nuclei per time points and cell type. Briefly, A549 cells were grown to confluence in 35 mm petri-dishes with 0.17 mm glass bottom. After treatment of cisplatin, oxaliplatin and 3 at 50 μM for 8 h and 12 h, cells were fixed in 4% paraformaldehyde / PBS for 10 min at room temperature and incubated in nuclear extraction buffer  sodium dodecylsulfonate, 20% (v/v) glycerol, 200 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 1 g/ml aprotinin). Lysates were centrifuged at 12,000 g for 0.5 h at 4°C. The concentrations of total proteins were measured using the BCA assay method with Varioskan spectrofluorometer and spectrophotometer  at 562 nm. Protein (20–100 μg) prepared from the indicated cells was loaded per lane and electrophoresed in 8% or 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto polyvinylidene difluoride (PVDF) Immobilon-P membrane  using a transblot apparatus . The membranes were blocked with 5% (w/v) non-fat milk at 0.5 h at 37°C, followed by overnight incubation at 4°C with primary antibodies diluted in PBST. After washing with PBST, the membranes were incubated for 1 h with an IRDye´800 conjugated secondary antibody diluted 1:20000 in PBST, and the labeled proteins were detected with an Odyssey Scanning System .Flow cytometry with acridine orange staining as described in a previous study was used to detect and quantify the AVOs, one of the characteristicsof autophagy . At the 2. Cells were subsequently stained with annexin V (2.5 μg/ml) and PI (2 ng/ml) for 20 min, followed by analysis by flow cytometry (Beckman Coulter). Cystograms of the four quadrants in the figure were used to distinguish normal (annexin V−/PI−), early apoptotic (annexin V+/PI−), late apoptotic(annexin V+/PI+), and necrotic cells (annexin V−/PI+). The sum of early apoptosis and late apoptosis is presented as total apoptosis.The mode of cell death was analyzed by flow cytometry with annexin V/propidium iodide (PI) double-staining to detect membrane events according to a previous study . After i7 cells per mouse) was injected subcutaneously into the right oxter of nude mice. When tumor grew to a size of 80–150 mm3 at 12 days, the mice were administrated via oxaliplatin (dosed intravenously at 5 mg/kg twice a week), complex 3 (dosed intravenously at 5 mg/kg once every three days), complex 3a (dosed intravenously at 5 mg/kg once every three days). The control group was administered glucose. The tumor growth was monitored by measuring the perpendicular diameter of the tumor using calipers every two days and calculated according to the formula:Thirty-five nude mice (BALB/c) with body weight range from 18–22 g, were supplied by Shanghai Laboratory Animal Center, China Academy of Sciences. Experimental protocols were in accordance with National Institutes of Health regulations and approved by the Institutional Animal Care and Use Committee. All animals were randomly divided into 5 groups. The A549 single-cell suspension in PBS (1×103) = 0.5 × length × width2Tumor volume , resulting in a final concentration of 50 μM. At varying time points  at 37°C, a 300 μL aliquot was removed from the incubating blood. The ICP-MS measurement was served to confirm the blood stability of complex 3 in rat plasma.Twenty-four SD rats (240–250 g) were supplied by Shanghai Laboratory Animal Center, China Academy of Sciences. Experimental protocols were in accordance with National Institutes of Health regulations and approved by the Institutional Animal Care and Use Committee. SD rats composed of half male and half female were randomly divided into 2 groups: 1) oxaliplatin (5 mg/kg); 2) complex 3 . 0.6 ml of blood compounds were collected from orbital cavity at 5 min followed by 0.5, 1, 2, 4, 8, 12 and 24 h after intravenous administration, heparinized and then centrifuged at 4000 rpm for 20 min at 4°C. The supernatant plasma was collected for measurement of Pt content using ICP-MS. The apparent plasma half-life time was calculated using a Phoenix Win Nonlin 6.3 Program .t-test. All comparisons are made relative to untreated controls and significance of difference is indicated as *P < 0.05 and **P < 0.01.All data in different experimental groups were expressed as mean ± SEM. These data shown in the study were obtained in at least three independent experiments. Statistical analyses were performed using an unpaired, two-tailed Student's


This observational study was designed to evaluate the efficacy of ultramicronized palmitoylethanolamide (um-PEA) (Normast®) administration, as add-on therapy for chronic pain, in the management of pain-resistant patients affected by failed back surgery syndrome.T0) and after one (T1), two (T2), and three (T3) months. A total of 35 patients were treated with tapentadol (TPD) and pregabalin (PGB). One month after the start of standard treatment, um-PEA was added for the next two months. Pain was evaluated by the Visual Analogue Scale (VAS) at the time of enrollment (T0) to 4.3 ± 0.11 (T1) (p < 0.0001); however, it failed to provide significant subjective improvement in pain symptoms. Addition of um-PEA led to a further and significant decrease in pain intensity, reaching VAS scores of 2.7 ± 0.09 (T2) and 1.7 ± 0.11  (p < 0.0001) without showing any side effects. After the first month with TDP + PGB treatment only, VAS score decreased significantly from 5.7 ± 0.12 at the time of enrollment  as part of a multimodal therapeutic regimen in the treatment of pain-resistant patients suffering from failed back surgery syndrome. Clinicarability .An important development in pain management has been the discovery that initiation and maintenance of neuropathic pain involve communication between neurons and nonneuronal immunocompetent cells, such as mast cells and microglia, together with a cascade of pro- and anti-inflammatory cytokines –14. Mastα) ligand that exerts anti-inflammatory, analgesic, and neuroprotective actions [α null mice [An innovative approach in the management of chronic pain is represented by palmitoylethanolamide (PEA), a member of the N-acylethanolamine family of fatty acid amide signaling molecules. PEA has an established history of antiallodynic and antihyperalgesic effects, which are mediated by downmodulation of proinflammatory mediator release from mast cells , 18 and  actions , 22. Furull mice . Severalull mice –26. The ull mice , 28. Intull mice . Based oT0) and after one (T1), two (T2), and three (T3) months , affiliated to the University of Perugia Medical School. Patients selected for the study were suffering from FBSS caused by laminectomy, discectomy, or vertebral stabilization, who came to our attention complaining of an increase in pain intensity compared to the immediate postoperative condition. See ) months . This stT−2) was 6.9 ± 0.14 and decreased significantly immediately after surgery (T−1) to 5.1 ± 0.13 (p < 0.0001). Subjects came to our attention after a median time of 6.5 ± 2.5 months (range: 2–8 months) after surgery and presented a considerable increase in the mean pain intensity at the time of enrollment, with a mean VAS score of 5.7 ± 0.12 (p = 0.0054) (Tables T0) to 4.3 ± 0.11 (T1); in the time periods following addition of um-PEA, VAS scores showed further decreases to 2.7 ± 0.09 (T2) and 1.7 ± 0.11  (p < 0.0001)  . At each 0.0001) .µ-opioid receptor agonist and noradrenaline reuptake inhibitor, is efficacious in patients with nociceptive and neuropathic low back pain, either alone [T1 − T0) of 1.48 ± 0.14 was comparable to that achieved in the month leading up to surgery (T−2 − T−1) (1.83 ± 0.17), even though the latter period encompassed the surgery variable. While encouraging, the VAS score at T1 exceeded 4, an indication still of moderate pain intensity. In the search for new molecules as add-on therapy in the treatment of FBSS, we decided to assess the potential of um-PEA. PEA is an endogenous fatty acid amide signaling molecule produced on demand in response to cellular stress or injury. The anti-inflammatory and analgesic effects of PEA are likely accounted for by several not mutually exclusive mechanisms. PEA acts by downregulating mast cell degranulation via an “autacoid local inflammation antagonism” (ALIA) effect [α, the latter mediating many of PEA's anti-inflammatory effects [The present observational study provides preliminary evidence suggesting that um-PEA (Normast) as add-on treatment to conventional pharmacological regimens in patients suffering from FBSS contributes to a significant pain intensity reduction. As the complex physiopathology of this pain syndrome often renders monotherapy inadequate to achieve meaningful pain relief, polytherapy may thus represent a more fruitful therapeutic approach. TPD, a dual er alone , 32 or ier alone , 34. Theer alone . In the ) effect . A “rece) effect , 38 or t effects . In vivo effects , 37, 39. effects , 40–42.  effects  evaluateT1 − T0). In the subsequent two months with um-PEA as add-on therapy, there was a further and significant decrease in pain intensity of 1.62 ± 0.15 (T2 − T1) after the second month and 0.97 ± 0.13 (T3 − T2) after the third month (Tables After the first month of TPD + PGB treatment, FBSS patients had a pain reduction of 1.48 ± 0.14 (h Tables . To asse


Streptococcus pyogenes, binds to extracellular histones, a group of danger signals released during necrotizing tissue damage. This interaction leads to the formation of large aggregates in vitro. Extracellular histones and SIC are abundantly expressed and seen colocalized in biopsies from patients with necrotizing soft-tissue infections caused by S. pyogenes. In addition, binding of SIC to histones neutralized their antimicrobial activity. Likewise, the ability of histones to induce hemolysis was inhibited in the presence of SIC. However, when added to whole blood, SIC was not able to block the pro-inflammatory effect of histones. Instead SIC boosted the histone-triggered release of a broad range of cytokines and chemokines, including IL-6, TNF-α, IL-8, IL-1β, IL-1ra, G-CSF, and IFN-γ. These results demonstrate that the interaction between SIC and histones has multiple effects on the host response to S. pyogenes infection.Innate immunity relies on an effective recognition of the pathogenic microorganism as well as on endogenous danger signals. While bacteria in concert with their secreted virulence factors can cause a number of inflammatory reactions, danger signals released at the site of infection may in addition determine the amplitude of such responses and influence the outcome of the disease. Here, we report that protein SIC, Streptococcal Inhibitor of Complement, an abundant secreted protein from  Streptococcus pyogenes  is an important human pathogen that may cause a number of conditions from harmless throat infections to invasive diseases such as septicemia, necrotizing soft-tissue infections (NSTI), and streptococcal toxic shock syndrome  (syndrome . The inn see Ref ). One of see Ref , 4, but  see Ref –9. Perha see Ref –9. The b see Ref , 7. This see Ref . The sicAS (mga) . Mga is AS (mga) .in vitro studies have shown that SIC can be considered as an anti-inflammatory and anti-cytolytic agent . Also included were biopsies of healthy skin tissue obtained at plastic surgery at Karolinska University Hospital, Stockholm, Sweden. The studies were conducted in accordance with the Helsinki Declaration and approved by the regional Ethical Review Board at the Regional Ethics committee in Stockholm as well as by the National Committee on Health Research Ethics in Copenhagen. Written informed consent was obtained from all patients or their legal surrogates.Tissue biopsies were collected from patients with NSTI enrolled at Rigshospitalet, Copenhagen, Denmark, as part of the EU-funded project INFECT during 2013–2014  and calf thymus histones (histones) from Roche . [Histone H4-derived peptides were synthesized at Biopeptide Co. .] Peroxidase-conjugated goat anti-rabbit and goat anti-mice immunoglobulin G were from Bio-Rad Laboratories . Antibodies against histone H4  were from Abcam . Rat polyclonal antibodies to human TLR4, TLR2 and isotype control and the TLR4-inhibitor CLI-095 were purchased from InvivoGen .S. pyogenes strain AP1 (M1 serotype) was from World Health Organization Collaborating Center for Reference and Research on Streptococci, Prague, Czech Republic. SIC was purified from S. pyogenes AP1 strain culture medium as described by Åkesson et al. . Molarity calculation of SIC was based on a molecular weight of 31,000 Da.The n et al.  by precin et al. . The SICBlood was drawn from healthy donors into 6.0-mL sodium heparin tubes  or into 2.7-mL 0.109-M sodium citrate tubes . Blood samples were stored at room temperature for 30 min before usage.Streptococcus pyogenes AP1 bacteria and SIC-mutant bacteria were grown for 9 h in Todd–Hewitt (TH) broth at 37°C. After 6 h, the bacteria were centrifuged at 2,000× g and the SNs were sterile filtered. SNs (1 mL) from SIC-producing AP1 and the SIC knockout were mixed with 250 µL of 100% trichloroacetic acid. Samples were incubated for 10 min at 4°C. All samples were centrifuged at 18 000× g for 5 min. The SN was removed from the white pellet in bottom of every test tube. Pellets were dissolved in 200 µL ice-cold 100% acetone. All samples were centrifuged again at 14,000 rpm for 5 min. SN was removed and the pellet was dried at 95°C in a heat block for 10 min. Each pellet was dissolved in sample buffer  and boiled for 10 min at 95°C. Samples were loaded into a 10% PAGE as described by Laemmli ( Laemmli . Protein125I-labeled SIC (2 × 105 cpm/mL) for 3 h in RT. The filter was washed three times for 20 min with PBS-T and autoradiography was carried out using Kodak x-Omat AR films and regular intensifying screens.A polyvinylidene difluoride filter  was activated in methanol, washed in destilled water, and exposed to different histones subtypes in three different concentrations using a Milliblot-D system . The filter was washed with 100-µL PBS, blocked with 5% non-fat milk powder in PBS and probed with 2CO3, 30-mM NaHCO3, pH 9.6) at 4°C. The next day, plates were washed three times in PBS containing 0.05% Tween-20 (PBST) and blocked in PBST containing 0.5% bovine serum albumin for 60 min at 37°C. After blocking and another washing step, plates were probed with 100 µL SIC (40 nM) for 1 h at 37°C and binding was detected with 100 µL rabbit SIC antisera  followed by 100 µL of a peroxidase-conjugated antibody against rabbit IgG . Stabilized tetramethylbenzidine Chromogen  was used as a substrate for the peroxidase-conjugated antibody. The reaction was terminated with 10% H2SO4 and measured at 450 nm for 0.1 s per measured well.Microtiter plates were coated overnight with 100 µL histone H4-derived peptides (1 µM) in coating buffer (15.9-mM Navia amine coupling to flow cell 2. Flow cell 1 was immobilized without protein and was used as a control for each experiment. SIC (400–25 nM) was injected in a flow (30 µL/min) over the Sensor Chip in HBS-EP buffer . Regeneration of the Sensor Chip was obtained by injection of 30 µL 50-mM NaOH. All experiments were run at 25°C in degassed buffers. Affinity constants were calculated using BIAevaluation software .All analyses were performed using a BIAcore X100 instrument . Histone H4 was diluted in sodium acetate  and immobilized to a Sensor Chip CM5 Binding between SIC and histone H4 was visualized using negative staining and electron microscopy as previously described . HistoneStreptococcus pyogenes AP1 strain and the SIC-mutant S. pyogenes strain were plated overnight at 37°C on blood agar plates and stored at 4°C. For each experiment, colonies from the blood agar plate were added to 10 mL in TH broth  and incubated at 37°C overnight. SIC mutant was grown in TH broth containing 0.15-mg/mL Kanamycin. The following day, the bacteria were mixed and 200 µL were transferred to 10-mL fresh TH broth and incubated to reach logarithmic phase (OD620nm 0.4) at 37°C. The bacteria were centrifuged at 2,000× g and washed in ice cold 10-mM PBS with 5-mM glucose (PBS-G). Bacteria were diluted to 2 × 106 colony-forming units (CFUs)/mL in PBS-G. Histones (5 µg/mL) or histone H4 (0.36 µM), with or without increasing concentrations of SIC , were preincubated for 30 min at 37°C in a total volume of 50 µL. The same volume of diluted bacteria was added to each sample, and the samples were incubated for 60 min at 37°C with 5% CO2. All samples were diluted in PBS-G and plated on TH agar plates overnight at 37°C. The following day, colonies were manually counted and the number of viable CFUs was described as a ratio of viable colonies compared with non-stimulated bacteria.g for 10 min to pellet cells. The blood plasma was discarded and replaced by an equal volume of PBS. This washing procedure was repeated twice. Histones and SIC were preincubated for 30 min at 37°C and erythrocytes were then added to each sample in a final dilution of 5% (v/v). After 60 min incubation at 37°C on rotation, all samples were centrifuged at 5,000× g for 10 min. The SN was transferred to a 96-well plate  and hemolysis was measured at 540 nm. Histone-induced hemolysis was presented as percentage of lysis buffer (0.1% Triton X-100)-induced hemolysis.Citrated blood (1 mL) was centrifuged at 2,000× g for 10 min. The plasma SN was removed and frozen at −20°C. IL-6 was measured in heparin plasma SN using Human IL-6 Quantikine ELISA kit  using the manufacturer’s instructions. Samples were discarded after the IL-6 measurements and were not repeatedly frozen.Histones (50 µg/mL), LL-37 (25 µg/mL), β defensin-3 (25 µg/mL), high-mobility group box 1 , heparin-binding protein , SIC (0.64 µM), or the AMPs in combination with different SIC concentrations were incubated with heparinized blood diluted 1:1 in PBS for 7 h at 37°C on rotation. For inhibition experiments, antibodies against human TLR4 (25 µg/mL), human TLR2 (25 µg/mL), isotype control (25 µg/mL), or the TLR4 inhibitor CLI-095 (1–10 µg/mL) were added simultaneously to histones and SIC. Blood aliquots were removed at different time points and centrifuged at 2,000× S. pyogenes strains were analyzed. The biopsies were cryosectioned to 8 µm and fixed in ice-cold acetone or 2% formaldehyde for immunofluorescent or immunohistochemical stainings, respectively. Stainings were performed in singlets on consecutive sections as described previously  FGF basic, Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, CXCL10, CCL2, CCL3, CCL4, PDGF-bb, RANTES, TNF-α, and VEGF. The cytokine panel was designed to provide a measure of cytokines, chemokines, and growth factors. All samples, standards, and controls were run in duplicates and data was acquired using Bio-Plex Manager Software (version 6.1).Data were analyzed using GraphPad Prism 7 . One-way ANOVA with Dunnett’s or Tukey’s test was used to determine significance between multiple comparisons.S. pyogenes isolates AP1 (wild-type) and the isogenic S. pyogenes-mutant strain-lacking SIC (SIC-mutant) were used throughout this study. The SNs from both bacterial strains were collected at 6 h, precipitated with trichloroacetic acid, and SIC was visualized using SDS-PAGE , is the only peptide that had affinity for SIC  as they can induce cytokine  and chemin vivo . Cytokinin vivo , 20, 21.in vivo . Howeverin vivo A, IL-8 . These findings indicate that the pro-inflammatory property of SIC applies to several cationic AMPs, including histones, with important functions in the innate immune system.S. pyogenes strains of the M1 serotype are among the most frequently identified serotypes isolated from patients with severe invasive infections including NSTI and toxic shock syndrome (S. pyogenes strains often belong to the M1 serotype and secrete large quantities of SIC under in vitro conditions (S. pyogenes M1 or M3 (a serotype negative for SIC) strains were analyzed with immunofluorescence staining. As shown in Table S. pyogenes (GAS). Moreover, a biopsy from a healthy donor was included with undetectable levels of HMGB1, IL-8, and S. pyogenes. The four biopsies were stained with antibodies against SIC (red) and histone H4 (green) as well as with the nuclear DNA stain DAPI (blue). Histone H4 was readily detected in tissue sections from both M1- and M3-infected patients, as well as in the biopsy from a healthy control . SIC showed a strong staining in the M1-infected tissue , whereas in the M3 only a background staining was noted . Moreover, we recorded a few areas of colocalization between SIC and histone H4 in the biopsy sections from M1 patients  at the Lund University Hospital (Protocol no. 790/2005).JW, LB, and HH conceived and designed the study. MM performed and analyzed the experiments in Figure The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To assess changes in oral cavity (OC) shapes and radiation doses to tongue with different tongue positions during intensity-modulated radiation therapy (IMRT) in patients with head and neck squamous cell carcinoma (HNSCC) but who refused or did not tolerate an intraoral device (IOD), such as bite block, tongue blade, or mouthpiece.3  in IMRT-S. Dmean of OC was 34.9 ± 8.0 Gy and 31.4 ± 8.7 Gy with IMRT-N and IMRT-S, respectively (p < 0.001). OC volume receiving ≥ 36 Gy (V36) was 40.6 ± 16.9% with IMRT-N and 33.0 ± 17.0% with IMRT-S (p < 0.001). Dmean of tongue was 38.1 ± 7.9 Gy and 32.8 ± 8.8 Gy in IMRT-N and IMRT-S, respectively (p < 0.001). V15, V30, and V45 of tongue were significantly lower in IMRT-S  than IMRT-N  . Positional offsets of tongue during the course of IMRT-S was –0.1 ± 0.2 cm, 0.01 ± 0.1 cm, and –0.1 ± 0.2 cm .Tongue volume outside of OC was 7.1 ± 3.8 cmt-test.13 patients with HNSCC underwent CT-simulations both with a neutral tongue position and a stick-out tongue for IMRT planning . Planning objectives were to deliver 70 Gy, 63 Gy, and 56 Gy in 35 fractions to 95% of PTVs. Radiation Therapy Oncology Group (RTOG) recommended dose constraints were applied. Data are presented as mean ± standard deviation and compared using the student IMRT-S for patients with HNSCC who refused or could not tolerate an IOD has significant decreased radiation dose to the tongue than IMRT-N, which may potentially reduce RT related toxicity in tongue in selected patients. Radiation therapy (RT) is part of the standard treatment for head and neck squamous cell carcinoma (HNSCC) together with surgery and chemotherapy . DespiteRadiation dose to salivary glands and mucosa lining in oral cavity and pharynx is closely related with decreased salivary flow, oral mucositis and dysphagia, which are exacerbated by concurrent chemotherapy during RT , 11. AdvThe oral cavity (OC) and its subsites, such as tongue, palate and buccal mucosa, contain minor salivary glands, taste receptors, and muscles coordinating speech and swallowing , 16, 17.The tongue is a mobile organ and can be easily elongated without causing discomfort even in patients with trismus or gag reflex. In the present cases report, authors demonstrate anatomical changes in OC and its subsites with different tongue positions, and dosimetric advantages by applying IMRT-S over IMRT-N in patients with HNSCC but refused or did not tolerate an IOD during RT.Of 13 patients, 8 patients were diagnosed with squamous cell carcinoma in oropharynx without tongue involvement. Trismus, severe gag reflex, and refusal to use IOD were the reasons why patients refused or did not tolerate IOD during CT-simulation with customized immobilization mask . However, geometrical shapes in OC changed with different tongue positions. As shown in Figures 3 with “stick-out” tongue position, which comprised 5.4 ± 2.6% of entire OC volume and 7.8 ± 3.1% of oral tongue volume. Positional offsets of tongue during the course of IMRT-S were –0.1 ± 0.2 cm in vertical, 0.01 ± 0.1 cm in longitudinal, and –0.1 ± 0.2 cm in lateral direction.The mean OC volumes were similar in IMRT-N and IMRT-S (129.2 ± 34.0 cmp < 0.001). Lip was located 0.7 ± 0.3 cm more anteriorly in IMRT-S than IMRT-N.By “stick-out” tongue, lip was pushed away from gingiva in mandible. Distances between anterior surface of mandible and anterior tip of lip was 0.9 ± 0.4 cm in IMRT-N and 1.5 ± 0.3 cm in IMRT-S (p < 0.001).Different tongue positions during CT-simulation also created different shapes in inside of OC. The dorsal surface of tongue was abutting the hard palate with 0.1 ± 0.2 cm of separation in IMRT-N Figure . In IMRTp < 0.001). Table Interestingly, BOT was also moved to anterior direction with “stick-out” tongue resulting more distance between BOT and pharyngeal constrictor (PC) in IMRT-S Figure  and 2F tmean to OC with IMRT-S (31.4 ± 8.7 Gy) comparing to IMRT-N (34.9 ± 8.0 Gy) (p < 0.001). OC volume receiving equal or greater than 36 Gy (V36) was 40.6 ± 16.9% and 33.0 ± 17.0% of entire OC .Changes in geometrical shapes in OC with different tongue position during CT-simulations have affected on radiation dose to OC and its subsites in IMRT plans. Although mean OC volumes were similar in IMRT-N and in IMRT-S Table , there wmean to tongue was decreased from 38.1 ± 7.9 Gy in IMRT-N to 32.8 ± 8.8 Gy in IMRT-S (14.8 ± 7.3% of reduction) (p < 0.001). Figure p < 0.001).By elongated and stretched out tongue from mouth, there were significant decreases in radiation dose to tongue in IMRT-S compared to those in IMRT-N. Dmean to lips with IMRT-S comparing to IMRT-N. Dmean to lips was 16.0 ± 3.7 Gy in IMRT-N and 13.5 ± 4.0 Gy in IMRT-S (p < 0.001).Changes in lips positions with different tongue positions also demonstrated that 16.0 ± 10.8% of reduction in Dn = 8) and laryngeal cancer (n = 5) patients  contains most of the minor salivary glands, which are mostly located in the buccal, labial, distal palatal, and lingual mucosa . The minAmong minor salivary glands, the lingual serous (von Ebner's) glands on the dorsal surface of the tongue secrete digestive enzymes and proteins with facilitate the perception of taste , 17. Radmean to oral tongue comparing to with neutral tongue position (Table p < 0.001). For the patients with HNSCC and who refuses or can't use bite-block or tongue blade, IMRT-S rather than IMRT-N can lower Dmean to OC and OT, and lower V30 to oral tongue, and therefore, may spare more minor salivary glands as well as taste receptors on the tongue.Authors in this report observed 5.4 ± 2.6% of OC volume and 7.8 ± 3.1% of oral tongue volume became in outside of mouth, i.e., away from RT targets, with “stick-out” tongue position, which has significantly decreased D2+, which creates less force in muscles on stimulation [mean to tongue was related to decrease in tongue movement, which adversely effecting on speech quality after RT [Changes in speech and swallowing function in patients with HNSCC after RT or CCRT are also related to radiation dose to OC, tongue and pharyngeal constrictor , 25, 26.mulation . Radiatimulation . Clinicaafter RT . Data suafter RT , 25, 26.In addition to decreased radiation dose to tongue with IMRT-S, this report also found an increased distance from BOT to pharyngeal constrictor from 0.9 ± 0.3 cm with neural tongue position to 1.5 ± 0.5 cm with “stick-out” tongue position Figures . For themean to lips by 16.0 ± 10.8% with IMRT-S than IMRT-N (p < 0.001). Distance from dorsal surface of oral tongue to hard palate was increased to 0.9 ± 0.4 cm with “stick-out” tongue position from 0.1 ± 0.2 cm with neutral tongue position  from “stick-out” tongue during RT needs further investigation, this report has showed lower radiation doses to OARs as low as reasonably achievable for the patients who refused or could not use bite-block or tongue blade during RT, comparing to neutral tongue position during RT Table . With thBetween January of 2013 and November of 2015, thirteen patients with histologically proven squamous cell carcinoma of the head and neck underwent CT-simulations: one with neutral tongue and one applying “stick-out” tongue position for planning IMRT. Due to the uncertainty of reproducible tongue position during daily RT, authors had limited applying “stick-out” tongue position only to the patients with oropharyngeal cancer but without oral tongue involvement. Therefore, daily variation of tongue position would not compromise radiation targets coverage.max) in 0.1 cc < 50 Gy; brain stem = Dmax in 0.1 cc < 54 Gy; mandible = Dmax in 0.1 cc < 70 Gy, and V50 < 30%; parotid glands = mean radiation dose (Dmean) < 26 Gy; submandibular gland = Dmean < 36 Gy; PC = Dmean < 50 Gy; larynx = Dmean < 36 Gy; upper esophagus = Dmean < 40 Gy; brachial plexus = Dmax in 0.1 cc < 64 Gy; and OC = Dmean < 36 Gy. Treatment planning aimed to reduce doses to OARs as much as possible without compromising the coverage of the PTVs. Pinnacle radiation therapy planning system  was used for IMRT planning. All plans were performed with 7- or 9-beam using 6 MV photon applied using a Varian iX Silhouette .Before the CT-simulation, patients were informed and guided to “stick-out” tongue with comfortable and reproducible ways. The thermoplastic mask was customized to create a marker that indicates the location of tip of the tongue in the “stick-out” tongue position Figure , and immDaily cone beam CT (CBCT) was performed before IMRT for image-guidance and checking tongue position. The tongue contoured on CT-simulation was compared with one on daily CBCT to measure daily offset of the tongue during the course of IMRT-S. Dosimetric comparison between IMRT-N and IMRT-S was performed using the overall target dose-volume histogram (DVH), quantitative values of the target minima, maxima, and mean dose and normal tissue mean dose.t-test. Data are presented as mean ± standard deviation. A probability level of a p value of < 0.05 was considered significant.Statistical analysis of dosimetric comparison between IMRT-N and IMRT-S was done using a Student In this cases report, IMRT-S for patients with HNSCC but who refused or could not tolerate an IOD have significant decreased radiation dose to OC and its subsites, specifically oral tongue and lips, and increased distance between base of tongue and pharyngeal constrictor, dorsal surface of tongue to palate comparing to IMRT-N. With customized thermoplastic mask, daily “stick-out” tongue position was reproducible. These dosimetric benefits with IMRT-S are noticed in both oropharyngeal and laryngeal primary HNSCC. To confirm decreased radiation doses to OARs with IMRT-S translating into clinical benefits, prospective trial with long-term clinical outcome is warranted.


P<0.05), while had no detectable effect on atrogin-1 protein (P>0.05). FoXO1/3a (Thr24/32) phosphorylation was enhanced (P<0.05), mTOR phosphorylation was suppressed (P<0.05), while Akt protein expression was not affected (P>0.05) by DEX. RU486 treatment inhibited the DEX-induced increase of FoXO1/3a phosphorylation (P<0.05) and MuRF1 protein; LY294002 (LY) did not restore the stimulative effect of DEX on the FoXO1/3a phosphorylation (P>0.05), but inhibited the activation of MuRF1 protein induced by DEX (P<0.05); rapamycin (RAPA) inhibited the stimulative effect of DEX on the FoXO1/3a phosphorylation and MuRF1 protein (P<0.05).The ubiquitin-proteasome system (UPS)-dependent proteolysis plays a major role in the muscle catabolic action of glucocorticoids (GCs). Atrogin-1 and muscle-specific RING finger protein 1 (MuRF1), two E3 ubiquitin ligases, are uniquely expressed in muscle. It has been previously demonstrated that GC treatment induced MuRF1 and atrogin-1 overexpression. However, it is yet unclear whether the higher pharmacological dose of GCs induced muscle protein catabolism through MuRF1 and atrogin-1. In the present study, the role of atrogin-1 and MuRF1 in C2C12 cells protein metabolism during excessive dexamethasone (DEX) was studied. The involvement of Akt/forkhead box O1 (FoXO1) signaling pathway and the cross-talk between anabolic regulator mammalian target of rapamycin (mTOR) and catabolic regulator FoXO1 were investigated. High concentration of DEX increased MuRF1 protein level in a time-dependent fashion ( Skeletal muscle is an essential component of the body. The maintenance of skeletal muscle mass is of paramount importance for systemic energy homeostasis . The musThe increased muscular loss during stress is mainly caused by activation of proteolytic systems . AmongstThe forkhead transcriptional factor subfamily forkhead box O1 (FoxO1) plays essential roles in the transcriptional cascades that are responsible for metabolism in the muscle, liver, brain, pancreas, and adipose tissues –19. BothIn this study, we investigated whether excessive GCs enhanced proteolysis through the MuRF1 and atrogin-1, and the involvement of Akt/FoXO1 signaling pathway was also evaluated. According to previous report  and our 2. When cells were 70% confluent, the proliferation medium was replaced with DMEM containing 2% horse serum  for inducing myogenic differentiation. After 84 h, the cultures were shifted to serum-free DMEM for 12 h [Murine C2C12 cells  were cultured in DMEM  supplemented with 10% FBS , 100 U/ml penicillin, and 100 µg/ml streptomycin  and maintained at 37°C in a humidified atmosphere containing 5% COfor 12 h .After a 12-h incubation in serum-free medium, cells were exposed to DMEM-LM  with or without 100 µmol/l DEX –25 and cAfter a 12-h incubation in serum-free medium, cells were exposed to one of the four following treatments: basal serum-free medium (Control); basal medium with 100 µmol/l DEX for 8 h (DEX); basal medium with 1.5 µmol/l epoxomicin (EPOX) for 5 h; basal medium with 1.5 µmol/l EPOX for 5 h prior to addition of 100 µmol/l DEX for 8 h (DEX + EPOX). Following this, all cells were collected and subjected to the further analysis.After a 12-h incubation in serum-free medium, cells were exposed to one of the four following treatments: basal serum-free medium (Control); basal medium with 100 µmol/l DEX for 8 h (DEX); basal medium with 10 µmol/l RU486 for 5 h (RU486); basal medium with 10 µmol/l RU486 for 5 h prior to addition of 100 µmol/l DEX for 8 h (DEX+RU486). Following this, all cells were collected and subjected to the further analysis.After a 12-h incubation in serum-free medium, cells were exposed to one of the four following treatments: basal serum-free medium (Control); basal medium with 100 µmol/l DEX for 8 h (DEX); basal medium with 10 µmol/l LY294002 for 5 h (LY); basal medium with 10 µmol/l LY294002 for 5 h prior to addition of 100 µmol/l DEX for 8 h (DEX+LY). Following this, all cells were collected and subjected to the further analysis.After a 12-h incubation in serum-free medium, cells were exposed to one of the four following treatments: basal serum-free medium (Control); basal medium with 100 µmol/l DEX for 8 h (DEX); basal medium with 25 µmol/l rapamycin (RAPA) for 5 h; basal medium with 25 µmol/l RAPA for 5 h prior to addition of 100 µmol/l DEX for 8 h (DEX+RAPA). Following this, all cells were collected and subjected to the further analysis.g for 5 min at 4°C. Protein concentration was determined using the BCA assay kit  according to the manufacturer’s protocol. After boiling at 100°C for 5 min, the protein extracts were electrophoresed in 7.5–10% SDS polyacrylamide gels  according to the procedure described by Laemmli [2448), anti-mTOR , anti-p-Akt (Ser473), and anti-Akt . Protein detection was performed using goat anti-rabbit IgG (H + L)-HRP conjugated secondary antibody  or HRP-labeled goat anti-mouse IgG (H + L) secondary antibody  with ECL using Western blot detection reagents . β-actin was used as a loading control . Western blots were developed and quantified using BioSpectrum 810 with VisionWorksLS 7.1 software .Cell samples were homogenized on ice in radioimmunoprecipitation lysis buffer  and centrifuged at 12000  Laemmli . The sep−ΔΔCT2) was used to quantitate mRNA expression, according to Livak and Schmittgen method [Total RNA extraction and real-time PCR were performed as described previously . Primersn method . All samP<0.05.The data are presented as the mean ± S.E.M. All the data were subjected to one-way ANOVA analysis performed with the Statistical Analysis Systems statistical software package  to test the main effect of treatments. The homogeneity of variances amongst treatments was confirmed using Bartlett’s test (SAS Institute). When the primary effect of treatment was significant, differences between means were assessed by Duncan’s multiple range analysis. Means were considered significantly different at P<0.05, P<0.05, P<0.05, P>0.05, P>0.05, P>0.05, P<0.05, P<0.05, P<0.05, P<0.05, P<0.05, P>0.05, The effect of high concentration of DEX on the UPS was investigated. Atrogin-1 mRNA level was significantly increased at 3 h P<0.05, A, decreaP<0.05, P<0.05, P>0.05, P<0.05, The ubiquitin ligase MuRF1 was dramatically regulated by DEX treatment at 8 h; therefore, we then detected the DEX effect at 8 h. The upstream pathway of atrogin-1 and MuRF1, Akt/FoXO1, was then measured. DEX treatment increased the FoXO1 mRNA and decreased FoXO3 mRNA P<0.05, A, while P<0.05, P>0.05, To further confirm the role of ubiquitin ligases in UPS pathway during GC treatment, EPOX was used to inhibit proteasome in C2C12 cells. MuRF1 protein expression was suppressed by DEX + EPOX compared with DEX treatment alone P<0.05, A. HoweveP<0.05, P>0.05, To further explore the mechanism underlying GC effect on UPS pathway, RU486 was used to inhibit GR in C2C12 cells. DEX-induced increase of FoXO1/3a phosphorylation was significantly inhibited by DEX+RU486 treatment P<0.05, A, and DEP>0.05, P<0.05, To further explore the effect of PI3K/Akt on UPS pathway, LY294002 was used to inhibit PI3K in C2C12 cells. DEX+LY294002 did not restore the stimulative effect of DEX on the FoXO1/3a phosphorylation P>0.05, A, but inP<0.05, P>0.05, To further explore the effect of mTOR on UPS pathway, RAPA was used to inhibit mTOR in C2C12 cells. DEX + RAPA inhibited the stimulative effect of DEX on the FoXO1 phosphorylation and MuRF1 protein P<0.05, A,B. HoweThe previous report  and our The catabolic effect and muscle atrophy caused by GCs have been well studied in mammals  and in cAdrenal GCs produce their actions via a signal pathway involving the ubiquitously expressed GR. Upon binding GCs, GR translocates into the nucleus and binds to the GC response element (GRE) in the promoters of target genes. It has been reported that the promoter regions of MuRF1 , but notSeveral studies have demonstrated that muscle-specific ubiquitin ligase MuRF1 is the target of FoxO1, and FoxO1 inhibition prevents MuRF1 activation ,33. The FoxO1 is an important downstream target of the PI3K/Akt pathways ,37. FoxOExcessive GCs cause muscular loss by impeding protein synthesis and enhancing protein breakdown . The mTOGiven GCs facilitated muscle proteolysis meanwhile blunting muscle protein synthesis, we speculate there are associations between atrophy-related genes and anabolic regulators. We thus investigated whether MuRF1 is affected by mTOR signaling, using mTOR inhibitor RAPA. Much of the evidence suggests that there is a cross-talk between mTOR and FoXO1-MuRF1 pathways. mTOR signaling could be inhibited directly by FoXO1 , whereasIn conclusion, the present study indicates that excessive GCs increased UPS-dependent protein proteolysis via stimulating MuRF1, but not atrogin-1. GCs induced MuRF1 overexpression independent of Akt/FoXO1 pathway.


Background: Hedyosmum brasiliense Mart. ex Miq. (Chloranthaceae) is a dioecious shrub popularly used in Brazil to treat foot fungi and rheumatism. This work investigated the chemical composition, antifungal, and antioxidant activities of flowers and leaves of H. brasiliense essential oils; Methods: H. brasiliense male and female flowers and leaves were collected at Ilha do Cardoso (São Paulo) and the essential oils were extracted by hydrodistillation and analyzed by GC/MS and their similarity compared by Principal Component Analysis. Antifungal activity was performed by bioautography and antioxidant potential by 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) free radical scavenging and β-carotene/linoleic acid system; Results: The major compounds for all oils were sabinene, curzerene, and carotol, but some differences in their chemical composition were discriminated by Principal Component Analysis (PCA) analysis. Bioautography showed two antifungal bands at Rf’s 0.67 and 0.12 in all samples, the first one was identified as curzerene. The oils presented stronger antioxidant potential in β-carotene/linoleic acid bioassay, with IC50’s from 80 to 180 μg/mL, than in DPPH assay, with IC50’s from 2516.18 to 3783.49 μg/mL; Conclusions: These results suggested that curzerene might be responsible for the antifungal activity of H. brasiliense essential oils. Besides, these essential oils exhibited potential to prevent lipoperoxidation, but they have a weak radical scavenger activity.  Sarcandra Gardner, Chloranthus Sw., Ascarina J.R. Forst. and G. Forst., and Hedyosmum Sw. The genus Hedyosmum comprises 40 species of shrubs and small trees distributed from Mexico, throughout Central America to Bolivia, east of the Guianas and the Antilles /Abs control}. All samples were tested in triplicate and the results are expressed by mean ± standard deviation. The amount of essential oil required to reduce the initial DPPH concentration in the reaction by 50% is referred to as the inhibitory concentration (IC50). The antioxidant activity was performed by the 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) free radical scavenging method , adapted50). The essential oil (10 μL) solubilized in methanol at concentrations between 10,400–325 μg/mL was added in a 96-well microplate, together with 250 μL of a reactive solution composed by β-carotene and linoleic acid in water saturated with O2. Methanol (10 μL) was used as the negative control and as the positive controls, butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA) at concentrations between 130–4.1 μg/mL. The microplate was incubated in the dark at 45 °C and subsequently the absorbance was read at 450 nm , immediately after adding the reactive solution and every 30 min for 2 h. The absorbance was converted to percentage of antioxidant activity (AA) using the AA% = 100 − {[ × 100]/Abs control}. All samples were tested in triplicate and the results are expressed by mean ± standard deviation.The antioxidant activity was also evaluated by the β-carotene bleaching test, which is based on spectrophotometric measurements of the β-carotene oxidation induced by the lipoperoxidation products from linoleic acid  to deterH. brasiliense flowers were 0.24 ± 0.01 and 0.38 ± 0.01%, respectively. The yield of leaf essential oils, both male and female, presented a yield of 0.33 ± 0.01% , followed by sesquiterpenes (39% ♂ and 41% ♀), and phenylpropanoids (2% ♀ and 5% ♂). Flower essential oils presented monoterpenes (54% ♀ and 45% ♂), sesquiterpenes (40% ♂ and 50% ♀), and phenylpropanoids (1% ♂ and 2% ♀). The leaf main constituents were sabinene (16%), β-pinene (5%), 1,8-cineole (3–7%), methyl eugenol (2–5%), curzerene (17–18%), and carotol (6%), while flowers presented sabinene (8–10%), α-phellandrene (1–8%), 1,8-cineole (2–7%), germacrene D (4–6%), curzerene (11%), and carotol (9%) as major compounds. Fifty compounds were identified from all essential oils analyzed, comprising 96–98% of the total components. The major compounds for all Principal Component Analysis (PCA) data from H. brasiliense presented clear inhibition zones corresponding to antifungal activity in the limit of detection against Cladosporium cladosporioides and C. sphaerospermum  of 0.67 and 0.12, were observed in all samples  were calculated and the IC50 values are presented in Ginkgo biloba extract were employed as positive controls showing IC50 2.5 ± 0.09 and 13.5 ± 0.5 μg/mL, respectively, for DPPH assay, while butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA) were the positive controls for β-carotene assay, showing IC50 0.08 ± 0.09 and 0.27 ± 0.02 μg/mL, respectively.Antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and β-carotene bleaching methods. The 50% inhibitory concentration  Thymus vuAnopheles subpictus (LC50 = 4.14 μg/mL), Aedes albopictus (LC50 = 4.57 μg/mL), and Culex tritaeniorhynchus (LC50 = 5.01 μg/mL) [50 = 47 μmol/L in 72 h (in vitro) and 135 mg/kg daily (in vivo) [Eugenia uniflora L. essential oil, rich in curzerene (42.6%), showed antifungal activity, inhibiting completely Paracoccidioides Braziliensis at 62.5 μg/mL [E. uniflora essential oil composed by 47.3% of curzerene presented anti-Leishmania activity, with IC50 = 3.04 μg/mL against promastigotes and IC50 = 1.92 μg/mL against amastigotes of L. amazonensis [Curzerene has been known for its toxic effects against 1 μg/mL) . In addiin vivo) . Eugenia.5 μg/mL . Moreovezonensis . H. brasiliense essential oils was determined by two different mechanisms of action: DPPH free radical scavenging, that works as electron transfer, and β-carotene/linoleic acid assay, measuring the suppression of lipoperoxides. In the DPPH assay, the IC50 values for all the oils ranged from 2516.18 to 3783.49 μg/mL (G. biloba extract, that presented IC50 values of 2.5 and 13.5 μg/mL, respectively. These results are comparable to those obtained for Salvia tomentosa Mill. essential oils [Artemisia spp. were tested by the same methods and showed weak activities, as their composition were markedly rich in non-phenolic components [H. brasiliense were much more active in the β-carotene bleaching assay, presenting IC50 values from 71.12 to 180.71 μg/mL (H. brasiliense essential oils might be explained by the low amounts of phenolic compounds present in the oil, only 1.11 to 3.63% of thymol, and the high amounts of mono- and sesquiterpene hydrocarbons (The antioxidant activity of essential oils is already well known ,35,36,3749 μg/mL , indicatmponents . The antmponents . In a temponents . On the 71 μg/mL , indicatocarbons . Since aThe leaf and flower essential oils were dominated by sabinene, 1,8-cineole, curzerene, and carotol. However, the contents of these constituents differed according to the plant part and gender.H. brasiliense essential oils by bioautography. Moreover, the complex mixture of α-terpineol, α-eudesmol, and ferula lactone I may have acted synergistically to the antifungal activity observed. Bioautography assay helped to detect antifungal fractions in a complex matrix of essential oils and guided to the isolation of target-directed constituents. Further studies are necessary to isolate higher amounts of pure curzerene, in order to test its limit of detection. This was the first report of antifungal activity of curzerene, explaining partially the activity of H. brasiliense was more effective by β-carotene discoloration assay than by DPPH assay, indicating their higher ability in quenching conjugated hydroperoxides than free radicals. The inhibition of lipid peroxidation may have been a result of the interaction of all constituents of the essential oils, since it was not possible to attribute antioxidant activity to a single compound. Antioxidant activity of essential oils from


The key proteins related the nectarine fruit senescence during storage were identified by two-dimensional electrophoresis and MALDI-TOF/TOF. Level and enzymatic activity of peroxidase were reduced, while both the protein levels and the enzymatic activities of superoxide dismutase, glutathione reductase, glutathione-s-transferase and monodehydroascorbate reductase were enhanced in nectarine fruit treated with CHA. In addition, levels of several pathogen-related proteins were also enhanced by CHA-treatment. Taking together, the present study showed that CHA could influence changes in defense related proteins and reduced oxidative damage in nectarine fruit during postharvest ripening.To study how chlorogenic acid affects changes of reactive oxygen species (ROS) and the proteins involved in ROS scavenging of nectarine during storage time, the fruits were treated with chlorogenic acid (CHA) then stored at 25°C for further studies. The CHA-treatment significantly reduced O Rosaceae family, is a typical climactic fruit. It originated from China and represents one of the most important species of the stone fruits  fruit at green mature stage were obtained from a commercial orchard in Beijing, China, and was selected for uniformity in shape, color, and size, and then were used for the experiments.Nectarine [-1 chlorogenic acid  solutions under vacuum (-0.02 M Pa) for 2 min and kept in the solutions for additional 3 min at 25°C without vacuum. The control nectarines also were vacuum infiltrated with distilled water. Fruits were air dried, and then were stored at 25°C with 80–90% relative humidity. Samples were conducted at 0, 2, 4, 6, 8 days of storage, respectively and the sampled tissues were immediately powdered in liquid nitrogen and stored at -80°C until further analysis. Three replications were conducted in this experiment, and there were 60 fruits in each replicate.Nectarine fruit were randomly divided into two groups. One group was provided as the untreated control and the other group was infiltrated with 50 mg L2-·) production rate was determined according to [2-·. The O2-· production rate is expressed as mmol min-1 kg-1 fresh weight.Superoxide radical (Ording to  with som2O2) content was determined according to the method previously described [2O2 content is expressed as mmol kg-1 fresh weight.The hydrogen peroxide : 2.0 g of pulp powders were homogenized with 2.0 ml of 0.1 M sodium acetate buffer (pH 5.5) containing 0.34% PEG 6000 (w/v), 4% (w/v) polyvinypyrrolidone (PVP) and 1% (v/v) Triton X-100.Superoxide dismutase  and catalase : 2.0 g of pulp powders were homogenized with 2.0 ml of 0.1 M sodium phosphate buffer (pH 7.5) containing 5% (w/v) PVP and 5 mM dithiothreitol.Ascorbate peroxidase : 2.0 g of pulp powders were homogenized with 2.0 mL of 0.1 M potassium phosphate buffer (pH 7.5) containing 2% polyvinlpyrrolidone cross linked (PVPP), 1 mM ascorbic acid and 1 mM ethylene diamine tetraacetic acid (EDTA).Glutathione reductase : 2.0 g of pulp powders were homogenized with 2.0 mL of 0.1 M sodium phosphate buffer (pH 7.5) containing 1 mM EDTA, 2 mM dithiothreitol (DTT).Glutathione-s-transferase : 2.0 g of pulp powders were homogenized with 2.0 mL 0.2 M sodium phosphate buffer (pH 8.0), containing 1 mM EDTA, 4% (w/v) PVPP, 1mM DTT.Monodehydroascorbate reductase : 2.0 g of pulp powders were homogenized with 50 mM Tris-HCl (pH 7.8).All the homogenates were then centrifuged at 10,000×g for 30 min at 4°C. The supernatants were used for the enzyme assays.POD activity was determined by the increase in absorbance at 470 nm according to former research . One uniSOD activity was determined by measuring its ability to inhibit the photoreduction of nitro-blue-tetrazolium (NBT) as described by previous researchers . U of SO2O2 at 240 nm following the method of previous study [CAT activity was measured by monitoring the decomposition of Hus study . U of CAAPX activity was determined as described in previous research . The actGR activity was determined by the increase in absorbance at 334 nm due to former scientists . U of GRGST activity was determined by the increase in absorbance at 334 nm due to formation of s- glutathione (DNP-GS) from 1-chloro-2,4-dinitrobenzene (CDNB) and GSH, according to former researchers . U of GSMDHAR activity was determined by following the decrease in absorbance at 340 nm due to NADH oxidation, according to previous study . U of MD-1 protein). Protein content in the enzyme extracts was determined according to the Bradford method [The activity of each enzyme is expressed on a protein basis  overnight at 4°C, then was concentrated by lyophilization. Protein content was determined by Bradford method [Briefly, 1.0 g of frozen sample was finely powdered in a mortar with liquid nitrogen and then homogenized with 150 μL ice-cold 1 M Tris (pH 11.2) and 30 mg PVPP. The homogenate was centrifuged at 10,000×d method , using BAfter extraction, the proteins were solubilized in lysis buffer  CHAPS, 1% (w/v) DTT and 0.5% (v/v) pH 3–10 IPG buffer). 1 mg proteins were applied to 17 cm pH 3–10 IPG strips, and isoelectric focusing was performed on a PROTEAN IEF system  for a total of 116.4 kVh at 20°C. Then, the strips were equilibrated for two periods of 15 minutes with 1% (w/v) 1,4-dithiothreitol and 2.5% (w/v) iodoacetamide in equilibration buffer. Following equilibration, the strips were run on 12% home-made gels with a vertical set . Then the gels were stained with 0.1% Coomassie Brilliant Blue R250. Three biological replicates were performed for each treatment and each biological replicate with three technical replicates.p < 0.05) were considered as differentially abundant proteins.The stained gels were imaged by a Versdoc 3000 scanner , and analyzed by PDQuest Version 8.0 . Images were properly cropped and optimized, and then subjected to gel-to-gel matching with standard protein maps. The abundance of each protein spot was estimated by the percentage volume (% volume), i.e. the spot volume was normalized as a percentage of the total volume of all spots in the gel. Finally, the spots that changed more than 2-fold and passed the Student's t-test (http://wolfpsort.org).Method for protein in-gel digestion and identification by MALDI-TOF-TOF/MS was accorded to former researchers . Trypticp < 0.05 were considered as significant.Data were evaluated by the analysis of variance (ANOVA) with Statistical Analysis System of SPSS Statistics 17.0 . Significant differences were performed by Duncan’s new multiple range tests, where differences at 2-· production rate in nectarine fruit increased from 0.80±0.03 μmol g-1min-1 to 0.96±0.06 μmol g-1min-1 and the H2O2 content increased dramatically from 0.28±0.05 μmol g-1 to 2.17±0.33 μmol g-1 during storage at 25°C for 8 days. Treatment with CHA significantly reduced the ROS generation. The O2-· production rate or H2O2 content in the CHA-treated nectarine was only 0.77 μmol g-1min-1 or 1.36 μmol g-1 at end of the storage   decreasehttp://www.ncbi.nlm.nih.gov/GO/) and eukaryotic orthologous groups . 50% of these proteins is involved in cellular process and signaling, including posttranslational modification, signal transduction mechanism, defense mechanism and cytoskeleton. Among these 18 proteins, 27.8% of them is involved in response to stimulus and 22.2% is involved in metabolism.As shown in Two lowly expressed small heat shock proteins   were ideth day and 6th day of observation, calmodulin was remarkably induced by CHA treatment, and profilin of CHA group kept higher levels throughout the whole storage time than the control group  and profilin (spot 18)  were ideoup Figs  and 3.Our results showed some allergen proteins and pathogen-related proteins were nearly all up-regulated by CHA during the observation. With CHA treatment, levels of putative allergen Pru p 1.06B (spot 14), PR 10 (spot 16), and putative allergen Pru p 1.06 A (spot 17) were all enhanced  and 3. LTwo hypothetical protein involved were successfully identified: hypothetical protein PRUPE_ppa006990mg (spot 8), hypothetical protein PRUPE_ppa008531mg (spot 13) . It seemvs. POD activity, GR vs. GR activity and MDHAR vs. MDHAR activity were 0.410 (p<0.05), 0.407 (p<0.05) and 0.526 (p<0.01), respectively. Although it was not significant, a positive correlation between protein expression accumulations of POD (spot 4) and GST and their enzyme activities was also existed.In this study, a significant positive correlation was found between protein expression accumulations of POD (spot 3), GR, MDHAR and their enzyme activities. As shown in http://wolfpsort.org/), the 18 identified proteins are assigned to five categories Click here for additional data file.


Plutella xylostella, revealed an intrinsic capacity to detoxify plant defense compounds, which is an important factor in its success as a pest. Here we expand on that work with a complete taxonomic and functional profile of the P. xylostella gut microbiota obtained by metagenomic sequencing. Gene enrichment in the metagenome, accompanied by functional identification, revealed an important role of specific gut bacteria in the breakdown of plant cell walls, detoxification of plant phenolics, and synthesis of amino acids. Microbes participating in these pathways mainly belonged to three highly abundant bacteria: Enterobacter cloacae, Enterobacter asburiae, and Carnobacterium maltaromaticum. Results show that while the gut microbial community may be complex, a small number of functionally active species can be disproportionally important. The presence of specific enzymes in the microbiota community, such as supporting amino acid synthesis, digestion and detoxification functions, demonstrates the beneficial interactions between P. xylostella and its gut microbiota. These interactions can be potential targets for manipulation to provide novel pest management approaches.Herbivore specialists adapt to feed on a specific group of host plants by evolving various mechanisms to respond to plant defenses. Insects also possess complex gut microbiotas but their potential role in adaptation is poorly understood. Our previous study of the genome of diamondback moth,  Nasutitermes) gut bacteria  gut bacteria  symbiont Buchnera aphidicola  symbiont Burkholderia  (Lepidoptera: Plutellidae), is one of the world's most destructive pests that has co-evolved with the cruciferous plants, such as cabbage, broccoli, and cauliflower  methods  was collected in July 2004 from a vegetable field in Fuzhou , Fujian province, south eastern China. All necessary permits were obtained from the Institute of Plant Protection of the Fujian Academy of Agricultural Sciences. The larvae were fed on radish seedlings at 25 ± 2°C, 70–80% relative humidity and a 16 h light/8 h dark photoperiod without exposure to insecticides. Pupae were collected and transferred to a 500 mL plastic bottle where emerging adults were fed 10% honey solution to mate and lay eggs. Neonates were able to pass through perforations in these bottles and drop onto radish plants below.A susceptible To collect the gut contents, 200 individuals each of 3rd-instar larvae, pupae, and adults were randomly sampled regardless of sex. The samples were surface-sterilized with 75% ethanol for 90 s and rinsed with sterilized-deionized water. After dissection, the gut contents were homogenized with 1 ml sterile deionized water and frozen at −80°C for DNA extraction. Total DNA from the larvae, pupae and adults samples was extracted using the QIAamp® DNA Stool Mini Kit  following the manufacturer's protocol. The DNA products were run on 1.0% agarose gels and recovered for library construction and sequencing.de novo assembler  to generate the taxonomic profile or KO functional profile. The raw sequences of gut microbiota of P. xylostella larvae, pupae, and adults were deposited to the SRA (Sequence Read Archive) database under accession number of SRP018285.DNA library construction was performed following the manufacturer's instructions . We constructed one paired-end (PE) library with insert size of 350 bp for each life stage sample. Then, high-throughput sequencing was performed using Illumina Hiseq 2,000 to obtain the metagenome data. The read length for each PE end was 100 bp. The raw reads that contained three or more “N” or adapter contamination were discarded. If there were continuous low-quality  bases at the end of one read, these bases were trimmed from the read. The trimmed reads with lengths below 30 bp were discarded, and host contaminated reads were removed; the remaining effective reads were used for further analysis. For each sample, the effective reads were assembled to obtain long contigs by using SOAP −4, 10−5, and 10−6, and 50 μL of the suspension was spotted onto the caboxymethyl cellulose (CMC), xylan, and pectin degrading enzyme isolation medium. The media for CMC, xylan, and pectin degrading enzyme isolations were: CPS1 (g/L): CMC-Na 10 g, peptone 1 g, (NH4)2SO4 4 g, NaCl 1 g, K2HPO4 2 g, MgSO4·7H2O 0.4 g, agar 20 g, pH 7.0; XPS1 (g/L): xylan 8 g, yeast powder 1 g, (NH4)2SO4 4 g, K2HPO4 2 g, NaCl 0.5 g, MgSO4·7H2O 0.5 g, agar 20 g, pH 7.0, and PPS1 (g/L): pectin 10 g, yeast powder 1 g, (NH4)2SO4 4 g, K2HPO4 1 g, KCl 0.5 g, MgSO4·7H2O 0.4 g, FeSO4 0.01 g, agar 20 g, pH 7.0. The plates were cultured at 30°C for 3 days. Bacteria grown on the plates were purified and identified by another identification medium , which contained less substrate than the isolation medium. The potentially degrading bacteria grown on the plates  at 30°C for 7 days were used for enzyme activity identification. The CPS2 plates were dyed with 0.1% Congo red for 10 min, the plates were floated with 1 mol/L NaCl for 5 min, and CMC degrading enzyme activity was determined by the visible clearance zones. The XPS2 and PPS2 plates were dyed by 0.1% Lugel solution and Bromophenol blue for 5 min, respectively, to see whether clearance zones became visible.Fifteen 4th-instar larvae were randomly sampled regardless of sex. The samples were surface-sterilized with 75% ethanol for 90 s and rinsed with sterilized-deionized water. After dissection with a sterilized scalpel, the gut contents were homogenized with 1 mL sterile deionized water. The gut content suspension was diluted to 102O 10.5 μL, 10 × PCR ExTaq Buffer 1.5 μL, DNA template (100 ng/μL) 1.0 μL, 27F (10 mmol/L) 0.5 μL, 1492R (10 mmol/L) 0.5 μL, dNTP 1.0 μL, ExTaq (5 U/μL) 0.15 μL. After initial denaturation at 95°C for 5 min, amplification was performed using 30 cycles of 30 s at 95°C, 30 s at 56°C, 60 s at 72°C, followed by a final extension at 72°C for 10 min. Amplification products were then run on 1.0% agarose gels and purified, and the products were sequenced by ABI PRISM 3730. The 16S rRNA sequences were submitted to the NCBI GenBank .The representative bacteria that contained cellulose, xylan and pectin degrading activities were purified and named as DBMG1, DBMG2, DBMG3, DBMG4, DBMG5, and DBMG6. Strains were cultured in LB medium (g/L): peptone 10 g, yeast powder 5 g, NaCl 10 g, pH 7.0. Genomic DNA of each strain was isolated by Rapid Bacterial Genomic DNA Isolation Kit . To amplify 16S rRNA of each strain for bacterial identification, universal primers targeting the 16S rRNA, 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were designed as previously described  (g/L): (NH4)2SO4, 0.4; K2HPO4, 0.4; KH2PO4, 0.2; MgSO4, 0.1; NaCl, 0.1; Fe2(SO4).H2O, 0.01; MnSO4.H2O, 0.01; for a 100 mg/L final concentration of filter-sterilized phenol as a carbon source, pH 7.5. The 100 mL phenol medium in a 250 mL conical flask with the bacteria was incubated in a rotary shaker at 150 rpm. Phenol degradation was monitored in the colorimetric assay according to established method , followed by Firmicutes , and to lesser extent, Cyanobacteria, Bacteroidetes, Actinobacteria, Nitrospirae, and the archaea species Euryarchaeota  and E. asburiae . A total of 47,872 effective reads from 9 samples with an average of 5,319 reads per sample were produced , followed by Firmicutes , glycosyltransferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), auxiliary activities (AAs), and carbohydrate binding modules (CBMs)  gut  contribute to plant cell wall degradation. This capacity enhances food utilization efficiency for the insect host  isolated from  Figures . The CMC Figures . The six Figures . DBMG1 a Figures . In hone Figures . Our resP. xylostella genome reveals the mechanism of detoxification of some plant defense compounds, particularly glucosinolates . We believe that these two species confer protection to the host insect against certain plant defense compounds of Brassica plants. Further functional analytical experiments indicated that the P. xylostella gut bacteria could degrade the phenol significantly in vitro  and glutathione S-transferase (GST)  when they are oxidized  from phosphoribosyl pyrophosphate (PRPP) as well as the pathway for the synthesis of threonine (Thr) from aspartate (Asp). We found that the P. xylostella gut bacteria metagenome possessed the whole pathway for synthesis of His and Thr , XX was supported by the project of the National Natural Science Foundation of China (Nos. 31501639).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Naturforsch. Teil A, 17, 630; Keller & Kanellakopulos  × 10−4 K−1. NaTcO4 adopts the scheelite (CaWO4) structure type in space group type I41/a with Na and Tc atoms (both with site symmetry -4) replacing Ca and W atoms, respectively.The redetermination of the title compound, sodium pertechnate, from single-crystal CCD data recorded both at 296 and 100 K confirms previous studies based on X-ray powder diffraction film data [Schwochau 1962. Z. Natuulos 1963. Radioch al. 2017. Inorg.  Since the inception of quantum chemistry, compounds of this type have been models  for which the validity of the proposed equations and approximations for the case of d-electrons are verified. It was believed that, owing to the d0 electronic state, they define the least complex class of compounds of d-elements. Such simplicity, due to the absence of d-electrons and their pseudospherical symmetry, does by far not imply that any of these compounds show no complex behavior under changing environmental conditions, e.g. by changing temperature and/or the strength of the crystal field, and publications on the discovery of a more complex behaviour and properties appeared periodically. For example, for sodium . The obtained bond lengths and angles are similar to those obtained from previous X-ray powder  Å, c = 11.874 (6) Å given by Weaver et al. (2017a = 5.2945 (2) Å, c = 11.7470 (5) Å . These values represent the thermal volumic expansion coefficient of 1.19 (12) × 10 −4 K−1. The c/a ratio in this structure changes from 2.2187 (7) to 2.2223 (4) as a function of the temperature change from 100 to 296 K.Lattice parameters determined here with the precision of 0.0002-0.0005 Å at 296 K Table 1 are clos al. 2017. The lat4 is isostructural to KTcO4 and RbTcO4  global, I, II. DOI: 10.1107/S2056989017008362/wm5391Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017008362/wm5391IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989017008362/wm5391Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017008362/wm5391IIsup5.cmlSupporting information file. DOI: 1554512, 1554511CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: