Alkaline α-amylases have potential applications for hydrolyzing starch under high pH conditions in the starch and textile industries and as ingredients in detergents for automatic dishwashers and laundries. While the alkaline α-amylase gains increased industrial interest, the yield of alkaline α-amylases from wild-type microbes is low, and the combination of genetic engineering and process optimization is necessary to achieve the overproduction of alkaline α-amylase.Bacillus alcalophilus JN21 (CCTCC NO. M 2011229) was cloned and expressed in Bacillus subtilis strain WB600 with vector pMA5. The recombinant alkaline α-amylase was stable at pH from 7.0 to 11.0 and temperature below 40°C. The optimum pH and temperature of alkaline α-amylase was 9.0 and 50°C, respectively. Using soluble starch as the substrate, the Km and Vmax of alkaline α-amylase were 9.64 g/L and 0.80 g/(L·min), respectively. The effects of medium compositions  and temperature on the recombinant production of alkaline α-amylase in B. subtilis were investigated. Under the optimal conditions (starch concentration 0.6% (w/v), peptone concentration 1.45% (w/v), soybean meal concentration 1.3% (w/v), and temperature 37°C), the highest yield of alkaline α-amylase reached 415 U/mL. The yield of alkaline α-amylase in a 3-L fermentor reached 441 U/mL, which was 79 times that of native alkaline α-amylase from B. alcalophilus JN21.The alkaline α-amylase gene from B. subtilis, and the obtained results make it feasible to achieve the industrial production of alkaline α-amylase with the recombinant B. subtilis.This is the first report concerning the heterologous expression of alkaline α-amylase in  The alkWhile the alkaline α-amylases have potential applications in textile and detergent industries, relatively few efforts have been made to improve the yield of alkaline α-amylases. The current studies mainly focus on the strain screening, enzyme purification, and properties characterization ,9,12-19.Bacillus halodurans MS-2-5 and Bacillus halodurans 38C-2-1 was expressed in Escherichia coli and the highest yield reached 52 U/mL under the optimal conditions [E. coli, Bacillus subtilis is non-pathogenic and bacteriophage-resistant, and has been extensively used for the overproduction of heterologous proteins [B. subtilis have not been reported.The yield of alkaline α-amylase from wild-type microbes, especially from extremophilic microbes, is usually low and hard to be improved, even with process optimization and control ,14,16. Inditions . Compareproteins -28. NeveBacillus alcalophilus JN21 (CCTCC NO. M 2011229), and the optimum pH and temperature of the alkaline α-amylase were 9.5 and 55°C, respectively [B. subtilis expression system was constructed to clone and express the alkaline α-amylase from B. alcalophilus JN21. The biochemical characterization of the recombinant alkaline α-amylase was also performed. To achieve the over-production of alkaline α-amylase in B. subtilis, the key media components  and temperature were optimized in shaker flask. The production of alkaline α-amylase by B. subtilis was conducted in a 3-L fermentor under the obtained optimal conditions. The results obtained here may be useful for the achievement of industrial alkaline α-amylase production.In our previous work, a strain capable of producing alkaline α-amylase was isolated and identified as B. alcalophilus JN21 by PCR and cloned into the B. subtilis expression vector, pMA5  secreted into the culture medium Figure . The recEffect of pH on the activity and stability of recombinant alkaline α-amylase was determined. As shown in Figure Figure Figure Km and Vmax valves were determined by nonlinear fit analysis based on Eadie-Hofstee plots [Km) of 9.64 g/L and maximum reaction rate (Vmax) of 0.80 g/(L·min) at 55°C  and the catalytic efficiency (kcat /Km) of the alkaline α-amylase were 7.53 min-1 and 0.78 L/(g·min), respectively.The kinetics of the recombinant alkaline α-amylase was analyzed using soluble starch as substrate. The ee plots . The ini+, indicating that the alkaline α-amylase activity was Na+ -dependent with a relative activity of 110%. The Ca2+ (1 mM) and K+ (10 mM) almost had no activation or inhibition on the alkaline α-amylase with a relative activity of 102% and 100%, respectively. The following metal ions had slight inhibitions on enzyme activity: Na+ (10 mM), Ca2+ (10 mM), Co2+ (1 and 10 mM), Zn2+ (1 and 10 mM), Mg2+ (1 and 10 mM), Cu2+ (1 and 10 mM), Fe3+ (1 and 10 mM), K+ (1 mM) and Fe2+ (10 mM). On the other hand, the stronger inhibitory effects were observed in the presence of Fe2+ (1 mM) and Mn2+ (1 and 10 mM), and only 26.5% of activity was retained in the presence of 10 mM Mn2+. The inhibitions of divalent metal ions on α-amylase from the other sources were also observed [Metal ions played a key role in protein folding and catalysis . As showobserved -35.Figure As shown in Figure The recombinant alkaline α-amylase activity increased with the increase of soybean meal concentration, and reached the highest level of 412 U/mL at 1.3% soybean meal Figure . The furB. subtilis also reached at 37°C. The yield of recombinant alkaline α-amylase significantly decreased at lower temperatures (below 37°C) or higher temperatures (above 37°C). The temperature was very important for the recombinant alkaline α-amylase expression in B. subtilis.As shown in Figure B. subtilis was further explored in a 3-L fermentor . As shown in Figure Bacillus alcalophilus JN21 and 8.5 times that of alkaline α-amylase from recombinant E. coli [B. subtilis was a suitable host for the industrial production of alkaline α-amylase.The expression efficiency of the engineered  E. coli . The higB. alcalophilus JN21 was cloned and expressed in B. subtilis WB600. The optimum pH of recombinant alkaline α-amylase was 9.5, and it displayed optimal stability at pH 9.0. The optimal temperature of the alkaline α-amylase was 50°C, and it was stable below 40°C. The recombinant alkaline α-amylase hydrolyzed soluble starch with Km of 9.64 g/L and Vmax of 0.80 g/(L·min). The alkaline α-amylase was slightly activated by 1 mM Na+, 1 mM Ca2+ or 10 mM K+ but inhibited by other metal ions. Under the optimal conditions , the highest yield of alkaline α-amylase reached 415 U/mL, which was 4.2-fold that before optimization. The yield of alkaline α-amylase in a 3-L fermentor reached 441 U/mL.In this work, the gene of alkaline α-amylase from Bacillus alcalophilus JN21 (CCTCC NO: M 2011229) was an isolate of Lab of Bioprocess and Biosystems Engineering, Jiangnan University. Bacillus subtilis WB600, a strain that contains deletions of all six extracellular protease genes, and the plasmid pMA5 were obtained from Invitrogen. The EZ-10Spin Column Plasmid Mini-Preps kit, agarose gel DNA purification kit, restriction enzymes, and T4 DNA ligase were obtained from TakaRa . Other chemicals were obtained from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. . The synthesis of DNA primers and DNA sequencing were performed by Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. .The strain of E. coli/B. subtilis shuttle vector (pMA5) was used to clone and express alkaline α-amylase. The mature alkaline α-amylase gene was amplified by polymerase chain reaction (PCR) using gene-specific primers as follows: 5'ACGCGTCGACATGGAGCATASGGCCATGACGA3' (forward primer) and 5'CGGGATCCTTATGACCGCCGAATCAGTGAAGC3' (reverse primer). The forward primer and the reverse primer contained a Sal I and a BamH I restriction sites (underlined), respectively. The amplification was carried out under the following conditions: the first step was at 94°C for 10 min, followed by 30 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 2 min, and the final extension was carried out at 72°C for 10 min. The PCR product was digested with Sal I and BamH I, gel-purified and then ligated into pMA5 which was subjected to a similar treation. The recombinant plasmid was identified by restriction analysis and sequencing.The A-amyQ was transformed into the competent cells of Bacillus subtilis WB600. The transformants were selected at 37°C on the LB agar plates containing 100 μg/mL Kanamycin and 1% soluble starch for 8 h. The transformants with transparent rings around colonies were positive. The presence of alkaline α-amylase gene in the transformants was confirmed by PCR. The colonies were grown in 25 mL of LB medium at 37°C for 36 h in conical flasks (250 mL).The recombinant plasmid pMA5/B. subtilis were examined. The starch of different concentrations ) on the production of alkaline α-amylase was added into the culture medium to examine the effects of starch on alkaline α-amylase production. The peptone of different concentrations ) was also added into the culture medium to examine the effects of peptone on alkaline α-amylase production. Soybean meal of different concentrations ) was added into the culture medium to investigate the effects of soybean meal on the alkaline α-amylase production. The effects of temperature  on the production of alkaline α-amylase were also investigated.The effects of culture conditions  on the recombinant production of alkaline α-amylase in B. subtilis with the highest alkaline α-amylase yield was used to scale up fermentation in a 3 L fermentor . The medium was supplemented with 1.45% peptone, 0.6% soluble starch, 1.3% soybean meal, 1.0% NaCl and 100 μg/mL Kanamycin. The fermentation was performed with a working volume of 1.6 L at 37°C for 60 h. The stirring speed of fermentation was 600 rpm, and the volume of ventilation was 1 vvm.The recombinant Alkaline α-amylase was determined by measuring the amount of reducing sugar released during enzymatic hydrolysis of 1% soluble starch in glycine/NaOH buffer (pH 10.0) at 55°C for 5 min. A control without enzyme addition was used. The amount of reducing sugar was measured by a modified dinitrosalicylic acid method . One uniSolid ammonium sulfate was added to the supernatant to 70% saturation at 0°C. The precipitate was collected and dissolved in glycine/NaOH buffer (pH 9.0) and dialyzed overnight against the same buffer. After dialysis, the enzyme solution was filtered using water film. And then the enzyme solution was injected into AKTA purifier  through the anion exchange (Q-Sepharose HP). The subunit molecular weight of recombinant alkaline α-amylase was determined by SDS-PAGE.Km and Vmax according to the enzyme reactions [The reaction was performed in glycine/NaOH buffer (pH 10.0) at 55°C for the determination of kinetic parameters. Assays were performed with active enzyme and soluble starch of different concentrations from 1 to 10 g/L. The Eadie-Hofstee plots were used to calculate kinetic parameters eactions .To determine the optimal temperature, the enzyme was analyzed from 30 to 90°C in glycine/NaOH buffer (pH 10.0). The thermal stability of alkaline α-amylase was determined at the indicated temperatures in glycine/NaOH buffer (pH 10.0). To estimate the optimal pH for the alkaline α-amylase, the purified protein was incubated in various buffers. The buffer used for determination of optimal pH was as follows: glycine/HCl buffer for pH 3.0-6.0, glycine/NaOH buffer for pH 6.0-11.0. The pH stability of alkaline α-amylase was determined at pH ranging from 3.0 to 11.0 at 25°C for 24 h. After incubation, the alkaline α-amylase activity was measured at pH 10.0 and 55°C.2, MgCl2, FeCl3, FeCl2, CoCl2, ZnCl2, MnCl2 and CuCl2, respectively. The relative activity was determined and compared with the activity obtained in glycine/NaOH buffer (pH 10.0) without the addition of any metal ions.Alkaline α-amylase was pre-incubated with 1 mM and 10 mM KCl, NaCl, CaClThe protein content of samples was measured by the Bradford method with BSA as a standard .The authors declare that they have no competing interests.HQY carried out the cloning and expression of alkaline α-amylase, and drafted the manuscript. JHL carried out the fermentation. LL participated in the statistical analysis of data. GCD and JC critically revised and corrected the manuscript. All authors read and approved the final manuscript.


Pediococcus acidilactici strain D3, a component of multistrain commercial cultures with biopreservative and biocontrol properties in food-based applications. Strain D3 encodes at least one antimicrobial peptide, pediocin AMPd3. The AMPd3-encoding operon exhibits high sequence similarity to the archetype pediocin, PA-1, encoded by P. acidilactici PAC 1.0.We describe a draft genome sequence for  Approximately 1.3 billion tons of edible food is either lost or wasted annually, which represents nearly one-third of the global food mass that is produced for human consumption each year . As the Pediococcus acidilactici D3 is a component of lactic acid bacterium (LAB)-based biocontrol cultures that are generally recognized as safe (GRAS) for use in meat products , a sequencing coverage of approximately 2,289-fold was inferred. The reads were assembled de novo with SPAdes version 2.2.1  by the Texas AgriLife Genomics and Bioinformatics Center . The size of the library inserts ranged from 197 to 557 nucleotides . Based on the size of the on 2.2.1 . In totaP. acidilactici neotype strain, DSMZ 20284T, a D3 pseudochromosome was generated using the FGD and ACT software packages (P. acidilactici MA18/5M (1.99 Mb and 42% G+C) (P. acidilactici DSMZ 20284T (1.93 Mb and 42% G+C) (accession no. NZ_AEEG00000000.1), P. acidilactici 7_4 (2.01 Mb and 42% G+C) (accession no. NZ_ACXB00000000.1), and Pediococcus pentosaceus ATCC 25745 (1.83 Mb and 37.4% G+C) (accession no. CP000422).Based upon alignments with the packages , 14. The42% G+C)   tentatively related to the immunity to and expression of an antimicrobial peptide (pediocin), designated AMPd3. The AMPd3-deduced protein sequence is identical to that of an archetype pediocin, PA-1, encoded by P. acidilactici PAC 1.0  version 2 was used to predict protein-coding domain sequences and to annotate the draft genome . Based o PAC 1.0 .AQGT00000000. The version described in this paper is the first version, accession no. AQGT01000000.The Whole-Genome Shotgun project was deposited at DDBJ/EMBL/GenBank under the accession no.


The reliability of the pulse contour methods (PCMs) in cardiac output (CO) monitoring has been questioned when changes in arterial tone occur spontaneously  or after a therapeutic intervention . The purpose of this study was to compare the CO values assessed with the MostCare system  (MC-CO) with those obtained with transthoracic echocardiography  (TTE-CO) in trauma patients treated with norepinephrine.Twenty-seven adult trauma patients admitted to a seven-bed ICU and requiring norepinephrine infusion were enrolled in the study. Inclusion criteria were: age >18, no aortic valve pathologies, sinus rhythm. TTE-CO and MC-CO were evaluated simultaneously at two different stable hemodynamic states: baseline (T1), and after raising mean arterial pressure to 90 mmHg by starting norepinephrine infusion (T2). The MostCare system, an uncalibrated PCM, was connected directly to the main monitor of the patient for the analysis of the radial artery pressure wave. Bland-Altman and linear regression analyses were performed.R = 0.9; at T2 the mean bias between the techniques was -0.13 l/minute (2SD = 0.83 l/minute), PE was 17% and R = 0.88. Overall, a good correlation between TTE-CO and MC-CO was observed , with a mean bias of -0.10 l/minute (2SD ± 0.76 l/minute), 95% limits of agreement of -0.86 to 0.66 l/minute, and a PE of 16%. Mean arterial pressure was 82.2 ± 11.6 mmHg at T1 and 94.1 ± 3.8 mmHg at T2 (P < 0.05). Heart rate did not change significantly from T1 to T2 . Mean dosage of norepinephrine was 0.22 ± 0.1 μg/kg/minute (range 0.1 to 0.65 μg/kg/minute).Fifty-four paired CO values were obtained; TTE-CO values ranged from 2.9 to 6.8 l/minute and MC-CO from 2.8 to 6.9 l/minute. At T1 the mean bias between the techniques was -0.07 l/minute (2SD = 0.69 l/minute), with a percentage of error (PE) of 15% and MC-CO values showed a good agreement with TTE-CO at the two different hemodynamic states of trauma patients. Under the studied conditions, the reliability of the MostCare system seemed not to be affected by the changes in vascular tone induced by norepinephrine infusion.


E. coli and polyinosinic:polycytidylic acid [poly (I:C)], a synthetic analog of dsRNA virus, aiming to be used as a non-specific vaccine nanocarrier in different fish species. This liposomal carrier showed high encapsulation efficiencies and low toxicity not only in vitro using three different cellular models but also in vivo using zebrafish embryos and larvae. We showed that such liposomal LPS-dsRNA cocktail is able to enter into contact with zebrafish hepatocytes (ZFL cell line) and trout macrophage plasma membranes, being preferentially internalized through caveolae-dependent endocytosis, although clathrin-mediated endocytosis in ZFL cells and macropinocytocis in macrophages also contribute to liposome uptake. Importantly, we also demonstrated that this liposomal LPS-dsRNA cocktail elicits a specific pro-inflammatory and anti-viral response in both zebrafish hepatocytes and trout macrophages. The design of a unique delivery system with the ability to stimulate two potent innate immunity pathways virtually present in all fish species represents a completely new approach in fish health.Development of novel systems of vaccine delivery is a growing demand of the aquaculture industry. Nano- and micro- encapsulation systems are promising tools to achieve efficient vaccines against orphan vaccine fish diseases. In this context, the use of liposomal based-nanocarriers has been poorly explored in fish; although liposomal nanocarriers have successfully been used in other species. Here, we report a new ∼125 nm-in-diameter unilamellar liposome-encapsulated immunostimulant cocktail containing crude lipopolysaccharide (LPS) from  The development of sustainable aquaculture, a strategic sector to feed the ever-increasing human population Prior to this study, some advances have been made on the encapsulation of vaccines for fish vaccination and immunostimulation. Some of these studies have suggested that microencapsulated vaccines significantly enhance the protection and immune response in various fish species 600 (Chol-PEG), lipopolysaccharides (LPS) from E. coli 0111:B4, TriReagent, insulin, EGF, chloroquine and all endocytosis inhibitors were purchased from Sigma-Aldrich. MarinaBlue-DHPE, fluorescein-DHPE, LPS-AlexaFluor594, antibiotic/antimycotic solution, TrypLE Express, Cell Mask Deep Red, Hoechst 33342 and Superscript III reverse transcriptase were purchased from Invitrogen. Poly(I:C) High Molecular Weight, poly (I:C)-Fluorescein and Primocin were purchased from InvivoGen, whereas ZFL cells were purchased from ATCC. Oligo-dT15, GelGreen and SYBR Green I were purchased from Promega, Biotium and Bio-Rad, respectively.1,2-didodecanoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dioleoyl-sn-glycero-3-phosphoric acid monosodium salt (DOPA), Cholesterol (Chol), 3β-N-(di-methyl-amino-ethyl)carbamate hydrochloride (Cholesteryl), Cholesterol-PEGOnchorynchus mykiss) and zebrafish (Danio rerio) were submitted and authorized by the Ethics Committee of the Autonomous University of Barcelona (CEEH number 1582) who agree with the International Guiding Principles for Research Involving Animals (EU 2010/63).All experimental procedures involving rainbow trout (600 were dissolved in chloroform solutions (100 mg/ml) and mixed at the desired molar ratios  of the final liposomal formulations were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS . The morphology was examined using Cryo-Transmission electron microscopy (Cryo-TEM) in a JEOL-JEM 1400 microscope . Liposome stability was followed (48 h at 28°C) by turbidity measurement in a Turbiscan Lab Expert .Liposomal formulations were prepared by the thin film hydratation method r ratios Table 1.IS,total-CIS,out)/CIS,total] x100, where CIS,total is the initial immunostimulant concentration and CIS,out is the concentration of non-encapsulated immunostimulant. To measure the CIS,out, all liposome suspensions were centrifuged at 110000 xg for 30 min at 10°C. Supernatant aliquots were taken to quantify the concentration of non-encapsulated poly (I:C) and LPS by UV-Vis spectroscopy using a Nanodrop ND-1000 . Poly (I:C) was linearly detected in a range from 2.5 µg/ml to 1 mg/ml , whereas LPS was linearly detected in a range from 4.0 µg/ml to 1 mg/ml . Liposomes that did not contain any encapsulated immunostimulant were also ultracentrifuged and their supernatant quantified (Abs at 220 nm) to verify that liposomes were properly precipitated. To calculate the EE of the liposomal IS-cocktail, the putative non-encapsulated immunostimulants in the supernatant were separated by aqueous Gel Permeation Chromatography  and quantified by UV-Vis spectroscopy, where poly (I:C) and LPS were linearly detected. All experiments were done in triplicate.Encapsulation efficiencies (EE) were calculated according to the equation EE(%) = [. Fluorescent LPS-AlexaFluor594 and poly (I:C)-Fluorescein were individually or simultaneously encapsulated in liposomes and the resulting liposomal formulations were examined using a Leica TCS SP5 confocal microscope .2 in Dulbecco's modified Eagle's medium (DMEM) 4.5 g/l glucose, supplemented with 0.01 mg/ml insulin, 50 ng/ml EGF, 5% (v/v) of antibiotic/antimycotic solution, 10% (v/v) heat-inactivated FBS and 0.5% (v/v) heat-inactivated trout serum (TS). HepG2 cells were grown at 37°C, 5% CO2 in DMEM 4.5 g/l glucose, supplemented with 5% (v/v) of antibiotic/antimycotic solution and 10% (v/v) heat-inactivated FBS. Adherent trout monocyte/macrophages were isolated as previously described Zebrafish ZFL cells  were cultured at 28°C, 5% CO5 cells/well. The medium was removed and fresh non-supplemented medium containing the liposome formulation at indicated concentration was added, incubating the cells for 24 h. Lactate dehydrogenase  activity in the medium and MTT assay on the cells were performed. Cell viability was expressed as a percentage of the control. All the measurements were done in triplicate in 3 independent experiments. Dose-response curves were fitted using a sigmoidal dose-response curve model provided in the GraphPad Prism 5.0 . EC50 value was derived from these fitted curves for single experiments. Differences among data were analyzed using One-way ANOVA followed by Tukey's post test p<0.001.Two different cell viability assays (MTT and LDH) were simultaneously performed using three cell lines . Cells were seeded at 2.5×10xg for 5 min. Pellets were resuspended in ice-cold PBS for FACS analysis using a BD FACSCanto cytometer . Experiments were performed in triplicate . The internalization of fluorescence was calculated as the mean fluorescence intensity (MFI). To compare membrane-bound versus endocyted liposomes, the medium was removed at different times , and the cells were washed either with ice-cold PBS (pH = 7.4) or with an ice-cold PBS-acetic acid (pH = 4.2) to remove the liposomes attached to the membrane. The remaining  fluorescence of the cells was then analyzed using the PBS washed cells as a total uptake. The uptake of liposomes at long incubation times was also studied. When needed, cells were pretreated for 1 h with 100 µM chloroquine. Then, fluorescent liposomes were added and left to incubate 15 min for the ZFL cells and 30 min for the trout macrophages. After 3× PBS washes, liposome-free medium was added and cells were incubated for 1, 6 or 16 h in the presence of chloroquine, when required. Finally, cells were routinely treated for flow cytometry analysis. To determine the liposome endocytosis pathways, the following inhibitors were used: methyl-β-cyclodextrin , 5-(N-Ethyl-N-isopropyl)amiloride , sucrose  and wortmannin . The inhibitor's toxicity was assessed  and working concentrations were selected. Cells were pretreated for 1 h with each inhibitor, and liposomes were added for 15 min (ZFL cells) or 30 min (trout macrophages). Finally, 1 h after adding the liposomes, cells were analysed by flow cytometry.To visualize liposome endocytosis, DHPE-fluorescein was incorporated at a 0.05 molar ratio into the liposomal IS-cocktail. Labeled liposomal IS-cocktail was added to either ZFL or trout macrophages to a final concentration of 750 µg/ml liposomal IS-cocktail (containing 25 µg/ml poly (I:C) and 12.5 µg/ml LPS) and incubated for selected times. After treatment, cells were cooled down, washed 3× with ice-cold PBS, trypsinized and centrifuged at 200 Cells were seeded one day before the endocytosis experiments. For short incubation times (from 30 min to 1.5 h), liposomal IS-cocktail was added at 750 µg/ml liposomal IS-cocktail (containing 25 µg/ml poly (I:C) and 12.5 µg/ml LPS). For the 16 h incubation time, liposomal IS-cocktail was added at 375 µg/ml liposomal IS-cocktail (containing 12.5 µg/ml poly (I:C) and 6.25 µg/ml LPS). After 3× PBS washes, cells were stained with CellMask and Hoechst and viewed under a Leica TCS SP5 confocal microscope . Image analysis was performed using Imaris software and z-stacks were analyzed to visualize the particle contact sites and location.Table S1 in File S1) and qPCR was carried out using SYBR Green I mix, 500 nM of primers and 5 µl of cDNA. Samples from 3 independent experiments were run in triplicate, and quantification was done according to Livak method Cells were stimulated for 16 h with 750 µg/ml of liposomal IS-cocktail containing 25 µg/ml poly (I:C) and 12.5 µg/ml LPS and 375 µg/ml of liposomal IS-cocktail containing 12.5 µg/ml poly (I:C) and 6.25 µg/ml LPS. Non-loaded liposomes and non-encapsulated IS were used as controls. Total RNA from the ZFL and trout macrophages cell cultures was extracted using TriReagent following manufacturer's instructions. The RNA quality and concentration was assessed and cDNA was synthesized with 1.0 µg and 0.5 µg of total RNA for ZFL cells and macrophages, respectively, using SuperScript III reverse transcriptase and oligo-dT15 primer. PCR was carried out with 1 µl of cDNA as a template with specific primers  and 6.25 µg/ml LPS). Non-loaded liposomes and free LPS were used as controls. Supernatants were collected, centrifuged and precipitated with 25% trichloroacetic acid. TNFα secretion was assessed by Western blot as previously described Danio rerio) were held in tanks with recirculating water under a photoperiod of 14 h light/10 h dark at 28°C. Embryos were obtained from random pair-wise mating collected, rinsed and kept in E3 medium at 28°C. Viable embryos and post-hatching larvae were plated in 96-well plates. Liposomal IS-cocktail (liposome concentrations from 0.75 to 6 mg/ml) were added to the wells (200 µl), and incubated for 120 h. The plate evaporation rate was minimized as previously described Adult AB zebrafish  in the different NL1,n to NL5,n formulations were studied, showing that a positively charged liposome surface, like in NL1,n (+23.47±0.40 mV) and NL2,n (+10.43±1.77 mV), favors the encapsulation of LPS and poly (I:C). In contrast, the encapsulation efficiency of both LPS and poly (I:C) in liposomes decreased as the surface charge became more negative like in NL5,n (−30.87±2.53 mV), as previously described by Balazs et al. and Nakhla et al.2,LPS and NL2,poly (I:C), respectively. The maximum loading efficiencies for LPS were 49.6±5.9% and 66.0±0.1% for NL2,LPS and NL1,LPS, respectively. Interestingly, loading efficiencies achieved for poly (I:C) were always higher, with values of 95.0±1.4% and 91.2±0.1% for NL2,poly (I:C) and NL1,poly (I:C), respectively . Both types of liposomes were in vitro assayed on ZFL and HepG2 cell lines using MTT and LDH assays. Interestingly, NL1,n and NL2,n liposomes showed similar cytotoxicity activity in HepG2 cells . However, the more cationic liposomes  clearly showed higher toxicity on ZFL cells (EC50 = 0.166 mg/ml) than the less cationic one . Because of their similar loading efficiencies but different cytotoxicity, the less toxic NL2,n formulation (DLPC 50%-Cholesteryl 10%-Chol 35%-Chol-PEG 5%) was finally chosen as the ideal liposomal composition to co-encapsulate LPS and poly (I:C) (c formulation) was composed of 125.8±6.6 nm-in-diameter liposomes that entrapped both LPS and poly (I:C) with loading efficiencies of 22.3±2.1% and 99.6±0.1%, respectively. Therefore, the NLc formulation was composed of a mixture of 15 mg/ml of liposomes containing 250 µg/ml and 500 µg/ml of LPS and poly I:C, respectively. Importantly, after co-encapsulating LPS and poly (I:C), such liposomes exhibited a slightly positive surface charge (1.37±3.58 mV), which was attributed to electrostatic interactions between their positively charged lipidic bilayer and the negatively charged immunostimulants. The occurrence of these attractive interactions was corroborated by co-encapsulating AlexaFluor594-labeled LPS and fluorescein-labeled poly (I:C) into cationic liposomes, from which the localization of both immunostimulants in the lipidic bilayer was observed . Three lipid mixtures were studied, NLChol-PEG Table 1.in vitro cytotoxic studies . The therapeutic immunostimulant doses were chosen according to our previous results on LPS and poly (I:C) responses in different fish species 2,n, NL2,LPS, NL2,poly (I:C) and NLc in ZFL cells at the indicated concentrations  that further improve its biocompatibility. The same results were obtained by using the LDH assay . Finally, toxicity studies were also carried out using trout primary cell cultured APCs . In this cells, we observed low toxicity levels of NLc formulations , but did not observe a LPS/poly (I:C) mediated toxicity at the indicated doses.To fully characterize the safety of our formulations, we carried out trations Figure 2c were probably metabolized by the endosomal/lysosomal system. Different studies have shown the ability of cationic liposomes to deliver different compounds to endosomal compartments c endocytosis in the presence of chloroquine (CQ), an inhibitor of lysosomal acidification, and we observed a significant increase of fluorescence in the presence of CQ . This observation confirmed the occurrence of NLc in the endosomal/lysosomal compartment (55.53±0.83% CQ-dependant endocytosis inhibition at 16 h). To discriminate between membrane-bounded and endocytosed NLc, we measured the total versus endocytosed fluorescence at different times, observing that around 80% of total fluorescence signal corresponded to endocytosed liposomes  known to block a particular endocytosis pathway  and efficient liposome uptake . Note that the intracellular liposomes, as in ZFL cells, were present primarily in the cytosol as agglomerates (1.09±0.37 µm), with no fluorescence in the nuclei  and GIG2  had higher expression levels in NLc formulation than in non-loaded liposomes . The chemokine CCL4, a chemotactic cytokine that is induced in fish after viral infection c treatment  were still able to induce low levels of gene expression  as compared to non-loaded liposomes  and to the free LPS/poly (I:C) mixture . The pro-inflammatory cytokines IL-6 and TNFα showed a slightly different pattern, achieving good stimulation levels after NLc treatment with respect to non-loaded liposomes, but similar or lower levels when compared to the free-LPS/poly (I:C) mixture . A NLc concentration range from an extremely high dose (NLc Dose 4 = 6 mg/ml) to a putative therapeutic dose (NLc Dose 1 = 0.75 mg/ml) was chosen. We did not observe significant differences in survival curves obtained with pre-hatched larvae incubated with NLc formulation at different doses (c Dose 4) caused a significant increase in mortality . In contrast, high LPS toxicity with free-LPS treatment both in pre- and post-hatching larvae was observed . A moderate poly (I:C) toxicity in pre-hatching larvae  versus control  was also recorded. Therefore, and in accordance with our previous in vitro toxicity studies . Importantly, the embryos incubated with NLc formulations were able to hatch and develop normally until 120 h with no morphological defects. The survival curves in post-hatching larvae incubated with these liposomal formulations were substantially different  showed less toxicity than that of the corresponding liposomal IS-cocktail , 25 µg/ml LPS). In addition, a dose-dependent toxicity for the NLc formulation after 48 h incubation was observed  and anti-viral responses (TLR3 and GIG2) , indicating a stimulation of the zebrafish larvae immune system. Finally, DLS measurements done using NLc and NL2,n formulations after 5 days incubation in E3 medium indicated a good stability after the in vivo challenge. We also characterized the NLc stability in in vivo experimental conditions by Turbiscan, and we found that the NLc stability index was not significantly changed after incubation in aquarium water or in E3 medium at 28°C for 2 days . These data further confirm that this liposomal IS-cocktail might be used for future in vivo immunization in aquatic species.We conducted different dose-response survival experiments with the NL studies Figure 2Listonella anguillarum causing vibriosis) have achieved good protection levels against different fish infections in vitro and in vivo in fish. As far as we know, this study is the first attempt to co-encapsulate two model immunostimulants specifically designed to target fish APCs in nanosized liposomes. To date, the unique attempt to vaccinate fish using liposomes was done by Irie et al., who explored the use of microsized liposomes containing A. salmonicida total extracts in carp et al. have also shown that a combination of poly(lactic-co-glycolic acid) microparticles loaded with β-glucan and human γ-globulins were able to target head kidney macrophages inducing an adaptive in vivo immune response in salmon in vitro toxicity of cationic liposomes has been reported by several groups  and in vitro and in vivo has been well documented in different vertebrates Vaccination and preventive immunostimulation has become the principal prophylactic tool for disease control in aquaculture. Some conventional vaccines made with inactivated bacteria  and zebrafish (Danio rerio) specific primers for PCR and Q-PCR. Figure S1. Evaluation of toxicity of cationic liposomes without encapsulated immunostimulants . Viability of ZFL cell line was assessed with the MTT assay (A) or LDH assay (B) after a dose response (0.1 µg/ml-10 mg/ml) with the two liposomal formulations . Viability of HepG2 cell line was determined with the MTT assay (C) and with the LDH assay (D) after a dose response (0.1 µg/ml-10 mg/ml) with the two liposomal formulations . Non-treated cells were used as 100% viability control (dotted line) in the MTT assays and non-treated cells were used as control of the basal death (dotted line) in the LDH assays. Data represent means ± SD of three independent experiments. Differences were analyzed using One-way ANOVA followed by Tukey's post test. *, p<0.05; **, p<0.01; ***, p<0.001. Figure S2. Cytotoxicity of NLc formulation in ZFL cells by LDH assay. (A) Viability of ZFL after 24 h incubation with the liposome-encapsulated LPS  at Dose 1 = 1 mg/ml liposome with 50 µg/ml LPS, Dose 2 = 0.5 mg/ml liposome with 25 µg/ml LPS and Dose 3 = 0.20 mg/ml liposome with 10 µg/ml LPS. The white bar is the control treatment with liposomes without encapsulated immunostimulants  and the blue bar is the non-encapsulated LPS control (50 µg/ml). (B) Viability of ZFL after 24 h incubation with the liposome-encapsulated poly (I:C) , green bars) at Dose 1 = 1.5 mg/ml liposome with 50 µg/ml poly (I:C), Dose 2 = 0.75 mg/ml liposome with 25 µg/ml poly (I:C) and Dose 3 = 0.375 mg/ml liposome with 10 µg/ml poly (I:C). The white bar is the control treatment with empty liposomes  and the red bar is the non-encapsulated poly (I:C) control (50 µg/ml). (C) Viability of ZFL cells after 24 h with liposomal LPS-poly (I:C) cocktail  at Dose 1 = 1.5 mg/ml liposome with 50 µg/ml poly (I:C) and 25 µg/ml LPS, Dose 2 = 0.75 mg/ml liposome with 25 µg/ml poly (I:C) and 12.5 µg/ml LPS and Dose 3 = 0.375 mg/ml liposome with 12.5 µg/ml poly (I:C) and 6.25 µg/ml LPS. The white bar is the control treatment with empty liposomes , the blue bar is the non-encapsulated LPS (25 µg/ml) and the red bar represents the non-encapsulated poly (I:C) control (50 µg/ml). Non-treated cells were used as 100% viability control (dotted line). Data represent means ± SD of three independent experiments. Differences were analyzed using One-way ANOVA followed by Tukey's post test. *, p<0.05; ***, p<0.001. Figure S3. In vitro cytotoxicity of NLc formulation in trout macrophages. (A) The cytotoxicity of NLc was assessed by the LDH assay. Viability of the trout macrophage primary cell culture after 24 h incubation with NLc encapsulating both poly (I:C) and LPS (green bars) at Dose 1 = 0.75 mg/ml liposome with 25 µg/ml poly (I:C) and 12.5 µg/ml LPS and Dose 2 = 0.375 mg/ml liposome with 12.5 µg/ml poly (I:C) and 6.25 µg/ml LPS. The white bar is the control treatment with non-encapsulating liposomes  and the grey bar is the non-encapsulated poly (I:C) and LPS control . Basal dead cells of the non-treated cells were used as control (dotted line). Data represent means ± SD of 3 independent experiments. Differences were analyzed using One-way ANOVA followed by Tukey's post test **, p<0.01. Figure S4. In vitro cytotoxicity of endocytosis inhibitors. (A) Viability of ZFL cells after 1 h exposure (16 h in the case of the chloroquine) to different endocytosis inhibitors, assessed by the MTT assay. (B) Viability of trout macrophages after 1 h exposure to different endocytosis inhibitors, assessed by the MTT assay. Non-treated cells were used as a 100% viability control (Control bar). Figure S5. Time-course of NLc uptake in vitro. (A) Flow cytometry time course of NLc uptake  and 12.5 µg/ml LPS) by ZFL cells. To study the metabolization of NLc, ZFL cells were also pretreated for 1 h with chloroquine at 100 µM (red bars). Then, liposomes were added (750 µg/ml liposome containing 25 µg/ml poly (I:C) and 12.5 µg/ml LPS), and left to incubate in the constant presence of chloroquine. (B) Flow cytometry time course of NLc uptake  and 12.5 µg/ml LPS) by trout macrophages. Cells not exposed to NLc were used as controls (white bars). Data represent means ± SD of triplicates of three independent experiments. Figure S6. In vivo NLc toxicity assay controls. Survival of zebrafish embryos was recorded every 24 h at 120 h post fertilization (hpf) (A) and 72 h post hatching (hph) (B) after exposure to non-encapsulated LPS , non-encapsulated poly (I:C)  and non-encapsulated LPS (25 µg/ml) and poly (I:C) (50 µg/ml) in combination (orange). Non-treated embryos (blue) were used as controls. Survival curves were analyzed using the log rank test . Figure S7. Analysis of gene expression in zebrafish larvae after time-course exposure to liposome preparation. NL2,n = liposomes without encapsulated immunostimulants (1.5 mg/ml), NLc = liposomes (1.5 mg/ml) with 50 µg/ml poly (I:C) and 25 µg/ml LPS and LPS+poly (I:C) = stimulation control (50 µg/ml poly (I:C), 25 µg/ml LPS). Non-treated embryos were used as control (Ctrl). Elongation factor (EF1) was the reference gene and TLR3, GIG2, TNFα and iNOS mRNA abundance was analyzed by conventional PCR (right panel). Representative images of three independent experiments are shown.(DOCX)Click here for additional data file.


Purpose. The present study investigated the potential effects of long-term T3 treatment on glioma tumor cell lines. Thyroid hormone action on cell growth, differentiation and survival during development may be of therapeutic relevance Methods and Results 1321N1 cell line, an astrocytoma grade II, and U87MG, a glioblastoma grade IV, were exposed for 2 and 4 days in medium deprived of T3 and in medium containing 1 nM T3. T3 promoted re-differentiation in both cell lines. However, T3 increased cell proliferation in 1321N1 (2 days) which declined thereafter (4 days) while in U87MG resulted in suppression of cell proliferation. At the molecular level, a 2.9 fold increase in the expression of TRα1 receptor was observed in U87MG versus 1321N1, P < 0.05. TRβ1 receptor was undetectable. These changes corresponded to a distinct pattern of T3-induced kinase signaling activation; T3 had no effect on ERK activation in both cell lines but significantly increased phospho-Akt levels in 1321N1. Conclusion. In conclusion, T3 can re-differentiate glioma tumor cells, whereas its effect on cell proliferation appears to be dependent on the type of tumor cell line with aggressive tumors being more sensitive to T3. TRα1 receptor may, at least in part, be implicated in this response. It is now recognized that thyroid hormone (TH) may have a critical role in the pathogenesis and the progression of the diseases due to its regulatory action on cell differentiation, proliferation, and survival . ExperimGliomas represent the most common primary brain tumor and are among the most aggressive of cancers. Patients with glioma typically relapse within a year of initial diagnosis . Althoug1321N1 cell line, an astrocytoma grade II, and U87MG, a glioblastoma grade IV, were used in this study. Glioma cell line U87MG was obtained from the American Type Culture Collection (ATCC) , and glioma cell line 1321N1 was obtained from the European Collection of Cell Culture (ECACC) . All cell lines were maintained in 150 cm² cell culture flasks (CORNING). U87MG was maintained in Eagle's Essential Minimum Medium (MEM) with Earle's salts supplemented with 10% fetal bovine serum (GIBCO), 1 mM sodium pyruvate, streptomycin and penicillin (5% v/v), 0.1 mM nonessential amino acids, 2 mM L-glutamine, and amphotericin B (5% v/v).1321N1 was maintained in Dulbecco's Modified Eagle's Medium (DMEM) with glucose and sodium bicarbonate supplemented with 10% FBS, 2 mM L-glutamine, 5% penicillin and streptomycin, and 5% amphotericin B.2. For all experiments, each of the glioma cell lines was used between passages 20–30. Once cells were 70–80% confluent, they were trypsinized using 1X Trypsin. Cells were settled for 24 h in stripped medium  before the initiation of treatment. Cells were cultured for 48 h and 96 h either in stripped medium only (nontreated) or in stripped medium in which 1 nM of T3 was added.All cell lines were maintained in a 37°C humidified incubator with 5% COCell morphology was used to assess cell differentiation. Cells were fixed in, before being viewed with an inverted light microscope fitted with phase contrast optics. Five random fields, each containing no more than 50 cells, were examined in each well, and the total number of cells as well as the total number of extensions that were greater than two cell body diameters in length were recorded. Data were derived from approximately 100 cells in each group. Cell morphology could not be reliably assessed at 96 h due to more than 80% confluency in non-treated cells.In order to measure cell proliferation, BrdU labeling reagent  was added to the medium. Cells were incubated for 30 min and then fixed using 4% paraformaldehyde for 15 min. Primary antibody  was applied for 1 h at room temperature. Samples were washed 3 × 5 min with PBS. Secondary antibody (peroxidase anti-mouse IgG2a) was then applied for 30 min at room temperature, followed by washing 3 × 5 min. Finally, BrdU-immunostained cultures were visualized using DAB and photographs taken with a digital camera (Zeiss Axiovert) attached to an inverted microscope fitted with phase contrast optics. BrdU-positive nuclei were counted as a percentage of total nuclei. Proliferation data are derived from between 450 to 600 cells measured in each group.μL trypsin 0.25% were added to each plate and incubated for 37°C until the cells were detached. A solution of 10% FBS in PBS was added to each plate to inhibit the trypsin action. Then, cells were harvested, and the cell number was determined after several counts of a certain volume in Neubauer hematocytometer.At 2 and 4 days after treatment, cells were washed twice with PBS, and 100 μg/mL) which stained the nuclei of all cells. Administration of doxorubicin is known to induce apoptosis and was used as a positive control in order to certify the selected method.Apoptotic cell nuclei were assessed by Tunnel staining using the In Situ Cell Death Detection Kit, according to standard protocol based on manufacturer's instructions . Cell cultures were counterstained with Hoeschst 33358 (5 Cellular injury was assessed by LDH enzyme release in cultured medium. Culture medium was collected at the end of the experiment for the measurement of lactate dehydrogenase (LDH) activity (IU/L) using an ELISA kit . Measurements were performed with Tecan Genios system. LDH release was expressed in each group as percentage of the non-treated group.μL lysis buffer containing 20 mM HEPES, pH 7.9, 10 mM KCl, 1 mM EDTA, 10% glycerol, 0.2% NP-40, 0.5 mM DTT, 0.5 mM PMSF, 5 μg/mL aprotinin, and 5 μg/mL leupeptin. A small quantity of total lysate was kept and the remainder centrifuged at 12000 g for 1 min at 4°C. The nuclear fraction was prepared by resuspension of the pellet in buffer containing 20 mM HEPES, pH 7.9, 0.42 M NaCl, 0.2 mM EDTA, 1.5 mM MgCl2, 25% glycerol, 0.5 mM DTT, 0.5 mM PMSF, 5 μg/mL aprotinin, and 5 μg/mL leupeptin and incubated with agitation for 1 hour at 4°C before centrifugation for 10 min at 12,000 g. The resulting supernatants were collected and used for protein analysis of the nuclear fraction. Protein concentrations were determined by the BCA assay method. After boiling for 5 min , a quantity of 15 μg protein from nuclear or total fraction was separated on 7.5% SDS-PAGE using a Bio-Rad Mini-Protean gel apparatus. For Western blotting, proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond ECL) at 100 V and 4°C, for 1.5 h using Towbin buffer. After Western blotting, filters were probed with specific antibodies against TRα1  and TRβ1 . Filters were incubated with appropriate anti-mouse (Amersham) or anti-rabbit  HRP secondary antibodies. Immunoreactivity was detected by enhanced chemiluminescence using Lumiglo reagents (New England Biolabs). Chemiluminescence was detected by the image analysis system FluorChem HD2  equipped with a CCD camera and analysis software. Five samples from each group were loaded on the same gel. Ponceau staining was used in order to normalize slight variations in protein loading.After washing twice with PBS, the cells were scraped into 400 n = 5 samples for each group.Filters were probed with specific antibodies. Membranes with total protein extracts were blocked with 5% nonfat milk in TBS-Tween for 60 min and then probed with specific antibodies against total and phospho-ERK  and total and phospho-Akt , overnight at 4°C. Filters were incubated with appropriate anti-rabbit HRP secondary antibody , and immunoreactivity was detected by enhanced chemiluminescence using Lumiglo reagents (New England Biolabs). Immunoblots were quantified using the FluorChem HD2 system . Data were obtained from t-test or nonparametric Mann-Whitney test was performed, as appropriate. A two-tailed test with a P value less than 0.05 was considered significant.Values are presented as mean (S.E.M.). Data were analyzed with single factor analysis of variance ANOVA across groups. An unpaired independent sample P < 0.05. In U87-MG cells, the ratio of total number of projections to total number of cells was 1.16 (0.14) for nontreated versus 1.83 (0.19) in T3 treated, P < 0.05.  for non-treated versus 1.9 (0.11) in T3 treated,  < 0.05. .P < 0.05. At 4 days, cell proliferation was shown to be 45.2% (5) in non-treated versus 40% (6) in T3 treated, P > 0.05  in non-treated versus 30.5% (3) in T3 treated, P > 0.05 . P < 0.05. In addition, after 4 days, cell proliferation was shown to be 36.5% (6) in non-treated versus 16.3% (4) in T3 treated, P < 0.05.  in nontreated versus 23.6% (4) in T3 treated,  < 0.05. .No change in LDH release was observed either in 1321N1 or U87MG cell cultures . ApoptosP < 0.05. At 4 days, total cell number was 381105 (4100) in non-treated versus 372433 (2595) in T3 treated, P > 0.05  in non-treated versus 232366 (2390) in T3 treated, P > 0.05 .P < 0.05. In addition, after 4 days, total cell number was 396866 (5791) in non-treated versus 331133 (11652) in T3 treated, P < 0.05  in non treated versus 186166 (3122) in T3 treated, P < 0.05 .α1 receptor was observed in U87MG cells as compared to 1321N1, P < 0.05. TRβ1 receptor was undetectable in both cell lines (A 2.9-fold increase in the expression of TRll lines .P > 0.05) as compared to non-treated cells. Furthermore, the ratio of phospho-Akt to total Akt was found to be 1.4 higher in T3 treated cells as compared to non-treated cells, P < 0.05. At 4 days, no differences in the ratio of p44 and p42 phospho-ERK to total ERK and phospho-Akt to total Akt were observed between the two groups  instead of being inactive exerts repressive or inducible effect on the transcription of T3 inducible or repressive genes by recruiting corepressor complexes with histone deacetylase [α1 to be overexpressed at early embryonic stages when TH is low, resulting in cell proliferation and declines thereafter with the rise of TH resulting in cell differentiation [α1 expression reemerges under pathological conditions and may lead to pathological hypertrophy [α1 in glioma cell aggressiveness and response to TH. However, this issue merits further investigation.Our study further showed that the two cell lines responded differently to TH treatment as regards cell proliferation with the more aggressive tumor cells to be more sensitive. Thus, in 1321N1, T3 treatment resulted in increased cell proliferation at two days which declined thereafter, while T3 had no effect on cell injury. In contrast, in the U87MG cell line, T3 markedly suppressed cell proliferation without increasing cell injury. The potential underlying mechanisms of this cell-type-dependent action of TH on cell proliferation are not fully understood. Long-term TH effects are mediated via thyroid hormone receptors (TRs). TRs are transcription factors which regulate important genes related to cell differentiation, proliferation, and survival . It is ncetylase . This isertrophy , 21 or pertrophy . The addertrophy  and suppertrophy . Taken tα1 had an impact on the activation of growth kinase (ERK and Akt) signaling activation induced by T3 treatment. These cascades are important regulators of cancer growth and cancer cell survival [Our study further explored whether this differential expression of TRsurvival –25. Bothsurvival –29. Intesurvival .α1 receptor may, at least in part, be implicated in this response.In conclusion, T3 can redifferentiate glioma tumor cells. However, the T3 effect on cell proliferation appears to be dependent on the type of tumor cell line with aggressive tumors to be more sensitive to thyroid hormone treatment. TR


Cephalotaxus oliveri is a scarce medicinal conifer endemic to the south central region of China and Vietnam. A small fragmented population presently exists due to anthropogenic disturbance. C. oliveri has been used for its alkaloids harringtonine and homoharringtonine, which are effective against leucocythemia and lymphadenosarcoma. Monoecious plants have been detected in nature, although they were understood to be dioecious. In order to study the mating system, population genetics and the genetic effects of habitat fragmentation on C. oliveri, 15 polymorphic and 12 monomorphic microsatellite loci were developed for C. oliveri by using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol. The polymorphisms were assessed in 96 individuals from three natural populations . The number of alleles per locus ranged from two to 33, the observed and expected heterozygosity per locus ranged from 0.000 to 1.000 and from 0.000 to 0.923, respectively. These loci would facilitate a comprehensive understanding of the genetic dynamics on C. oliveri, which will be useful for establishing effective conservation strategies for this species. Cephalotaxus oliveri Masters  is a scarce medicinal conifer endemic to the south central region of China and Vietnam  at 48 °C for 2 h, 55 °C for 3 h, and 52 °C for 2 h, respectively. DNA fragments containing targeted repeats were selectively captured by magnetic streptavidin-coated particles . Recovered microsatellite DNA fragments were amplified with MseI-N by 30 cycles according to the conditions described above. About 20 ng PCR product with a range of 200 to 800 bp purified via the E.Z.N.A. Gel Extraction kit  were directly ligated into a pEASY-T1 Cloning vector , and then transformed into Escherichia coli DH5a competent cells . Positive clones were selected using blue/white screening after growing on IPTG-XGal media, and further tested by PCR with M13 primers (5′-GTAAAACGACGGCCAGT-3′/5′-CAGGAAACAGCTATGAC-3′) and the corresponding primer-mix (AG)15 and (AC)15, or (CT)15, and or (AAG)10, respectively, for confirming positive clones containing microsatellites. PCR amplification was performed in a 15 μL reaction mixture containing 2 μL of bacterial suspension, 2.0 mM MgCl2, 0.2 μM each primer, 0.2 mM dNTP, 1 U Taq DNA polymerase . PCR amplification was as follows: 95 °C for 3 min, 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, followed by 72 °C for 10 min. Detected microsatellite clones were sequenced by an ABI3730xl Genetic Analyzer . Microsatellite sequences with sufficient flanking regions were used to design primers with Primer 5 [L.X.D. 09-103; Pingbian: L.X.D. 09-275; Luxi: L.X.D. 10-366) deposited in Yunnan University Herbarium Laboratory of Pteridophyta (PYU), Kunming, Yunnan, China. We labeled 6-FAM at the 5′ end of each forward primer for the loci showed clear and reliable bands.Three microsatellite-enriched libraries were constructed following the FIASCO (Fast Isolation by AFLP of Sequences Containing repeats) protocol . Genomic) method . About 52, 0.5 mM each primer, 0.25 mM dNTP, 1.5 U Taq DNA polymerase . PCR amplification was as follows: 95 °C for 3 min; 35 cycles of 94 °C for 30 s, annealing at optimal temperature 49 °C–66 °C for 30 s  per population were estimated using FSTAT 2.9.3 (A program to estimate and test gene diversities and fixation indices) [Ho) and expected (He) heterozygosities were calculated by ARLEQUIN 3.01[The linkage disequilibrium between loci and number of alleles per locus (indices) . The obsQUIN 3.01. The sigQUIN 3.01. Lastly,QUIN 3.01.C. oliveri in this study. These markers will facilitate the further studies on the mating system, population genetics, and genetic effects of habitat fragmentation of the species. Such information will be useful for developing sound and effective conservation strategies for this endangered species. Furthermore, these developed SSR markers for C. oliveri would also be potentially useful for exploring population genetics in the Cephalotaxus species.In summary, 15 polymorphic microsatellite markers have been specifically developed for the


E. coli lipopolysaccharide (LPS) as a standard TNF-α release agent. Cells were exposed either to 0, 2.5 or 12.5 µmol/L OTA, or to 0.1 µg/mL LPS, for up to 24 h. OTA at 2.5 µmol/L and LPS at 0.1 µg/mL were not toxic to the tested cells as indicated by viability markers. TNF-α was detected in the incubated cell medium of rat Kupffer cells, peritoneal rat macrophages, and the mouse monocyte macrophage cell line J774A.1: TNF-α concentrations were 1,000 pg/mL, 1,560 pg/mL, and 650 pg/mL, respectively, for 2.5 µmol/L OTA exposure and 3,000 pg/mL, 2,600 pg/mL, and 2,115 pg/mL, respectively, for LPS exposure. Rat liver sinusoidal endothelial cells, rat hepatocytes, human HepG2 cells, and mouse L929 cells lacked any cytokine response to OTA, but showed a significant release of TNF-α after LPS exposure, with the exception of HepG2 cells. In non-responsive cell lines, OTA lacked both any activation of NF-κB or the translocation of activated NF-κB to the cell nucleus, i.e., in mouse L929 cells. In J774A.1 cells, OTA mediated TNF-α release via the pRaf/MEK 1/2-NF-κB and p38-NF-κB pathways, whereas LPS used pRaf/MEK 1/2–NF-κB, but not p38-NF-κB pathways. In contrast, in L929 cells, LPS used other pathways to activate NF-κB. Our data indicate that only macrophages and macrophage derived cells respond to OTA and are considered as sources for TNF-α release upon OTA exposure.The release of tumor necrosis factor α (TNF-α) by ochratoxin A (OTA) was studied in various macrophage and non-macrophage cell lines and compared with  OTAochratoxin ATNFtumor necrosis factorLPSlipopolysaccharideNF-κBnuclear factor kappa BAspergillus and Penicillium species. OTA causes diverse toxicological responses: it is genotoxic, carcinogenic, nephrotoxic, hepatotoxic, embryotoxic, teratogenic, and immunotoxic. Previous risk assessments have studied its genotoxicity and OTA-mediated nephrotoxicity [Ochratoxin A (OTA) is a natural mycotoxin produced from toxicity ,2,3,4. Itoxicity . The immtoxicity . In additoxicity . Furthertoxicity . A marketoxicity . This ratoxicity . Its reltoxicity .E. coli lipopolysaccharides (LPS) has been observed [In blood-free perfused rat livers, a synergistic effect on OTA-mediated TNF-α release by co-addition of low doses of observed  Thus, thobserved ,12 ThereOur previous studies have shown that OTA releases TNF-α from blood-free perfused rat livers in a dose- and time-dependent fashion without effects on liver vitality ,9,13. Frin vitro by 15 µmol/L gadolinium chloride or when rats were pretreated in vivo with the Kupffer cell depleting clodronate liposomes [In perfused rat livers, several types of cells may serve as a source of TNF-α, e.g., hepatocytes, sinusoidal endothelial cells, and Kupffer cells. In a previous study, OTA failed to induce TNF-α release in the blood-free perfused isolated rat liver when Kupffer cells were blocked iposomes . Additioiposomes . In ordei.e., Kupffer cells and peritoneal macrophages, were exposed to the vehicle or a standard dose of 2.5 µmol/L OTA or 0.1µg/mL LPS, respectively, during 24 h in culture. Kupffer cells  may have caused the discrepancies of OTA effects on monocytes.Our observation of TNF-α release from macrophage cells by OTA contrasts with a report by Heller in vitro model for human liver parenchymal cells, were used. TNF-α basal levels in the culture medium treated with vehicle increased only from 30 pg/mL to 36 pg/mL after 24 h. Neither 2.5 µmol/L OTA nor 0.1 µg/mL LPS altered the TNF-α concentrations in HepG2 cells when compared with untreated controls at any time point indicated. Thus, isolated hepatocytes and HepG2 cells failed to show any TNF-α release under OTA.Hepatocytes represent the major cell mass of the liver. To examine the possibility of hepatocyte participation in OTA-mediated TNF-α release from blood-free perfused rat liver ,8, primai.e., from 49 pg/mL at time point zero to 230 ± 10 pg/mL after 24 h, indicating a lack of effect of OTA. In contrast, after adding 0.1 µg of LPS/mL into the incubation medium, a significant TNF-α release occurred as early as 4 h (P < 0.001) and reached 2000 ± 70 pg/mL after 24 h. Sinusoidal endothelial cells, together with Kupffer cells, line the liver sinusoids. The basal TNF-α release from separately prepared and cultured endothelial cells treated with vehicle  induced NF-κB activation in J774A.1 cells starting from even 5 min after cell exposure [In all positive cells responding to both stimuli, the effects occurred more rapidly under LPS than under OTA. This also indicates that different signal cascades are involved for each of these two stimuli. Although far from being understood, earlier experiments have indicated that OTA-induced TNF-α release from the liver is likely to occur via the NFκB transcription factor pathway ,9. For uexposure . The signaling of cells upon LPS stimulation is quite well understood. To date, pure LPS is known to induce TNF-α by Toll like receptor TLR4 . TLRs mai.e., extracellular signal-regulated kinase 1 and 2 (ERK1/2)), c-jun amino-terminal kinase (JNK), and extracellular-regulated protein kinase 38 (p38) in proximal tubular cells [OTA, on the other hand, activates mitogen-activated protein kinases , collagen type VII from a rat tail, heat fetal calf serum, trypan blue, modified HANK's Ca2+ free balanced salt solution and modified HANK's balanced salt solution with Ca2+ were purchased from Sigma-Aldrich Co. Steinheim, Germany. Collagenase type CIS 212 U/mg and RPMI 1640 medium were purchased from Biochrom AG, Berlin, Germany. HEPES was purchased from SERVA Electrophoresis GmbH, Heidelberg, Germany. Penicillin, streptomycin, trypsin/ EDTA, and Dulbeccos modified Eagles medium (DMEM) were purchased from GIBCOTM, Paisley, Scotland. DMSO and collagenase type NB 4 from Cl. histolyticum 0.161 PZU/mg was from SERVA Electrophoresis GmbH, Heidelberg, Germany. DNAase I was obtained from Boehringer-Mannheim GmbH, Mannheim, Germany, and Nycodenz® 5--2,4,6-tri-iodo-N,N'-bis  isophthalamide was from AXIS-SHIELD PoCAS, Oslo, Norway. Pronase E was from E. Merck, Darmstadt, Germany. The enzyme-linked immunosorbant assay (ELISA) kit, Cytoscreen®, was purchased from BioSource International, Camarillo, Canada, with antibodies selective for detection of rat TNF-α., LIVE/DEAD® Reduced Biohazard Viability/Cytotoxicity Kit No.1. (L-7013) was purchased from Invitrogen Molecular Probes, Karlsruhe, Germany. Cell line L929 cells , J774A.1 cells (mouse monocyte macrophage cell line), and HepG2 (human hepatoma cell line) were generously donated by Dr. Ayub Darji, Institute of Medical Microbiology, University of Giessen, Germany. Ochratoxin A was purchased from CSIR Food Science and Technology, Pretoria, South Africa; it was free of lipopolysaccharide contamination as tested using the Limulus Amebocyte Lysate Endosafead libitum with Altromin® standard diet and received water ad libitum. They were kept under 12 h light-dark cycles at 22 °C with ventilation. The health of the rats was routinely tested by sentinel animals, and the animals were found to be free of chronic infections and parasites.Primary cells from the liver  and peritoneal macrophages were isolated from male Wistar rats (200–280 g). The animals were fed 2+ free balanced salt solution at 7.5 mL/min for 5–10 min. Then livers were re-perfused with 80 mL RPMI 1.640 medium containing 34.4 mg/mL pronase E for 10 min at a flow rate of 10 mL/min. The medium was replaced by 100 mL RPMI 1640 medium containing 40 mg collagenase type CIS and 22.6 mg pronase E, and the livers were re-circulated for 30 min at 20 mL/ min with gassing by air containing 5% CO2. After that, the livers were dissected, and the tissues were digested for 15–30 min in 120 mL RPMI 1.640 medium containing 20 mg collagenase type CIS, 5.3 mg/mL pronase E, and 5 mg/mL DNase. During theincubation, the pH was carefully controlled to 7.4–7.6 by gassing with air containing 5% CO2 and by adding small amounts of 0.1 M NaOH. Subsequently, the cell suspension was filtered through sterile mesh 300 µm into three tubes and centrifuged . The pellets were re-suspended in 10 mL HANK's balanced salt solution with Ca2+, and then centrifuged . The cell pellets were re-suspended in up to 6 mL maximum of HANK's balanced salt solution with Ca2+; 14 mL of Nycodenz® was also added to this suspension, which was then divided into two test-tubes. One mL of fresh HANK's balanced salt solution with Ca2+ was dropped carefully on the tube’s wall to form a clear layer above the suspension surface. The tubes were subjected to a density centrifugation at 3,500 rpm for 20 min. Afterwards, the upper three of four formed layers from each test-tube were collected, and the volume was replenished to 10 mL with cold RPMI 1640 medium, mixed, and injected into the JE-6B elutriation system and rotor . The speed was gradually raised from 19 to 27 mL/min. The outlets were collected in tubes, which contained pure endothelial sinusoidal cells. The outlets starting from 32 to 51 mL/min contained pure Kupffer cells. The cells were collected separately and centrifuged at 1800 rpm for 10 min at 4 °C. Finally, the pellets were re-suspended in the RPMI 1640 medium containing 10% heat-inactivated fetal calf serum and penicillin (100 IU/mL) and streptomycin (100 µg/mL). The cells were counted with a Bürker-Türk counter, adjusted to 2 × 106 cells/mL, and seeded into tissue culture plates. Experiments were carried out under cell culture conditions.Kupffer cells and sinusoidal endothelial cells were isolated according to the collagenase/pronase digestion method previously described . Briefly2+ free Krebs-Henseleit solution containing 60 mg collagenase type NB 4 for 15 min in a temperature-controlled hood at 37 °C under 95% O2/5% CO2 gassing. After that, the livers were carefully dissected, and the tissue was digested for 5 min in the perfusate solutions by bubbling gas. The cell suspensions were filtered through double-layer sterile gauze into four test-tubes. The volume was adjusted by Ca2+-containing Krebs-Henseleit solution and centrifuged at 300–400 rpm (~50 grav) for 10 min. The pellets were re-suspended in Tyrode buffer containing 5.5 mM glucose, then washed three times, and filteredthrough four to six layers of gauze. The cells in suspension regenerated during 30 min in a shaker water bath adjusted to 37 °C and were gassed with 95% O2/5% CO2 gas. Finally, the cells were suspended in Dulbeccos Modified Eagles Medium (DMEM) containing 10% heat-inactivated fetal calf serum, penicillin (100 IU/mL), and streptomycin (100 µg/mL). The cells were counted under a microscope with a Bürker-Türk counter, adjusted to 2 × 106  cells/mL, and then seeded in collagen pre-coated tissue culture plates. Experiments were carried out under cell culture conditions.Hepatocytes were isolated by a collagenase perfusion method as previously described . Briefly2 atmosphere for 2 h to allow the macrophages to adhere on the flask bottom. The culture medium with floating cells was discarded. Adhered cells were collected with trypsin and re-suspended in the culture medium. Then 5 mL of 2 × 106 cells/mL were again seeded into culture plates and incubated at 37 °C with a humidified 5% CO2 atmosphere for 2 h to allow the purified macrophages to adhere on the flask bottom again. Experiments were carried out under cell culture conditions.Peritoneal macrophages were prepared as described previously  with som2 atmosphere. The cells were monitored during growth with replacement of the culture medium every 72 h until the plates became overcrowded with cells. Then, the cells were collected with trypsin, resuspended in culture medium, and transferred to several culture flasks. When these plates contained about 10 × 106 cells/plate, then 5 mL of new medium was added. Experiments were carried out under the cell culture conditions.Connective-tissue-derived mouse cell line (L929), human hepatoma cell line (HepG2), and mouse monocyte macrophage cell line (J774A.1) were cultured under routine conditions. Briefly, the cells were taken out from liquid nitrogen and thawed at 37 °C in a water bath. Next they were suspended in DMEM supplemented with 10% fetal calf serum, 100 IU/ ml penicillin, and 100 µg/mL streptomycin. The cells were seeded in tissue culture plates and incubated at 37 °C in a humidified 5% CO6 cells/mL, were cultured at 37 °C in a humidifiedatmosphere of 5% CO2 in the air and incubated in 5 mL media supplemented with 10% heat-inactivated fetal calf serum and containing penicillin/streptomycin (100 IU and 100 µg/mL). Freshly prepared hepatocytes and sinusoidal endothelial cells were transferred to tissue culture plates pre-coated with collagen, while Kupffer cells, peritoneal macrophages, and cell lines L929, J774A.1, and HepG2 were seeded into uncoated tissue culture plates. Primary cells and cell lines were incubated without OTA or LPS during a pre-experimental period of 2 h. After that, at time point zero, medium samples were taken. One μg/mL (2.5μmol/L) OTA or 0.1 µg/mL of LPS were added to culture media 1 h after the zero-time sample. Whereas, the control cells or untreated were exposed to OTA vehicle.Single cell preparations, each at 2 × 10Culture medium samples were collected at 0, 0.5, 1, 1.5, 2, 4 and 24 h (end point). Samples were stored at −20 °C until they were analyzed by a rat TNF-α ELISA test system according to the company’s instruction.6 cells/mL) was seeded in 24 well tissue culture plates coated with collagen type I. The cells pre-incubated 2 h prior to the addition of OTA at concentrations 0, 2.5 and 12.5 µmol/L or 0.1 µg/mL LPS into incubation media up to 24, 48, and 72 h. After that, the cells were incubated with the medium containing MTT for 4 hours. The cells were then lysed in DMSO. Finally, the absorbance was measured at 570 nm. Cytotoxicity of OTA and LPS was determined by 3--2,5-diphenyltetrazolium bromide (MTT) assay, detecting the cellular mitochondrial capacity to convert MTT tetrazolium salt to formazan. 300 µL of different cell suspensions  is a cell-impermeant red fluorescent nucleic acid stain that labels only cells with compromised membranes. In brief, 300 µL of the cell suspensions (1 × 106 cells/mL) were seeded on sterile glass cover slips, then cells were treated with; 0, 2.5 and 12.5 µmol/L OTA or 0.1 µg/mL LPS for 24 h. After that, cells were washed with HBSS and then the dye mixture of the LIVE/DEAD kit was added for 15 minutes at room temperature in the dark. Finally, cells were washed with HBSS again and were visualized under a fluorescence microscope. Fluorescence imaging was performed on a Leica DM IRE2 fluorescence microscope supplied with FITC filter  and Texas Red filter  from Leica, Wetzlar, Germany. The captured images were analyzed with the Leica Fluorescence Workstation software (FW4000). Live cells appeared fluorescent green and dead cells fluorescent red.The applied concentrations of the toxins came from dose-finding experiments where concentrations of OTA between 0.5 and 75 µmol/L were selected; the concentrations used here were found not to significantly alter the viability of cells but effectively provoke TNF-alpha release. The viability of HepG2, L929 and J774A.1 cells were measured using LIVE/DEAD kit according to the company instructions. SYTOSubcellular fractionation was performed as following: cells were washed with cold PBS, then 0.5 mL sucrose (0.25 M) containing a protease inhibitor cocktail was added for 5 min at 4 °C. After scraping, the content of plates was transferred into tubes. Cells were homogenized by a glass stick and their nuclei were pelleted by centrifugation . The supernatant was used as cytosolic fraction, and nuclear pellets were re-suspended in IBP.7. containing protease inhibitor cocktail. The nuclear suspension was destructed by sonicating. The purity of fractionation was evaluated via detection of α-tubulin. TM Protein Assay Reagent . The samples were subjected to SDS-PAGE. The separated proteins were transferred to a polyvinylidene difluoride membrane , and the protein bands were subsequently probed with antibodies.The cells were washed with phosphate-buffered saline and lysed in IPB.7 containing protease inhibitor cocktail . The protein was quantified by using Coomassie PlusTM GE Healthcare Bio-sciences AB, Uppsala, Sweden). Actin was used as a loading control. The results were analyzed with Image J version 1.34s software. Primary antibodies used in the analyses were phospho-NF-κB p65 (C22B4), phospho-MEK1/2 (Ser217/221), phospho-Raf (Ser-259) ; Actin (c-11): sc-1615 and phospho-p38 (Thr180/Tyr182)-R: sc-17852-R . Secondary antibodies used in the analyses were goat anti-rabbit IgG sc-2004, goat anti-mouse IgG sc-2005 (Santa Cruz Biotechnology). The results were visualized by using the ECL or ECL plus detection kits , <0.01 (**), and <0.001 (***) compared with control values were considered statistically significant. Data was analyzed with Graphpad Prism 5.0 software .The data are presented as mean ± SEM for at least three separate trials for each experiment, obtained from different animals. A two-way ANOVA statistical test was used for analysis of variance, followed by a Bonferroni versus LPS with respect to TNF-α release for the different cells and cell lines. Macrophages and macrophage-derived cell lines were most sensitive to both stimuli. Sinusoidal endothelial cells were less sensitive, and HepG2 cells and hepatocytes the the least sensitive. Inter-individual cell differences between OTA and LPS could be explained by different signaling pathways required for the stimuli of TNF-α release.Our data indicate significantly different cell sensitivities to OTA


Paracetamol given for fever is associated with hypotension . Spectran = 12) were medical, 31% (n = 9) surgical, and 28% (n = 8) neurological. APACHE II score was 17.2 ± 8.3. Frequency domain analyses of the laser Doppler flowmetry (LDF) waveforms of two patients were undertaken. Both patients (A and B) had good LDF waveforms, both increased skBF whilst BP fell in patient B.Twenty-nine adults, 58 ± 15 years, were treated with enteral or intravenous paracetamol for fever. Forty-one percent (P < 0.05). Systolic BP decreased (P < 0.01) at all post-administration times and was 90 ± 13% of T0 at 60 minutes  and then every 15 minutes for 60 minutes. Thirty datasets were recorded. Temperature at T0 was 38.7 ± 0.6°C. BP decreased over the study period whilst skBF and cutaneous vascular conductance  increased (repeated-measures ANOVA, Paracetamol induced increases in skBF consistent with its antipyretic action. Changes in PSD and wavelet analysis were consistent with cutaneous vasodilation.


Agarivorans albus is a Gram-negative, strictly aerobic, and agar-hydrolyzing marine bacterium. We present the draft genome sequence of the A. albus strain MKT 106T, which is composed of 67 contigs (>500 bp) totaling 4,734,285 bp and containing 4,397 coding DNA sequences (CDSs), four rRNAs, and 64 tRNA sequences. Agarivorans, which is currently composed of two species, are Gram-negative, strictly aerobic, and agar-hydrolyzing bacteria , National Institute of Technology and Evaluation (NITE), and whole-genome shotgun sequencing was performed on the Roche 454 GS-FLX+ Titanium sequencing platform. A total of 180,812 reads  were obtained and assembled using Newbler v2.6 (Roche Diagnostics). The assembled genome is composed of 67 contigs (>500 bp) totaling 4,734,285 bp, with a G+C content of 44.2%. The N50 contig size is 124,617 bp and the largest contig is 357,826 bp.A. albus MKT 106T was annotated using the Rapid Annotations using Subsystems Technology (RAST) server v4.0 , followed by Shewanella loihica strain PV-4 . Both of these neighbors and A. albus belong to the order Alteromonadales.The draft genome sequence of ver v4.0  with SEEver v4.0 , 14. Thever v4.0 , 14. A cAgarivorans. This genomic information will provide a reference genome data set of Agarivorans, as well as help define the phylogenic relationships between this genus and other species of bacteria in future studies.This is the first whole-genome sequence for a member of the genus A. albus MKT 106T was submitted to DDBJ under accession no. BARX01000001 to BARX01000067.The draft genome sequence of


Formulation and evaluation of the selective medium "This compound was initially solubilized in 98% ethanol, sterilized using 0.2 μm filters, added to 45°C YEM medium, and homogenized to obtain a final concentration of 0,2 g.l-1 of PCNB into the P-YEM medium". Must be replaced by the following "A solution of 10% PCNB in acetone, was diluted 1/5 in Tween 80 0,5%v/v in water, sterilized using 0.2 μm filters, added to 45°C YEM medium, and homogenized to obtain a final concentration of 0,04 g.l-1 of PCNB into the P-YEM medium".The authors regret that an error appears in


Stellera chamaejasme L, a traditional Chinese herb, has long been used for treatment of various tumors in the Chinese population. In our previous study, we paid an attention to the cytotoxic and proapoptotic effects of Stellera chamaejasme L extracts  on 4 various tumor cells  in vitro. ESCs showed significantly inhibitory effects on the 4 tumor cells. ESC-2 had the strongest inhibitory effect and the broadest sensitive cell spectrum. ESC-2 and ESC acted in a similar way against tumor cells, which suggested anti-tumor active fraction of ESC might exist in ESC-2. Here, we further observe the inhibitory and proapoptotic effects of Stellera chamaejasme L extracts in vivo.Stellera chamaejasme L extracts. Apoptotic rates of the cancer cells retrieved from the hollow fibers were measured with flow cytometric analysis, caspase 3, 8, 9 enzyme activities were detected by colorimetric assay, Fas, Fas-L, TNF-R1 and TNF-α expression were determined with elisa assay and radioimmunoassay respectively.In this study, we used hollow fiber tumor model to evaluate the inhibitory and proapoptotic effects of The results showed that ESC, ESC-2 all had inhibitory effects on 4 tumor cells. According to the effect strength, dose and antitumor spectrum, the order of antitumor effects of ESCs was: ESC-2 > ESC > ESC-1. NCI-H460 cells were the most sensitive to ESCs. ESC, ESC-2 increased greatly the apoptotic rate and caspase 3, 8 enzyme activities in NCI-H460. ESCs had no significant effects on expression of Fas, Fas-L protein, but TNF-α/TNFR1 protein expression in NCI-H460 cells changed significantly after ESC and ESC-2 treatment.in vivo and further confirmed that ESC-2 may be the main antitumor active fraction of ESC, which was consistent with our previous results in vitro.ESC-2 had the similar antitumor effect to that of ESC  In vitro cytotoxicity experiment is the first step for screening or discovery of anti-tumor drug. It has the advantages of reducing substantially the amount of test drugs, determining the tumor specificity of the test drugs, simple experiment, low cost, etc. So, it is also easily adopted as the approach for pharmacological researchers to tracing active ingredients or fractions of most anti-tumor nature products, especially medicinal herbs. However, each herb in body is subjected to process of transport and metabolism, which makes the experimental results in vitro and in vivo always inconsistent. In particular, distinctive action characteristics of traditional Chinese herbs based on its integrity and interaction with the body determine the importance and necessity of investigating the efficacy in vivo. Therefore animal experiments must be conducted to verify the validity of the test samples, which is the critical step for active ingredients or fractions screening of traditional Chinese herbs. Xenograft is once the main the model for anti-tumor drug screening. But this kind of model is difficult to be as the large-scale screening method because it demands a large number of experimental animals and the test samples, consuming large amounts of time and manpower. Hollow fiber assay (HFA) is a high-throughput in vivo screening system built up by US National Cancer Institute (NCI) in 1995 In vitro products , p16 /4 and the low dose = 0.67 × the high dose [36 male nude mice (body weight range was 18-20 g), according to the above method, were charged hollow fibers with NCI-H460 lung cancer cells intraperitoneally and subcutaneously. Mice were divided into control, 20 mg/kg 5-Fu, 4.5 mg/kg ESC, 3.0 mg/kg ESC, 1.5 mg/kg ESC-2, 1.0 mg/kg ESC-2. The administration method was same as that mentioned above. On the collection day, the fibers were removed from sacrificed mice and transferred to 6-well plates added with 2 ml preheating acutase to digest for 30 min. Then the cells were flush out from fibers to be wait for detection of indexes related to apoptosis.The cancer cells were retrieved from the fibers and prepared for flow cytometric analysis to measure apoptotic rate. Briefly, the cells were washed with phosphate balanced solution (PBS) and fixed with 70% ethanol for 30 min on ice. After centrifugation, the pellet was resuspended in 400 ml hypotonic propidium iodide (PI) solution  and incubated on ice in the dark for at least 15 min. Then the cells were analysized by flow cytometer .6 cells were collected from fibers and lysised with lysate on ice for 10 min and centrifuged at 20,000 g for 15 min. The supernatant was transferred to a pre-cooled centrifuge tube and determined the activities of caspases immediately. Reaction system was set according to kits and incubated at 37°C for 120 min. Absorbance value was measured at 405 nm when the reaction system color obviously changed. If the color did not change significantly, incubation time of reaction system may be extended, even overnight incubation. Deference of A405 absorbance between treatment and the control was p-nitroaniline (pNA) absorbance of sample catalyzed by caspases. Catalytic amount of pNA of can be calculated through the pNA standard curve. Enzyme activity unit was defined as the amount of caspase enzyme catalyzing 1 nmol pNA substrate when the substrate was saturation. Each sample protein concentration was determined by Bradford method to calculate the enzyme activity in unit weight of protein of sample.Caspase 3, 8, 9 activities were measured with caspase assay kits. 1 × 10Cells removed from fibers were collected after conventional digestion and cleavage. The standard was diluted according to the Elisa kit. 100 μl standard or each test sample was added into wells of reaction plates and incubated for 120 min at 37°C. Plates were washed with washing liquid 5 times, and then first antibody working solution of 100 μl was added and incubated for 60 min at 37°C. Plates were washed another time. 100 μl Horseradish Peroxidase (HRP) working solution was added and incubated for 30 min. Plates were washed again. 100 μl substrate working solution was added and reacted in the dark for 15 min at 37°C, and then 100 μl of Stop Solution was added. Absorbances of samples were measured at 450 nm in microplate reader within 30 min.Cells flushed from the hollow fibers were washed with PBS, and then cells were lysised with ultrasonic cell crasher. Using liquid competitive inhibition principle, the cell samples were determined with balance method. The samples or standard and limited antisera or antigen together generated competitive binding reaction. After the reaction, added immune separating agent to isolating the antigen-antibody complex, radioactivity of compound was determined and the standard combination rate was calculated. Make the standard curve and determine the sample concentration.All results were expressed as mean ± SD. One way analysis of variance (ANOVA) with multiple comparison tests was used. P < 0.05 was considered as significant. Statistical tests were performed using SPSS version 12.0.For the NCI-157 cells: In the SC (subcutaneous) fibers, the IRs of 4.5,3.0 mg/kg ESC and 1.5 mg/kg ESC-2 were 43.05%, 77.15% and 43.13% respectively; In the IP  fibers, the IRs of 4.5 mg/kg ESC and 1.0 mg/kg ESC-2 were 96.33% and 49.78% respectively. For the NCI-H460 cells: In the SC fibers, the IRs of 4.5,3.0 mg/kg ESC, 1.5,1.0 mg/kg ESC-2 and 16.75 mg/kg ESC-1 were 98.62%, 60.01%, 79.71%, 100.84% and 45.54% respectively; In the IP fibers, the IRs of 4.5 mg/kg ESC and 1.5,1.0 mg/kg ESC-2 were 48.70% and 68.71%,108.34% respectively. For the Bel-7402 cells, In the SC fibers, the IRs of 1.0 mg/kg ESC-2 and 25 mg/kg ESC-1 were 43.91% and 55.03% respectively; In the IP fibers, the IR of 4.5 mg/kg ESC was 43.38%. For the SK-HEP-1 cells: In the SC fibers, the IR of 4.5 mg/kg ESC was 40.24%; In the IP fibers, the IRs of 4.5, 3.0 mg/kg ESC, 1.5,1.0 mg/kg ESC-2 were 46.78%, 52% and 44.42%, 47.33%. The results showed that ESC and ESC-2 have obvious toxicity to 4 tumor cells in hollow fibers loaded in nude mice  respectively compared to that of control group (23.32 ± 2.26); 4.5 mg/kg ESC and 1.5 mg/kg ESC-2 can also significantly improve caspase 8 enzyme activity of NCI-H460 cells and increase 1.49 and 1.47 folds  compared to that of control group (13.40 ± 4.17); but ESCs did not increase the caspase 9 enzyme activity of NCI-H460 cells  significantly improved expression of TNF-α in NCI-H460 cells, whereas 1.5, 1.0 mg/kg ESC-2  decreased significantly in NCI-H460 cell compared with the control group (0.78 ± 0.36); 1.0 mg/kg ESC-2 (39.10 ± 10.15) increased significantly TNFR1 expression in NCI-H460 cell compared with the control group(20.07 ± 3.02) to evaluate the anti-tumor pharmacological effects of in vivo inhibition rate was more than 40% according to evaluation standard of in vivo antitumor nature product crude extract [in vivo cytotoxic effect and both produced >40% growth inhibition on four tumor cell lines, ESC-1 only one dose had certain inhibition on NCI-H460 and BEL-7402 cells. Especially, the cell killing effect (a reduction in cell mass below the input mass and inhibition rate was more than 100%) was observed in NCI-H460 cell line in mice of ESC-2 group. Therefore, the order of antitumor effects of ESCs was comprehensively evaluated according to the effect strength (ESC-2 had cell killing effect), effect dose (administration dose of ESC-2 was 1/3 of ESC) and the antitumor spectrum (ESC-2 had obvious inhibitory effects on 4 cell tumor lines): ESC-2 > ESC > ESC-1.It was considered positive that  extract . The holComparison of sensitivity of ESCs to tumor cells, NCI-H460 cell line was the most because that all ESCs had inhibition on it. ESC-2 showed the strongest inhibition and inhibition rates of two sites (SC and IP) of NCI-H460 cells were more than 100%. Follow the ESC-2 was ESC and inhibition rate of two sites (SC and IP) were nearly 100%. Furthermore, ESC-1 showed certain inhibition in SC site on NCI-H460 cells.in vitro experiment, we found that ESCs could induce the tumor cells apoptosis by activating death receptor pathway. Therefore we selected the most sensitive NCI-H460 cell line to further confirm the apoptotic effect of ESCs in vivo based on HFA. The flow cytometry results showed that 4.5 mg/kg ESC, and 1.5 mg/kg ESC-2 increased significantly apoptosis rates in NCI-H460 cells in vivo. Enzymatic activity assay also confirmed the ESC and ESC-2 could significantly increase the activities of caspase 3 and caspase 8 in NCI-H460 cells. These results suggested that ESC and ESC-2 induce tumor cell apoptosis through activation signal of the death receptor pathway in vivo which was consistent with the in vitro results [In our previous  results ,15.The death receptor families contained Fas, TNFR1, death receptor , TNF related apoptosis inducing ligand receptor , etc. . Which pin vitro results. Maybe after the metabolism in vivo, the targets of ESCs changed. TNFR1/ TNF-α signal system was another important death receptor pathway which was not observed in our in vitro experiment. TNF-α was a multifunctional cytokine, mainly involving in inflammation and immune responses. It had a direct cytotoxic effect on tumor cells and could cause tumor necrosis. Recent researches had demonstrated that TNF-α inhibited certain tumor cell proliferation and induced apoptosis of tumor cells [We tested the classic death receptors, Fas, TNFR1 and their ligands Fas-L, TNF-α. The Fas/Fas-L system was a key regulator of apoptosis. The results showed that ESC, ESC-2 had no significant effects on Fas/Fas-L pathway which was not consistent with the or cells . But fewor cells ,20 and wor cells . Other ror cells ,23. VisiTNFR1, another death receptor protein, combining with TNF-α might induce typtical apoptosis of caspase dependent pathway. On the other hand, this combination might activate the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and c-Jun-N terminal kinase (JNK) signal pathway by recruitment of TNF receptor-associated factor (TRAF2) and Receptor interacting protein (RIP) -26. 1.0 in vivo and further confirmed that ESC-2 may be the main antitumor active fraction of ESC, which was consistent with the in vitro result.The results above showed ESC-2 had the similar antitumor effect to that of ESC The authors declare that they have no competing interests.XL was the main experimental investigator and had drafted the manuscript. QY, GZ and YL helped to establish the hollow fiber tumor model. YC and XW helped to complete the experiments of caspases acticities and elisa assay. YW helped to correct the language of the manuscript. YW helped to analysis the data. XZ supervised the study and the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/14/116/prepub


When validating new methods of cardiac output, measurement comparisons are made using Bland-Altman and percentage errors are generated that rely on a precision error for thermodilution of ± 20% ; data con = 6) and Edwards (n = 6) 7F pulmonary artery catheters and a Siemens SC9000 monitor were used. Sets of cardiac output readings were taken (three to six pairs). Catheters were changed frequently and cardiac output increased  and decreased  using drug infusions. Baseline and drug treatment data were compared.Four domestic pigs, weight 30 to 32 kg, were anaesthetized. An aortic flow probe was placed via a left thoracotomy. Both Arrow (Forty-five sets (259 pairs) of averaged data (21 baseline and 24 following treatment) were collected. Baseline cardiac outputs (mean (SD)) were 1.9 (0.4) and 1.8 (0.3) l/minute for flow meter and thermodilution readings, respectively. MAP (mean (range)) was 82 (69 to 95) mmHg. Following circulatory treatment, cardiac output ranged from 0.5 to 3.4 l/minute and from 0.7 to 3.5 l/minute, respectively. MAP ranged from 44 to 118 mmHg. For baseline data, bias was 0.0 l/minute, limits of agreement ± 0.45 l/minute and percentage error ± 24.3%. Following treatment, the bias was unchanged at 0.0 l/minute, but the limits of agreement widened to ± 0.78 l/minute and percentage error widened to 42.0%  precision error, thus the baseline percentage error of 24.3% is in keeping the quoted precision error for thermodilution of ± 20%. However, under more extreme circulatory conditions thermodilution behaved less reliably with widened limits of agreement and precision errors (42.0%). Thermodilution is less accurate than originally thought in haemodynamically unstable patients.