Euglena gracilis, a unicellular flagellated microalga, is regarded as one of the most promising species as microalgal feedstock for biofuels. Its lipids (mainly wax esters) are suitable for biodiesel and jet fuel. Culture of E. gracilis using wastewater effluent will improve the economics of E. gracilis biofuel production. Enhancement of the productivity of E. gracilis biomass is critical to creating a highly efficient biofuels production system. Certain bacteria have been found to promote microalgal growth by creating a favorable microenvironment. These bacteria have been characterized as microalgae growth-promoting bacteria (MGPB). Co-culture of microalgae with MGPB might offer an effective strategy to enhance microalgal biomass production in wastewater effluent culture systems. However, no MGPB has been identified to enhance the growth of E. gracilis. The objectives of this study were, therefore, to isolate and characterize the MGPB effective for E. gracilis and to demonstrate that the isolated MGPB indeed enhances the production of biomass and lipids by E. gracilis in wastewater effluent culture system.Emticicia sp. EG3, which is capable of promoting the growth of microalga E. gracilis, was isolated from an E. gracilis-municipal wastewater effluent culture. Biomass production rate of E. gracilis was enhanced 3.5-fold and 3.1-fold by EG3 in the co-culture system using a medium of heat-sterilized and non-sterilized wastewater effluent, respectively, compared to growth in the same effluent culture but without EG3. Two-step culture system was examined as follows: E. gracilis was cultured with or without EG3 in wastewater effluent in the first step and was further grown in wastewater effluent in the second step. Production yields of biomass and lipids by E. gracilis were enhanced 3.2-fold and 2.9-fold, respectively, in the second step of the system in which E. gracilis was co-cultured with EG3 in the first step.A bacterium, Emticicia sp. EG3 is the first MGPB for E. gracilis. Growth-promoting bacteria such as EG3 will be promising agents for enhancing E. gracilis biomass/biofuel productivities. Microalgae are widely recognized as alternative, renewable, and sustainable feedstocks for biofuels. The use of wastewater effluent as a medium for microalgal culture will no doubt improve the economics and sustainability of microalgal biomass production , 2. MicrEuglena gracilis, a unicellular flagellated microalga, is regarded as one of the most promising species as microalgal feedstock for functional foods and biofuels , nitrite-N [NO2-N], nitrate-N [NO3-N], phosphate [PO4-P]), and total bacteria concentrations). TOC was measured using TOC-LCSH . For NH4-N, the indophenol method was used; for NO2-N, the N-(1-naphthyl) ethylenediamine method was used; for NO3-N, the reduction-N-(1-naphthyl) ethylenediamine method and UV adsorption (at 220 and 275 nm) method was used; and for PO4-P, the molybdenum blue method was used. Total culturable bacteria in effluent were quantified using R2A agar plates . In some experiments, sterile effluent samples were prepared by autoclaving  for heat sterilization.Municipal wastewater effluent was collected from the final sedimentation process of a conventional activated sludge municipal wastewater treatment plant in Kofu City, Yamanashi, Japan. The effluent sample was first passed through a glass microfiber filter  and then through a membrane filter  to remove suspended solids and organisms larger than bacteria—including microalgae—from the effluent sample. The effluent filtrate, therefore, included the indigenous bacterial community. For each of the following experiments, a fresh effluent sample was collected and used each day. Table E. gracilis prepared by the above method was added to 100 mL of (non-sterilized) effluent filtrate in a 200-mL glass flask. The flask was incubated in the growth chamber (28 ± 1 °C with fluorescent lamps at 80 μmol photons m−2 s−1 and 16-h photoperiod) for 10 days, and then, about 10 mL of this culture was transferred to 100 mL of fresh effluent and incubated for another 10 days. This transfer and 10-day batch culture growth was repeated once more. During the three repeated growth periods, the flasks were shaken for 1 min three times a day to disperse and aerate the E. gracilis. The chlorophyll concentration in each flask was measured daily. E. gracilis was also cultured in 100 mL of autoclaved effluent without living indigenous bacteria. The culture was used as a control treatment to determine the effect of the indigenous bacteria in the effluent on the growth of E. gracilis. The initial chlorophyll a + b concentration of each culture was adjusted to about 0.5 μg/mL.A cell suspension (10 mL) of E. gracilis-effluent culture was transferred into a 50-mL tube, vortexed at maximum speed for 3 min, ultrasonicated (40 kHz) for 1 min, and vortexed again to disperse the E. gracilis and bacterial cells. The sample was then filtered through a GF/B glass microfiber filter to remove E. gracilis cells. The filtrate-containing bacteria was serially diluted and spread on R2A agar plates. The plates were then incubated at 28 °C. A total of ten bacterial strains were obtained from the isolation culture. A pure culture of each strain was maintained on an R2A agar plate.After the third 10-day batch culture growth period, 20 mL of the non-sterile g, room temperature, 5 min) and washed twice with 50 mM potassium phosphate buffer (pH 7.5). E. gracilis was inoculated into 100 mL of autoclaved wastewater effluent in 200-mL glass flasks. Optical densities of cultures of isolated bacterial cells were measured at 600 nm (OD600), and each bacterial cell suspension was then added into the E. gracilis culture at bacterial cell density at OD600 of 0.05. The co-cultures of E. gracilis and each isolated strain were incubated in a growth chamber (28 ± 1 °C with fluorescent lamps at 80 μmol photons/m2/s and 16-h photoperiod) for 7 days. At 7th day, concentrations of chlorophyll a + b were measured. E. gracilis was also cultured in autoclaved wastewater effluent without a bacterial inoculation. The growth-promoting ability of MGPB was assessed by comparing the chlorophyll a + b concentration at the end of 7-day cultures with and without bacterial inoculation.Six isolated strains grew well in R2A liquid medium. Each strain was cultured in R2A liquid medium for 24 h at 28 °C. Cells were harvested by centrifugation . A comparative 16S rRNA gene sequence analysis was performed as follows. Partial 16S rRNA genes were amplified by PCR using primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3′)  and 1510g, room temperature, 5 min), washed twice with phosphate buffer, and then suspended in the phosphate buffer. Highly concentrated EG3 cell suspension in phosphate buffers  were prepared for the following experiments. For each experiment, 1% (v/v) of the EG3 cell suspension was inoculated into the experimental culture (sterilized or non-sterilized effluent). Thus, the initial EG3 cell density of each experiment was adjusted as OD600 = 0.05, 0.1, or 0.2. To convert OD600 value into EG3 cell dry weight concentration (mg dry weight/mL) and cell count (CFU/mL), dry weight of cells was measured after drying at 90 °C for 3 h, and CFU of EG3 suspensions  was measured by R2A agar plate method in triplicate. An OD600 = 1.0 of EG3 cells was equivalent to about 0.45 ± 0.03 mg dry weight/mL. Also, OD600 = 0.05, 0.1, and 0.2 of EG3 cells were equivalent to about 2 × 106, 8 × 106, and 4 × 107 CFU/mL on R2A agar plate. In this manuscript, cell density of EG3 was described using CFU/mL.Strain EG3 was grown in R2A liquid medium for 24 h at 28 °C with shaking at 150 rpm. Cells were harvested by centrifugation  for 7 days. E. gracilis was also cultured without EG3 in 1 L of autoclave sterilized wastewater effluent under the above conditions as the control experiment.E. gracilis growth. E. gracilis was inoculated into 1 L of wastewater effluent in 2-L glass bottles. EG3 was then inoculated into the E. gracilis culture at 8 × 106 CFU/mL, because the growth-promoting ability of EG3 was highest at this inoculation cell density. The co-cultures of E. gracilis with EG3 were incubated in the growth chamber (28 ± 1 °C with fluorescent lamps at 80-μmol photons/m2/s and 16-h photoperiod) for 7 days.Both sterilized and non-sterilized effluent samples were used to examine the effects of co-existing indigenous bacteria in wastewater effluent on the ability of EG3 to promote a + b concentration was monitored daily. After 7 days, the dry weight of E. gracilis was measured. The experiments were conducted in triplicate.During the experiments, chlorophyll E. gracilis with strain EG3 to promote the growth of E. gracilis. The E. gracilis cells produced in this first step were used as the E. gracilis inoculum for the second step. In the second step, the productions of biomass and lipids by the E. gracilis inoculum from the first step were measured . After 7 days, 2–4 L of culture was collected, and E. gracilis was harvested by low gravity centrifugation . The E. gracilis cells were washed twice with distilled water to remove EG3 cells and harvested by centrifugation. We confirmed that the collected E. gracilis pellets contained only little EG3 cells  for 7 days. Aeration and mixing were achieved by bubbling with atmospheric air at a flow rate 1 L/min. Chlorophyll a + b and biomass were measured every day. The lipid content of E. gracilis was measured at the beginning and end of the second step.The production of biomass and lipids in the second step culture was performed in 15-L polycarbonate culture vessels. Fifteen liters of unautoclaved wastewater effluent was added to the culture vessel. The E. gracilis growth. Chlorophyll concentration was measured spectrophotometrically after extraction in 100% methanol for 30 min [665) and 650 nm (A650) with a spectrophotometer . The total chlorophyll (chlorophyll a + chlorophyll b: Chl a + b) concentration (μg/mL) was calculated as follows [In this study, chlorophyll concentration was monitored as an indicator of r 30 min . Absorba follows :1\documeE. gracilis was measured as follows. Fifty milliliters of the culture was collected and vortexed for 30 s to uniformly suspend the bacterial and microalgal cells. The mixture was centrifuged ; the pellet was washed with 20 mL of distilled water; and the pellet was then suspended in 20 mL of distilled water. The E. gracilis cells in the suspension were collected on a pre-weighed GF/B filter , dried , and then weighed. We confirmed that this method could collect E. gracilis cells with little interference from co-existing EG3 cells  of E. gracilis cells was quantified in terms of the percent of dry biomass accounted for by lipids. E. gracilis was harvested by centrifugation , washed with distilled water, dried, and then powdered. The E. gracilis powder (20 mg) was crushed with 1 mL of n-hexane in a BioMasher . The E. gracilis sample was transferred into a 50-mL tube, and 9 mL of n-hexane and 6-mL isopropanol were added to the tube. The tube was shaken at 225 rpm for 24 h. Thirty milliliters of distilled water was then added to the tube. The tube was shaken for 1 min and then centrifuged . The n-hexane layer containing the lipids was collected on a pre-weighed aluminum tray, dried at room temperature overnight, and then dried at 90 °C for 3 h. Finally, the collected lipids were weighed.The lipid content of the Biomass production rate (mg/L/day) and lipid production rate (mg/L/day) were calculated as follows:n = 3 replicates) per experiment. All results are expressed as mean ± SD. Significance (p < 0.05) was analyzed using the t test in SPSS Statistics v. 22.0 .Each value used in the statistical analysis represents the results from three samples  or without living bacteria (autoclave heat-sterilized effluent) in three sequencing batch cultures. After 2 days of each batch culture, the chlorophyll a + b concentration was higher (p < 0.05) in the E. gracilis-effluent culture with living indigenous bacteria than in the culture without living bacteria  of EG3 was very similar to the analogous sequences of Emticicia fontis strain IMCC1731T (97.9% sequence similarity), Emticicia ginsengisoli strain Gsoil 085T (97.6%), Emticicia soli strain ZZ-4T (97.5%), Emticicia oligotrophica strain GPTSA100-15T (94.4%), Emticicia aquatica strain HMF2925T (93.9%), Emticicia sediminis strain JBR12T (93.7%), and Emticicia aquatilis strain THG-DN6.14T (93.7%). We, therefore, identified strain EG3 as Emticicia sp.Colonies of strain EG3 on R2A agar were circular, smooth, convex, and orange color. EG3 showed positive for oxidase, glucose fermentation, and aesculin hydrolysis, but negative for nitrate reduction, indole production, arginine dihydrolase, urease, gelatinase, and β-galactosidase activities. EG3 utilized glucose, E. gracilis in wastewater effluent, E. gracilis was grew in heat-sterilized wastewater effluent with various EG3 cell densities, 0 (control), 2 × 106, 8 × 106, and 4 × 107 CFU/mL. The chlorophyll a + b concentrations and biomass production rate (mg/L/day) of E. gracilis were significantly higher (p < 0.05) in E. gracilis cultures with EG3 at 2 × 106, 8 × 106, and 4 × 107 CFU/mL than in the E. gracilis control culture  than those of E. gracilis cultures without EG3  concentration of E. gracilis in the second culture system were significantly higher (p < 0.05) in the system inoculated with E. gracilis that had been co-cultured with EG3 in the first step than in the system inoculated with the control E. gracilis culture  was 3.2 times of that of the control E. gracilis (27 ± 2.7 mg/L/day). The lipid content of E. gracilis-EG3 (28.4% ± 0.7%) was slightly but significantly lower than that of the control E. gracilis (30.9% ± 0.8%). The lipid production rate of E. gracilis-EG3 (25 ± 0.8 mg/L/day) was 2.9 times that of the control E. gracilis (8.5 ± 0.8 mg/L/day).We operated a two-step lis Fig. . First, Candidatus Phycosocius bacilliformis” BOTRYCO-2 and P. bermudensis KCTC 13073BP increase the rate of biomass production by B. braunii and T. striata 1.8-fold [E. gracilis-wastewater effluent culture was important for enhancing the biomass/lipid productivity of E. gracilis in a wastewater effluent culture system. The MGPB for three microalgae—C. reinhardtii, C. vulgaris, and E. gracilis—have recently been found to be present in common wastewater effluent [E. gracilis, Emticicia sp. EG3, was isolated from an E. gracilis-municipal wastewater effluent culture and was found to be capable of promoting the growth of E. gracilis. Strain EG3 is the first MGPB for E. gracilis. Members of the genus Emticicia have been isolated from various environments, including freshwater [Emticicia and its potential for industrial use are still unclear, our results reveal that one of its ecosystem services may be to function in a symbiotic relationship that enhances the growth of microalgae such as E. gracilis.Co-culture with MGPB is useful for enhancing microalgal growth in mass culture systems. For example, “1.8-fold  and twof1.8-fold , respecteffluent . In thiseshwater –37, wetleshwater , seawateeshwater , the sedeshwater , and soieshwater , 42. AltE. gracilis with strain EG3 at 8 × 106 CFU/mL, EG3 enhanced the production rate of E. gracilis biomass 3.7-fold and 3.1-fold in heat-sterilized and non-sterilized wastewater effluent, respectively  enhanced nutrient removal in synthetic wastewater compared to immobilized C. vulgaris alone, and (2) enhanced biomass, lipid, and starch productivities in synthetic media [E. gracilis. E. gracilis was first co-cultured with strain EG3 at 8 × 106 CFU/mL to promote the growth of E. gracilis, and the production of biomass and lipids was then enhanced by growing the stimulated E. gracilis in wastewater effluent culture. In the second E. gracilis culture step, E. gracilis rapidly grew over a period of 7 days  production rate twofold in CM medium under high-light and high CO2 conditions by overexpressing the cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase gene, which is involved in the Calvin cycle. The highest biomass concentration of the transgenic E. gracilis was 631.1 ± 89.9 mg/L. Zhu and Wakisaka [E. gracilis culture at 500 mg/L can increase E. gracilis cell density 2.5-fold and E. gracilis biomass (dry weight) production rate 2.2-fold. The maximum E. gracilis biomass concentration in CM medium with 500 mg/L ferulic acid was 670 ± 40 mg/L. Noble et al. [E. gracilis; a combination of trans-zeatin (10−7 M) and abscisic acid (10−9 M) produced optimal conditions for growth of E. gracilis and increased growth rate about threefold compared to an E. gracilis culture without any phytohormones. Enhancement of biomass production rate (3.2-fold) and highest biomass concentration (702 ± 23 mg/L) of E. gracilis by EG3 co-culture at 8 × 106 CFU/mL in real wastewater effluent were comparable to or higher than the results of these previous enhancement methods. Our results indicate the potential utility of a two-step E. gracilis culture system with EG3—the first step being co-culture of E. gracilis with EG3, and the second step being a large biomass production culture system for enhanced productivities of biomass and lipids by E. gracilis in wastewater effluent. A schematic design of the two-step E. gracilis culture system coupling with wastewater treatment plant is shown in Fig. E. gracilis. Optimization of the two-step culture system and better understanding of the growth-promoting effect of EG3 are the next challenges for commercial application of this strategy.Our study is the first to demonstrate the potential of the MGPB a et al.  were ablWakisaka  have repe et al.  have repEmticicia sp. EG3, which is capable of promoting the growth of E. gracilis, was isolated from an E. gracilis-municipal wastewater effluent culture. In a co-culture of EG3 and E. gracilis, EG3 significantly increased the biomass productivity of E. gracilis, not only in heat-sterilized wastewater effluent, but also in non-sterilized effluent that contained an indigenous wastewater effluent bacterial community. A two-step E. gracilis culture system was proposed to enhance E. gracilis biomass/lipid productivities by exploiting the stimulatory effect of EG3 on E. gracilis. First, E. gracilis was co-cultured with EG3 to promote E. gracilis growth in 5 L of effluent. Second, biomass and lipids were produced using the promoted E. gracilis in a 15-L wastewater effluent culture. Biomass and lipid productivities of E. gracilis were increased 3.2-fold and 2.9-fold, respectively, compared to a culture without EG3. Growth-promoting bacteria such as EG3 are promising agents for enhancing microalgal biomass/biofuels productivities.Additional file 1: Figure S1. Summary of E. gracilis collection method and fate of EG3 cells during this method.Additional file 2: Figure S2. Effects of isolated strains EG3, EG5, EG6, EG8, EG9, and EG10 on E. gracilis growth in autoclave heat-sterilized wastewater effluent.


KFD18. The structures of the compounds, including the absolute configurations of their stereogenic carbons, were determined by spectroscopic data and single-crystal X-ray diffraction analysis. Compounds 1–4 showed cytotoxic activity against the human gastric cancer cell line SGC-7901, the human leukemic cell line K562, the HeLa cell line, and the human lung carcinoma cell line A549, with 1 being the most active while compounds 5 and 6 were inactive against all the tested cell lines. Compounds 1 and 3 showed very potent and specific cytotoxic activities  against the SGC-7901 cells. Further, the apoptosis-inducing effect of 1 and 3 against SGC-7901 cells was demonstrated by two kinds of staining methods for the first time.Four new ansamycins, named divergolides T–W ( Streptomyces sp. HKI0576 and reported in 2011. Until now, a total of 19 members (divergolides A–S) of this family has been reported +. The UV spectrum showed characteristic absorption bands around 221 and 240 nm. The IR absorptions at 3414 and 1663 cm−1 revealed the presence of a hydroxy and carbonyl group, respectively. The 1H and 13C NMR spectra , twelve non-protonated carbons . Comparison of the above data with those of the known analogue 5 + .Divergolide T (2): White powder; + .Divergolide U (3): White powders; + .Divergolide V (4): White powders; − .Divergolide W  with graphite monochromated Cu Kα radiation (λ = 1.54178 Å). Crystallographic data for 1 were deposited in the Cambridge Crystallographic Data Center as supplementary publication number CCDC 1893418. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.X-ray Crystal Data for 1: Colorless crystals of 1. Monoclinic, C31H37NO7; space group P 1 21 1 with a = 12.5723(5) Å, b = 14.6723(6) Å, c = 17.4900(8) Å, V = 3226.3(2) Å3, Z = 1, Dcalcd = 1.109 g/cm3, μ = 0.691 mm−1, and F(000) = 1077. T = 296.15 K. R1 = 0.0526 (I > 2σ(I)), wR2 = 0.1480 , S = 1.021. Absolute structure parameter: −0.05(8). The structures were solved using ShelXS. The structural solutions were found by direct methods and refined using the ShelXL package by least squares minimization. The final structures were examined using the Addsym subroutine of PLATON to assure that no additional symmetry could be applied to the models. All non-hydrogen atoms were refined with anisotropic thermal factors.Crystal data for 1–6 were tested in vitro by using the MTT method optimized by Chuan et al. [The cytotoxic activities of compounds n et al. . Imatini1–4) and two known analogs (5 and 6) were isolated from the fermentation broth of mangrove-derived actinomycete Streptomyces sp. KFD18. Compounds 1–4 exhibited cytotoxic activity against SGC-7901, K562 , HeLa , and A549  cell lines, with 1 being the most active while compounds 5 and 6 were inactive against all the tested cell lines. The two most active compounds, 1 and 3, could induce apoptosis of SGC-7901 cells.In conclusion, four new ansamycins (


Oxidative stress plays a role in the onset and progression of a number of diseases, such as Alzheimer’s disease, diabetes and cancer, as well as ageing. Oxidative stress is caused by an increased production of reactive oxygen species and reduced antioxidant activity, resulting in the oxidation of glutathione. The ratio of reduced to oxidised glutathione is often used as a marker of the redox state in the cell. Whereas a variety of methods have been developed to measure glutathione in blood samples, methods to measure glutathione in cultured cells are scarce. Here we present a protocol to measure glutathione levels in cultured human and yeast cells using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS). The interest in studying oxidative stress mechanisms at the cellular level is increasing, primarily because of the current understanding that oxidative stress plays a role in the onset and progression of ageing and a variety of human diseases, including Alzheimer’s disease, diabetes and cancer ,2,3. BesThe cells were incubated for the indicated times  (see the Procedure section for details). Similarly, we recommend the use of appropriate negative controls, i.e., cells with vehicle treatment using equal amounts of solvents used for incubation with chemicals. In addition, we recommend conducting every experiment at least in triplicate.See 10H17N2NO6S), ≥98.0% peptide purity L-Glutathione reduced (C20H32N6O12S), ≥98.0% peptide purity L-Glutathione oxidized  (C813C2H17N215NO6S), ≥98 atom % 15N, ≥99 atom % 13C, ≥95% (CP) Labelled reduced glutathione-, ≥98.0% peptide purity DL- Buthionine--sulfoximine (BSO) Menadione Dimethyl sulfoxide (DMSO) Acetonitrile (99.9%) Ethanol absolute (Dehydrated) AR Formic acid (98–100%)   Natrium hydroxide ® IQ 7000)Ultrapure water (Milli-Q)  Corning Cell scrapers Eppendorf Safe-Lock Tubes, 2.0 mL Refrigerated microcentrifuge suitable for 2.0 mL tubes (Centrifuge 5425R) Dri-block (sample concentrator), Techne DB-3 Acquity UPLC HSS T3 1.8 µm column (2.1 × 100 mm) Acquity UPLC system consisting of an Acquity Solvent Manager with degasser and an Acquity Sample Manager with column oven Masslynx V4.1 software Quattro Premier XE triple quad , serial no.: VAB964PEX1), and patients diagnosed with 5-oxoprolinuria due to 5-oxoprolinase deficiency and glutathione synthetase-deficient cell lines which were obtained from Coriell Cell Repositories . Fibroblasts were cultured in 25-cm2 flasks in Ham’s F-10 Medium with L-glutamine, supplemented with 10% foetal calf serum , 25 mM Hepes, 100 U/mL penicillin and 100 µg/mL streptomycin and 250 µg/mL amphotericin in a humidified atmosphere of 5% CO2 at 37 °C.We used anonymised primary skin fibroblast cell lines from healthy controls, Zellweger Spectrum Disorder patients with a severe phenotype  as wild-type strain. The eviously . Wild-tyPrepare the NEM stock solution freshly on the day of experiment. Dissolve NEM in Milli-Q water to a final concentration of 20 mM.S. cerevisiae, prepare wash solutions containing NEM in Milli-Q water. Per sample, prepare 300 µL of wash solution containing 200 mM NEM, and 2 mL of wash solution containing 50 mM NEM.When using Prepare the precipitation solution freshly on the day of the experiment. Per sample, the precipitation solution consists of 280 µL NEM and 700 µL acetonitrile. Cool on ice prior to use.Prepare the internal standard mix. Per sample, the internal standard mix contains 10 µL labelled GSH  and 10 µL labelled GSSG  .Re-suspend the pellet in 100 µL of mobile phase A.PAUSE STEP The sample can be stored at −20 °C until analysis.Dry the cell pellet at room temperature.Re-suspend the cell pellet in 200 µL NaOH in Milli-Q water (0.2 M concentration).PAUSE STEP The sample can be stored at −20 °C until analysis.Determine the protein concentration of the cell pellet, e.g., using the bicinchoninic acid assay .On the day prior to the experiment, shift the yeast cells from minimal ethanol growth medium .Mix briefly by vortexing or shaking.Centrifuge at 7000 rpm for 1 min at 4 °C.Remove the supernatant.Add 1 mL of wash solution (50 mM NEM) to the cell pellet.Vortex for 10 s.Centrifuge at 7000 rpm for 1 min at 4 °C.Repeat step 7–10 for a second wash.Remove the supernatant.Add ice-cold precipitation solution and internal standard.Add glass beads.Vortex for 5 min at 4 °C.Centrifuge at 7000 rpm for 1 min at 4 °C.Transfer the supernatant to a new microcentrifuge tube.Evaporate the supernatant under a nitrogen stream at 45 °C (duration ca. 00:30 hours).Re-suspend the pellet in 100 µL of mobile phase A.PAUSE STEP The sample can be stored at −20 °C until analysis.Use 10 µL of sample to inject onto a UPLC HSS T3 1.8 µm column (2.1 × 100 mm) using a UPLC system.Set the column temperature to 50 °C and the flow rate to 400 µL/min.Use the following chromatographic conditions: 99.9% A to 2% B/98% A in 2 min., 2–3.5 min. to 30% B/70% A, to 95% B/5% A in 0.1 min., 3.51–4.5 min. isocratic 95% B/5% A, to 99.9% A in 0.1 min. The equilibration time with solvent A is 1.5 min.Use the triple-quadrupole mass spectrometer in positive electrospray ionization (ESI) mode.3 mbar.Use nitrogen as nebulizing gas and argon as collision gas at a pressure of 2.5 × 10Set the capillary voltage to 3.5 kV and the source temperature to 120 °C. Set the desolvation gas flow to 900 L/h and the desolvation temperature to 350 °C.m/z 311 > 165 , m/z 308 >162 (GSH), m/z 433 > 304 (GS-NEM), m/z 436 > 307 , m/z 613 > 355 (GSSG)**, and m/z 619 > 361   in the positive ionization mode to detect GSH-NEM*, GSSG and the labelled internal standards. Use the following transitions for detection: C4,15N2) . *NEM res GS-NEM A. **For s GS-NEM A.13C2,15N (619.1 > 361.1) and GS-NEM (433.3 > 304.4)/GS-NEM-13C2,15N (436.3 > 307.3), respectively. Subsequently, we divided the obtained value by the slope of the corresponding calibration curve. Finally, we divided the obtained concentration for GSH and GSSG, respectively, in the sample by the protein concentration of the cell pellet (in mg/mL) to adjust for differences in fibroblast cell confluency per culture well.The use of stable isotope-labelled internal standards and hyphenated UPLC-MS/MS in combination with multi reaction monitoring (MRM) allowed for highly selective measurement of each analyte A. We intFor the determination of linearity, we performed a serial dilution of resuspended fibroblast pellets, using Milli-Q as diluent. The limit of quantification (LOQ) was determined as signal-to-noise (S/N) ratios > 10, the limit of detection (LOD) was determined as an S/N ratio > 3. The limit of quantification  ratio >10) was 0.33 nmol for GSSG, and 1.50 nmol for GS-NEM. The limit of detection  was 0.10 nmol for GSSG, and 0.45 nmol for GS-NEM, and the S/N ratios for the respective peaks was greater than 10.We investigated the effect of oxidative stress inducers on the cellular gluthatione-redox state. For this, we used primary control fibroblasts and cells with different metabolic defects. First, we used two chemical compounds, menadione and buthionine sulfoximine (BSO), to study the glutathione levels in control fibroblasts with and without an oxidative challenge. Menadione increases ROS production by redox cycling, and high concentrations of menadione have been reported to induce apoptosis . BSO depPEX1 gene. In these cells, peroxisome biogenesis is disturbed, and cells accumulate very long-chain fatty acids such as cerotic acid (C26:0), because of a defective peroxisomal fatty acid ß-oxidation. When we incubated PEX1-deficient fibroblasts with 40 µM of me-C26:0 for 96h, we detected normal concentrations of GSH, while GSSG concentrations were significantly increased when compared to untreated PEX1-deficient fibroblasts  in patients . We deteS. cerevisiae. To this end, we modified the sample preparation protocol by already including NEM in the wash buffer when harvesting the yeast cells in order to immediately block thiol groups. As a proof-of-principle, we determined the levels of GSH and GSSG in wild-type yeast and in a mutant strain lacking the glr1 gene, which encodes glutathione reductase. This enzyme is crucial for the reduction of GSSG to GSH, and yeast mutants lacking glr1 (glrΔ) have been described to have increased levels of GSSG [To determine the applicability of our method to other cell types, we also measured glutathione levels in the yeast  of GSSG . Using o13C2,15N) by oxidation using potassium iodide and hydrogen peroxide [13C2,15N) was dissolved in Milli-Q water (stock concentration: 6.5 mM), and incubated with a solution of potassium iodide in Milli-Q water  and hydrogen peroxide (30%) for 120 min at 50 °C, followed by incubation for 5 min at 65 °C to inactive hydrogen peroxide activity. We prepared stock solutions of labelled standards in Milli-Q water, and stored samples at −20 °C.We prepared labelled oxidised glutathione-(glycine-peroxide . In brie


Leutea avicennia Mozaff. That belongs to Apiaceae family is an endemic species distributed in the west of Iran. The aim of this study was to investigate the antioxidant activity and acetylcholinestrase (AChE) inhibition of the crude extract, fractions, and isolated compounds from methanol fraction of L. avicenniae. Five compounds were detected from methanol fraction; three phenolic compounds as p-coumric acid, caffeic acid, salicylic acid and also, two flavonoids as quercetin and astragalin. These structures were identified by spectroscopic techniques such as 1H-NMR, 13C-NMR, and UV. Antioxidant activity was evaluated by the free radical scavenging assay using 2, 2-diphenyl-1-picryl- hydrazyl (DPPH) method. Ellman colorimetric method was used to determine acetylcholinestrase (AChE) inhibition. In the DPPH assay, Quercetin exerted the highest antioxidant activity (IC50 = 10.24 ± 1.3 µg/mL). Caffeic acid inhibited AChE with IC50 = 12.06 ± 2.01µg/mL which were comparable to Galanthamine as positive control (IC50 = 62.44 ± 2.2µg/mL). In conclusion, methanol extract of L. avicenniae contains bioactive components with antioxidant and AChE inhibitory effects.  Free radicals, along with oxidative stress reactions can lead to damage biological molecules like DNA, proteins, and lipids , 2. TherPlants supply a valuable resource, functional as leads to the extension of therapeutic compounds . RegularLeutea is represented by 11 taxa widespread throughout central Asia and Iran   . Since Drst time , 13.Ferula lutea were examined for antioxidant activity and AChE inhibitory effects. The ethyl acetate extract had the strongest antioxidant activity (IC50 = 12.8 ± 1.29 µg/mL) (Ferula gummosa Boiss.and its extract exerted moderate antioxidant activity (IC50 = 579.6 ± 19.4 µg/mL) which was comparable with vitamin C (The extracts and diastereoisomers of 9 µg/mL) . Anotheritamin C .Gossypium herbaceum were examined showing strong antioxidant effects (IC50=0.84 µg/mL) and acetylcholinesterase inhibitory (IC50 = 50.99 µg/mL)  . L. avicennia extract and it’s identified compounds isolated from the methanol fraction.In this study, we report the results of the antioxidant and AChE inhibitory effects ofChemicalsAcetylcholinesterase (AChE) and Acetylthiocholin iodide (ATCI) was purchased from Sigma (Germany). 5, 5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and other chemicals and solvents were provided from Merck (Germany).Plant materialL. avicennia were collected during flowering period from Kaboudarahang Mountain (2200 m) in Hamadan Province (West Iran), in August 2012 and identified by botanist Majid Aghaahmadi. The voucher specimen of the plant (Voucher No. 6761-TEH) was kept in the Herbarium of the Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.Aerial parts of MethodsExtraction and fractionation700g of the air dried aerial parts of plant was macerated with 80% methanol (4 L ×5) at room temperature and was concentrated with an evaporator in low temperature (40 °C). The crude extract (160 g) was sequentially fractionated with petroleum ether, chloroform, ethyl acetate, and methanol (each 4 L) respectively, to obtain four fractions.Acetylcholinestrase (AChE) inhibitory activity of FractionsAChE inhibitory effect of petroleum ether, chloroform, ethyl acetate, and methanol fractions were carried out by the Ellman′s method . Galanta50) was calculated , 20 µL DTNB  20 µL of sample solution, 2 µL of AChE solution were added into 96 micro plates and were incubated for 15 min at 25 °C and then acetylcholine iodide (100 µL of 0.05 mM water solution) was added as substrate. Acetylcholinestrase activity was evaluated by measuring the absorbance at 412 nm by an ELISA plate reader for 3.0 min at 25 °C. The concentration of the compound which inhibited 50% of acetylcholinestrase activity  was prepared and added to 1 mL of different concentrations of sample solution in methanol . The mixture was kept in the dark for 30 min at room temperature. The antioxidant activity was measured by Shimadzu, UV/VIS model 160A spectrophotometer at 517 nm.The DPPH method is based on the free radical scavenging activity . briefly50 values were calculated as means ± SD and butylated hydroxytoluene (BHT) was used as positive control.All the measurements were done in triplicate. The ICIsolation and identification of compounds1-M2-M3). Fraction M1 (1.5g) was re-chromatographed over a Sephadex LH-20 and eluted with MeOH-H2O (6:4) to collect compound 1 (8 mg). Fraction M2 (700 mg) was run on a C18 reversed-phase  column chromatography using MeOH-H2O (8:2) as eluent, and compound 2 (11 mg) was achieved.The potent methanol fraction (20 g) was subjected to Sephadex LH-20  column chromatography using 100% methanol as eluent. This was followed by increasing water up to 50% resulting in 3 fractions  and solvent system BAW . For further purification the subfractions were subjected to PC on Whatman No.1 with 15% acetic acid, then, they were re-chromatographed over sephadex LH-20 to earn compound 3 , compound 4 , and compound 5  .The isolated compounds were identified with different spectroscopic methods  as quercetin, astragalin, caffeic acid, salicylic acid and p-coumric acid  compared to the other fractions. Quercetin exhibited the highest free radical scavenging activity with IC50 = 10.24 ± 1.3 µg/mL compared to BHT as positive control (IC50=19.5 ± 2.8 µg/mL) while the lowest activity belonged to salicylic acid (IC50=114.57 ± 1.0 µg/mL). Regarding the inhibition of acethylcholinestrase, methanol fraction showed inhibitory activity (205.10 ± 3.3 µg/mL) among other fraction and caffeic acid (IC50 = 12.06 ± 2.01 µg/mL) was stronger than other isolated compounds.The antioxidant activity and AChE inhibitory assay of the total extract, fractions, and compounds are listed in the 1H- NMR, 13C-NMR, and UV) and were compared with the reported data, including quercetin . Finally, five compounds were isolated and identified with different spectroscopic methods . and 3-methoxy quercetin (IC50=37.9 µg/mL) isolated from Agrimonia had remarkable AChE inhibitory effect (In previous study, quercetin(ICy effect . Also, ey effect . In anoty effect .Leutea avicennia, and showed this plant is rich of phenolic compounds that exhibited appropriate antioxidant and anti- acetylcholinesterase properties. Caffeic acid is one of the substances which showed potent activity in both assays, therefore methanol fraction of L.avicennia and their components probably could be used in treatment of neurodegenerative disease, so in-vivo animal model is worthwhile and recommended. This study was conducted for the first time on


Current studies have demonstrated that DODAC/PHO-S (Dioctadecyldimethylammonium Chloride/Synthetic phosphoethanolamine) liposomes induces cytotoxicity in Hepa1c1c7 and B16F10 murine tumor cells, with a higher proportion than PHO-S. Therefore, our aim was to evaluate the potential of DODAC/PHO-S to elucidate the mechanism of cell death whereby the liposomes induces cytotoxicity in hepatocellular carcinoma Hepa1c1c7, compared to the PHO-S alone.Liposomes (DODAC/PHO-S) were prepared by ultrasonication. The cell cycle phases, protein expression and types of cell’s death on Hepa1c1c7 were analyzed by flow cytometry. The internalisation of liposomes, mitochondrial electrical potential and lysosomal stability were also evaluated by confocal laser scanning microscopy.2/M phases, and this treatment was significantly more effective to promote cell death by apoptosis. There also was a decrease in the mitochondrial electrical potential; changes in the lysosomes; nuclear fragmentation and catastrophic changes in Hepa1c1c7 cells. The liposomes additionally promoted increases in the expression of DR4 receptor, caspases 3 and 8, cytochrome c, p53, p21, p27 and Bax. There was also a decrease in the expression of Bcl-2, cyclin D1, CD90 and CD44 proteins.After treatment with liposomes (DODAC/PHO-S), we observed a significant increase in the population of Hepa1c1c7 cells experiencing cell cycle arrest in the S and GThe overall results showed that DODAC/PHO-S liposomes were more effective than PHO-S alone, in promoting cytotoxicity Hepa1c1c7 tumor cells, activating the intrinsic and extrinsic pathways of programmed cell death. Despite great advances in the research and the development of new therapeutic strategies, cancer remains one of the leading causes of death worldwide. In 2014, there were an estimated 1,665,540 new cancer cases diagnosed and 585,720 deaths were expected in the United States of America in 2014, and it is expected to increase to over 24 million by 2035 [With regards to the limiting factors of the therapies currently available for the treatment of cancer, new treatments that are more effective and less harmful are necessary. Therefore, antineoplastic phospholipids (AFTs) and lipid precursors have emerged as a promising new classes of antitumor agents that do not target the DNA, however they change the plasma membrane turnover, inducing cell death, with a high selectivity for cancer cell , 4. Edel2/M cell cycle arrest [Synthetic phosphoethanolamine (PHO-S), an lipid precursor, amino-ethyl phosphoric ester, has been previously synthesized by our group –13. We de arrest , 12.In vitro studies demonstrated that PHO-S induced cytotoxicity and apoptosis via mitochondrial pathways, in leukemia cells. The results showed that PHO-S was able to provide antiproliferative effects on acute promyelocytic leukemia (APL) cell lines. PHO-S demonstrated its antiproliferative effect on APL cell lines, decreasing CD177+ and Gr-17+ in immature myeloid cells in bone marrow, spleen and liver . Additio50% values significantly lower than PHO-S treatment. It was observed that Hepa1c1c7 cells display greater sensitivity to the DODAC/PHO-S formulation when compared with B16F10 and HUVEC cells. However, the molecular mechanism responsible for the anti-tumor properties of DODAC/PHO-S has not been demonstrated [Recently, PHO-S was encapsulated in DODAC (Dioctadecyldimethylammonium Chloride) liposomes by our group and the liposomes were physico-chemically characterised , 15. In nstrated , 15. ConLiposomal formulation DODAC/PHO-S were formulated (1:1) in water, in accordance with procedures previously published , 12, 13.2. The cell viability was verified using Trypan Blue exclusion test. Hepa1c1c7 was chosen for the study because of its easy reproduction in vivo studies.Hepa1c1c7 murine hepatocellular carcinoma (ATCC® CRL 2026) was cultured in αMEM medium  and supplemented with 10% fetal bovine serum in a humidified incubator at 37 °C and 5% CO5 cells/well (80 to 90% confluence) were treated with PHO-S (0.3–2.0 mM), DODAC/PHO-S 1:1 (0.3–2.0 mM), and empty DODAC (0.3–2.0 mM), for 12 h. After treatment, the cells were washed with PBS  and fixed by the addition of 3 ml of ice cold 70% ethanol. The cells cycle was evaluated after cell incubation with 1.8 μg/ml propidium iodide (PI) solution , for 30 min. in the dark. The analysis was performed using Biosciences FACSCalibur flow cytometer  and 10,000 events were collected. Subsequently, the data was analysed by ModFit LT 3.2 software .The Hepa1c1c7 cells at a cell density of 1 × 105 cells/well in 6 well plates. After 24 h, the cells were treated with PHO-S and DODAC/PHO-S, for 12 h. Then, the cells were washed three times with PBS at 4 °C and resuspended in 100 μL of PBS, following this, were incubated with 4 μg/mL of AnnexinV  and 1.8 μg/μL of PI , for 1 h at 37 °C.The Hepa1c1c7 cells were plated at 1 mL 1 × 10Following incubation, the cells were centrifuged at 2000 rpm for 10 min at 4 °C and resuspended in 400 μL of binding buffer 1×, provided by the manufacturer. The analysis were performed using Biosciences FACSCalibur flow cytometer  and 10,000 events were collected. Following this, the data was analysed using ModFit LT 3.2 software .5 cells/well, the cell density reached 80 to 90% confluence), were treated with PHO-S (0.3–2.0 mM), DODAC/PHO-S 1:1 (0.3–2.0 mM), and empty DODAC (0.3–2.0 mM), for 12 h, were washed with PBS and resuspended in FACS buffer with 2.5% paraformaldehyde for 1 h. After washing, cells were again resuspended in a primary antibody specific for the proteins anti-CD44, anti-CD90, anti-p53, anti-p21, anti-p27, anti-Bax, anti-Bcl-2, anti-caspase-3, anti-caspase-8 ; anti- cytochrome c, anti-DR4  and anti-cyclin D1 , at a concentration of 1 μg/ml at 4 °C, for 1 min. The corresponding isotope antibody was used as a negative control and as a secondary antibody was used Goat anti Mouse IgG (H/L): FITC . The cells were pelleted, washed twice with PBS, then, fluorescence-activated cell sorting (FACS) analysis was performed on BD Biosciences FACs Calibur flow cytometer  using Cell Quest and Win MDI 2.9 softwares.The analysis of protein expression were performed as previously published , 16. In DODAC/PHO-S liposomes 1:1 were prepared according to the protocol as described previously, adding 0.25 mol%, in relation to DODAC, of the fluorescent probe (Dil): 1,1′-dioctadecyl-3,3,3′,3′- tetramethylindocarbocyanine perchlorate .5 cells/well, the cell density reached 80 to 90% confluence), were plated on sterile glass slides. After adhesion, cells were treated with DODAC/PHO-S fluorescent liposomes (1:1) 0.3 and 2.0 mM. Then, cells were fixed by adding ProLong® and incubated at − 20 °C, in the dark. The analysis was realised by confocal laser scanning microscopy .Aliquots of Hepa1c1c7 cells  at 0.3 and 2.0 mM, for 6 h. Then, the incubation with rhodamine-123 and analyses were perfomed as previously published [The analysis of the ΔΨm was realised by confocal laser. The Hepa1c1c7 cells were plated on sterile glass slides in 24-well plates, at concentration of 10ublished , 16.5 cells/well. After 24 h, the cells were treated with PHO-S and DODAC/PHO-S, for 6 h. The cells were then incubated with OA and analysed as previously published [The Hepa1c1c7 were plated on sterile glass slides in 24-well plates, at concentration of 1 × 10ublished , 16.p-values * < 0.05, ** < 0.01, and *** < 0.001 are statistically significant.Statistical analyses were perfomed as previously detailed , 16. In The compounds were evaluated for their ability to induce changes in the cell cycle. The Hepa1c1c7 cells were treated with PHO-S, DODAC/PHO-S (1:1) at a concentration of 0.3 and 2.0 mM, for 12 h. The exposure time was determined by taking into account the results of the cytotoxicity, as previously published, and performed simultaneously with this study , 14.1 phase, among the treatments and control group , presenting a percentage of 53.2 ± 1.2% (0.3 mM) and 48.9 ± 3.5% (2.0 mM)  Fig. .Fig. 1An2/M phase showed a significant increase when treated with DODAC/PHO-S liposomes, with values of 14.3 ± 1.7% (0.3 mM) and 14.3 ± 2.1% (2.0 mM)  and 9.1 ± 0.8% (2.0 mM) Fig. . The permM) Fig. .The Hepa1c1c7 cells were treated with PHO-S, DODAC/PHO-S (1:1) and DODAC (0.3–2.0 mM), for 12 h. Subsequently, cells in apoptosis, late apoptosis and necrosis were quantified by flow cytometry using FITC Annexin V/PI apoptosis detection Kit. The treatment with PHO-S (0.3 and 2.0 mM) significantly increased the number of apoptotic and necrotic cells, with values of 13.9 ± 3.4% (0.3 mM) and 7.5 ± 0.3% (0.3 mM), respectively. In the highest concentration (PHO-S 2.0 mM), the percentage of apoptotic cells was 21.2 ± 5.4% and 7.5 ± 0.3% of necrotic cells. Only in the highest concentration (2.0 mM) was it possible to observe an increase in the number of cells in late apoptosis, presenting a percentage value of 9.7 ± 4.6% Fig. .Fig. 2EvThe treatment with DODAC/PHO-S (0.3 and 2.0 mM) significantly increased the number of necrotic cells, with values of 9.6 ± 0.14% (0.3 mM) and 12.3 ± 0.3% (2.0 mM). There was also an increase in late apoptosis, with a percentage of 30.4 ± 0.4% (0.3 mM) and 31.0 ± 1.3% (2.0 mM). The treatment with DODAC (0.3 mM) increased the number of necrotic cells to a lesser extent when compared to the other treatments, with a percentage of 6.7 ± 0.9% Fig. .The internalisation of DODAC/PHO-S liposomes with fluorescent probe (Dil), was evaluated by confocal laser scanning microscopy. The Hepa1c1c7 cells were treated with DODAC/PHO-S liposomes (0.3 and 2.0 mM). The fluorescently labeled liposomes were observed and easily distinguishable by their green colour. After treatment, the presence of the DODAC/PHO-S fluorescently labeled liposomes (0.3 and 2.0 mM) were observed in the cytoplasm of the Hepa1c1c7 cells, confirming that internalisation of the liposomes occurred, in a short period of time  and DODAC (0.3 and 2.0 mM), for a period of 6 h. The cells treated with PHO-S and DODAC/PHO-S, demonstrated a significant decrease in ΔΨm, when compared to the control group. The cells were treated with DODAC (0.3 and 2.0 mM) and presented an attenuation of fluorescent intensity, compared to the control group. However, the positive staining demonstrated that cells had functional mitochondria Fig. .Fig. 4PhThe Hepa1c1c7 cells were treated with PHO-S, DODAC and DODAC/PHO-S liposomes (2.0 mM), for a period of 6 h. The effects of these compounds in lysosomes were then evaluated with AO (acridine orange) assay, by confocal laser scanning microscopy. The nuclear DNA was labeled with DAPI (blue emission). For this assay, only the highest concentration (2.0 mM) was used, because it showed the greatest effectiveness in inducing cytotoxicity.The control group cells demonstrated intense labeling of some lysosomes with a granular appearance, evenly distributed into cytoplasm Fig. . The samHepa1c1c7 tumor cells were treated with PHO-S (2.0 mM), DODAC and DODAC/PHO-S (0.3 and 2.0 mM), for 24 h. The expression of proteins involved in checkpoint, cell adhesion, regulation of the cell cycle and apoptotic pathway were quantified after the treatment. These proteins are as follows: TRAIL receptors 1 (DR4), active caspases-3 and -8, free cytochrome c, p53, p21, p27, CD44, CD90, Bax, Bcl-2 and cyclin D1.The Hepa1c1c7 cells treated with 2.0 mM DODAC/PHO-S, showed a significant increase in the expression of DR4 receptor, active caspases-3 and 8 and cytochrome c, with percentage values of 11.4 ± 0.1% Fig. , 13.5 ± Bax protein had an expression increase after treatment with 2.0 mM PHO-S (9.1 ± 1.2%), DODAC/PHO-S (11.9 ± 0.1%) and DODAC (6.5 ± 0.2%) Fig. . In contThe expression of p21 protein increased with 2.0 mM PHO-S (1.9 ± 0.1%); DODAC/PHO-S (0.3 mM - 4.3 ± 0.3% and 2.0 mM - 25.5 ± 0.7%) and empty DODAC (0.3 mM - 1.6 ± 0.3% and 2.0 mM - 18.8 ± 0.5%) Fig. . P27 proIt was noticed that p53 protein increased with 2.0 mM DODAC/PHO-S (38 ± 1.7%) and 2.0 mM empty DODAC (22.9 ± 1.3%) Fig. . In contSeveral liposomal formulations containing antitumor agents are already clinically available or are under advanced stages of development, for example, Doxil® , Lipoplatin® (cisplatin) DepoCyte® (cytarabine), DaunoXome® (daunorubicin), MBP-426 , BIND (docetaxel) and MM30 (doxorubicin) –20. Cati50% values significantly lower than PHO-S treatment. It was also observed that the liposomes were able to induce their highest level of cytotoxicity in Hepa1c1c7 tumor cells [In a recently published study, PHO-S was encapsulated in DODAC liposomes by our group , 15. Theor cells , 13. Thi2/M phase [2/M phases. The treatment at the lowest and highest concentrations provided the same effect on the cell population, in these phases in the cycle. Therefore, demonstrating once again the sensitivity of Hepa1c1c7 cells to the treatment with DODAC/PHO-S liposomes, in relation to the effects observed in another study with B16F10 cells, performed simultaneously [Recent studies have demonstrated that PHO-S inhibits cell proliferation and migration, leading to the accumulation of B16F10 and MCF-7 cells in the G/M phase , 13. As aneously , 15. The2/M phase, which is a specific targeted antitumor effect mediated by PHO-S [The treatments performed only with empty DODAC liposomes resulted in a lesser increase in the number of Hepa1c1c7 cells in the S phase, however it did not increase the number of cells stopped in the Gby PHO-S . This deby PHO-S , 22.1/S checkpoint), at the entrance into mitosis (G2/M checkpoint), as well as during replication (intra-S-phase checkpoint) [The induction of cell cycle arrest after DODAC/PHO-S treatment is very important, as one of the aims of the development of novel anticancer drugs is to formulate compounds that can act on cell cycle checkpoints, which are responsible for controlling cell cycle progression. The checkpoints occur at the entrance into the S phase (Gckpoint) . A largeckpoint) . To evalckpoint) , 27. Recckpoint) , 28, 29.The treatments with empty DODAC liposomes did not trigger cell death by apoptosis, with only a small increase in the number of cells killed by necrosis (with treatment at lower concentrations).2/M phase. The expression of proteins involved in checkpoint, apoptosis and cell cycle regulation was greater in cells treated with the 2.0 mM DODAC/PHO-S than in the cells treated with PHO-S only. Thus indicating that treatment with the formulation is more effective than treatment with PHO-S.In Hepa1c1c7 cells, the liposomal formulation promoted cell death by inducing the activation of the intrinsic death pathway modulated by p53 activity. This protein is likely to have induced the activity of Bax and p21 proteins, and consequently the decreased expression of cyclin D1 protein, as shown in our results. Moreover, an increase of the pro-apoptotic p27 protein and decrease of the anti-apoptotic Blc-2 protein were observed. These results demonstrate the path or route that the DODAC/PHO-S liposomes possibly uses in order to induce cellular arrest in the GThere was no significant increase in p53 protein in the Hepa1c1c7 cells treated with PHO-S 2.0 mM. This effect was not observed probably by the exposure time of the cells to PHO-S because the expression of the proteins, which are regulated by transcriptional activation of p53, such as p21 and Bax, was changed. However, in the cells treated with the DODAC/PHO-S, the regulatory effect on p53 was sustained. This demonstrates the effectiveness of DODAC/PHO-S mediating mechanisms that cause cell cycle arrest and consequently die.2/M phase involves the regulation of Cdc2 kinase, which is essential for cells to enter mitosis [It is known that the mechanism through which p53 protein regulates the transition between the S and G mitosis . Cdc2 is mitosis –32. Simi mitosis .1 to S. In many neoplasms, the nuclear levels of p27 are reduced [The significant increase in the expression of protein p27 in the treatment with the DODAC/PHO-S (2.0 mM) shows an important action mechanism of this formulation, because this protein acts as an inhibitor of kinases that are dependent on cyclins and thereby negatively regulates the progress of the cell cycle from G reduced . The dow reduced . The indConsidering the importance of cyclin D1 reduction in antitumor treatment, 2.0 mM liposomal formulation proved effective for this purpose because caused a decrease in cyclin expression in Hepa1c1c7 cells. Recent studies demonstrated the ability of PHO-S in reducing mRNA of cyclin D1 in MCF-7 cells and in reducing the expression of CDK4/6 in the model H460/Bcl-2 cells , 28. TheThe checkpoint mechanism mediated by p53 protein may promote the repair of damaged DNA or lead to the activation of the mechanism of programed cell death. This is mediated by pro-apoptotic proteins such as Bax, PUMA and Noxa and decreased expression of anti-apoptotic proteins, including Blc-2. It was observed that treatment with DODAC/PHO-S liposomes promoted the activation of programed cell death mechanism, since there was an increase in the expression of Bax and decrease in the Bcl-2 proteins.The treatments with empty DODAC liposomes promoted an increase in the expression of p53, p21 and Bax, and a decrease in Blc-2 proteins, though to a lower percentage that the treatment with the DODAC/PHO-S liposomes. DODAC can interact with the DNA and other negatively charged molecules, it is probably by inducing p53 protein activity and therefore the activity of p21, Bax and Blc-2. However, the use of this carrier was not cytotoxic to normal HUVEC endothelial cells, under the same proportions as compared to the B16F10 and Hepa1c1c7 tumor cell lines . So, proThe induction of cell death by the intrinsic pathway in Hepa1c1c7 cells exerted by the DODAC/PHO-S (0.3 mM and 2.0 mM), was confirmed by the evaluation of ∆Ψm. The cells treated with the liposomal formulation decreased the ∆Ψm. Cells treated with only empty DODAC liposomes showed functional mitochondria. Recent study demonstrated that MCF-7 cells treated with PHO-S displayed disorders in their ∆Ψm .After the treatment with liposomes DODAC/PHO-S the protein expression of extrinsic pathway was also evaluated, since PHO-S has been stated as a compound capable of forming a complex with DR4 and DR5 receptors. This represents a mechanism by which the AFTs are internalized into the cytoplasm by forming coaggregation with the TNF family receptors, in particular FasL , 35. In The increased expression of pro-apoptotic proteins, as caspase 3 and 8, in the cells treated with DODAC/PHO-S, demonstrated that the encapsulation of PHO-S was able to potentialise its antitumor activity. Caspases are a family of cysteine proteases that serve as primary effectors during apoptosis, and have been categorised into two groups: initiator caspases  and effector caspases . The cleavage of different substrates by caspases is mechanically responsible for the morphological and biochemical characteristics of apoptotic cell death , 37. In Recent studies have shown that apoptosis triggered by TRAIL in hepatocellular carcinoma cells  was associated with the lysosomal permeabilization and consequently the increase of cathepsin B in the cytosol. The internalisation of TRAIL and its receptors can enable factors that induce permeabilization of the lysosomal membrane and culminate in the release of cathepsin B to the cytosol –40. In tThe internalisation of the liposomes DODAC/PHO-S (0.3 and 2.0 mM), with only 3 h of treatment, was confirmed by confocal laser microscopy. It was observed that the liposomes are rapidly internalised, which makes it a great tool for antitumor therapy. After 6 h, it was still possible to observe liposomes in the cytosol of the cells. However, within this treatment time some cells already presented morphological changes corresponding to the apoptotic process.+ were present. The CD44+/CD90+ cells showed a more aggressive phenotype than the CD90+/CD44− cells and formed metastatic lesions in the lungs of immunodeficient mice. The blockade of CD44 expression avoided the local and metastatic formation of tumor nodules by CD90+ cells [The potential of liposomes DODAC/PHO-S in inducing cell death in Hepa1c1c7 cells, was confirmed by significant reduction in the expression of CD90 and CD44 markers. It demonstrated the ability of this formulation in the promotion of cytotoxicity in these cells. The cells treated with empty DODAC liposomes (0.3 and 2.0 mM) did not significantly reduce the expression of the marker CD90, therefore, these cells still display tumorigenic potential. Studies of patients with liver and in 91.6% of the blood samples taken CD45 and CD900+ cells .The set of results demonstrate that electrostatic interaction between DODAC/PHO-S and cell membrane maximises the antitumor effects mediated by PHO-S. Thus, the overall results show that the liposomal formulation DODAC/PHO-S was effective in promoting cytotoxicity in Hepa1c1c7 cells, activating the intrinsic and extrinsic pathways of programmed cell death.


N2) reaction between hydride and methane, for which we have obtained reference energies at the coupled cluster toward full configuration-interaction limit (CC-cf/CBS). Several wavefunction  and density functional methods are compared for their reliability regarding these reference data.We report here a benchmark study on the bimolecular nucleophilic substitution (S N2) reactions play an important role in organic chemistry and in biochemistry (DNA replication mechanism). Interestingly, there is a profound solvent effect present which has a major effect on the reaction barriers and intermediates. For example, the prototypical SN2 reaction of chloride with methyl chloride shows in the gas phase a double-well potential , where N2 reaction possible, between hydride and methane:Here we have studied the smallest S3, [3, [3, [2, [3, [For this reaction, we have been able to perform coupled cluster calculations  up to th3, ,51 PBE0,3, ,53,54 PB3, [3, ,53,54 PW3, [3, ,56 BP86,3, [3, ,58 revPB3, [3,  OPBE, [53, [3, ,60,61 OL3, [3,  B3LYP, [3, [3, ,64 B3LYP [3, [3, ,64 BLYP, [3, [3, ,62 B2PLY [3, [3,  M06, [66 [3, [3,  B97, [68 [3, [3,  B97-D2,  [3, [2,  TPSS-D3, [2, [3,  TPSSh, [ [2, [3,  SSB-D, [ [2, [3,  S12g, [4 [2, [3,  S12h, [4 [2, [3,  CAM-S12h [2, [3,  CAM-B3LY [2, [3, ), among  [2, [3,  and the  [2, [3, .N2 reaction of H−+CH4 was studied using both wavefunction and density functional methods. The reaction proceeds from the reactants  even though the size of the nucleophile is probably much smaller here [note however that Pauling [− (2.08 Å) than for Cl− (1.81 Å), while Frecerreported a larger ionic radius for H [−) and 2.30 Å (Cl−) respectivelythrough Monte Carlo obtained values of 2.28 Å  to −0.69 kcal·mol−1 at CCSD, −0.90 kcal·mol−1 at CCSD(T), −0.93 kcal·mol−1 at CCSDT and −1.22 kcal·mol−1 at CC-cf. Likewise, the barrier continues to drop from 62.6 kcal·mol−1 at RHF to 49.4 kcal·mol−1 at CCSDT, and reaches 49.0 kcal·mol−1 at CC-cf. It should be noted that the perturbative triples approach in CCSD(T) gives a good approximation for the CCSDT energies . Based on the extrapolation towards full-CI and complete basis set with CC-cf at increasingly larger basis sets (CC-cf/CBS), final reference energies of -1.20 kcal·mol−1 (RC) and +48.90 kcal·mol−1 (TS) are obtained.The results of i.e., CCSD, CCSD(T), CCSDT and CC-cf, together with RHF and MP2 (see −1), and overestimates the barrier by ca. 3−4 kcal·mol−1. MP2 works in this respect even better with deviations from CC-cf that are twice as small, even though the computational effort is more or less the same as CCSD. As already noted often before, RHF cannot be trusted for these energy profiles as it gives barriers which are too large.The results obtained with the different levels of coupled cluster method,  MP2 see  indicate−1 with the largest basis set aqz), and a substantial barrier . Nevertheless, the results for the 28 density functionals show quite a diversity in the accuracy for the energy profile, even though some general trends are obvious: they tend to overestimate the RC well depth, and  underestimate the reaction barrier. The least reliable functional is not surprisingly LDA, which for the RC predicts a well depth of −2.74 kcal·mol−1, and places the TS at +34.16 kcal·mol−1 . Early GGA functionals  for the most often used hybrid functionals . The recently developed S12h and M06−2X bring the deviation from CC−cf down to ca. 2 kcal·mol−1, while excellent results  are obtained with four functionals: B2PLYP , M06−L , B97−3  and CAM−S12h . Of these four, two give an excellent description of the RC well depth: M06−L  and CAM−S12h , while the other two underestimate it slightly  or show a non-existent RC . The non−existence of a RC happens also for OPBE and OLYP with the augmented basis set , where the optimization proceeds towards reactants.All density functionals show the correct energy profile with a shallow well for the RC  iAll methods used here confirm the early character of the RC, with a distance between the nucleophile (Nu) and the central carbon observed within the range of 2.97 Å (B2PLYP) to 4.73 Å (RHF) see . These tThe comparison of the energy profiles for the different methods is of course influenced to some extent by the different geometries used with the different methods. Therefore, and in order to make an honest comparison between the different methods we also performed single-point energy calculations using the CCSDT/atz geometries. Given in −1 . However, the typical deviation is less than 0.1 kcal·mol−1 ; for instance, PBE−D3 shows differences of 0.06 and 0.08 kcal·mol−1 for the RC and TS energies with the two different geometries. All of this indicates that the energy surface is quite flat, as was already obvious from By comparing the results from 2:An alternative reaction is possible in which the hydride abstracts a proton from methane. This leads to the formation of a methyl anion and H−1 at S12h/aqz. This pathway is beyond the scope of the present investigation which focuses on the thermoneutral identity SN2 reaction.This alternative process is however associated with an endothermic reaction energy of +21.35 kcal·molabbreviated as dz, tz and qz respectively) and aug−cc−pVXZ . The continued-fraction approximant [All wavefunction based calculations  have been performed with the Coupled-Cluster techniques for Computational Chemistry (CFOUR) ,82 progrroximant  for obtaroximant  version N2) reaction possible: H−+CH4→ CH4+H−, for which we obtained reference data at the near-full-CI coupled cluster limit using the continued−fraction approximant (CC-cf). Unlike typical SN2 reactions in the gas phase, which usually show a double-well potential, the current reaction shows an energy profile that resembles more the unimodal profile of the SN2 reaction in solution, with a relatively shallow reactant-complex well of only −1.19 kcal·mol−1 and a high barrier amounting to 48.87 kcal·mol−1. All other computational methods also clearly show the steep reaction barrier that needs to be overcome , and the very shallow wells of the (ion-dipole) reactant complex. All density functionals have the tendency to underestimate the reaction barrier, while for the RC the deviations compared to CC-cf are much smaller . Excellent results have been obtained with B97-3, M06-L and the newly developed CAM−S12h functional.We have performed a benchmark study on the smallest bimolecular nucleophilic substitution (S


PBMCs were stimulated with TLR agonists LPS (TLR4), R848 (TLR7/8), and Pam3CSK4 (TLR1/2), and then infected with HIV NL4-3 AD8. Multiplexed ELISA was used to measure 28 cytokines in cell culture supernatants. Flow cytometry was used to measure the activation state (CD38 and HLA-DR), and CCR5 expression on CD4+ and CD8+ T cells. Although TLR agonists induced higher cytokine and chemokine secretion, they did not significantly activate CD4+ and CD8+ T cells and showed decreased CCR5 expression relative to the unstimulated control. Despite several classes of inflammatory cytokines and chemokines being upregulated by TLR agonists, CD4+ T cells were significantly less infectable by HIV after TLR4-stimulation than the unstimulated control. These data demonstrate that the inflammatory effects that occur in the presence TLR agonist stimulations do not necessarily translate to the activation of T cells. Most importantly, the finding that TLR4-stimulation reduces rather than increases susceptibility of CD4+ T cells to HIV infection in this in vitro system strongly suggests that the increased chemokine and possible antiviral factor expression induced by these TLR agonists play a powerful although complex role in determining HIV infection risk.Genital inflammation is associated with increased HIV acquisition risk. Induction of an inflammatory response can occur through the recognition of pathogenic or commensal microbes by Toll-like receptors (TLRs) on various immune cells. We used a  HIV and AIDS is a global epidemic that affects approximately 37 million people worldwide, with an additional 1.8 million new HIV infections documented in 2017  stimulated with LPS induced the production of IL-1β, TNF-α, IL-6, and IL-22  Biomedical Research Ethics Committee (BREC). All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the UKZN BREC (BE433/14).C10 media was used for all cell culture experiments. C10 media consisted of RPMI 1640 with L-glutamine  containing 10% FCS , 2% L-glutamine, 1% HEPES, 1% NaPy, 1% NEAA . Media was sterile filtered through the Filtermax 500 ml . IL-2 , was added to C10 media prior to use at a final concentration of 0.01 μg/ml.The TLR agonists LPS TLR4), R848 (TLR7/8), and Pam3CSK4 (TLR1/2)  concentrations were previously optimized in TLR titration experiments. As no significant differences were observed in HIV infections  or cytok, R848 T  or stimulated immediately after plating with TLR agonists or PHA, which was used as a positive control. Following stimulation, the PBMCs were cultured at 37°C 5% CO2 for 48 h. Following this incubation, the contents of each well was collected into 15 ml falcon tubes and an aliquot of 500 μl was removed for multiplexed ELISA (culture supernatants) and flow cytometry (PBMCs) for the day 3 time-point . The 15 ml falcon tubes were centrifuged, supernatants were discarded, and media replacement was performed with fresh C10 media. PBMCs were plated at 1 × 106 cells/ml into 24-well cell culture plates, no further TLR or PHA stimulations were performed. Subsequently, HIV infection was done by the addition of 250 μl at a 1:20 dilution of the HIV-1 NL4-3 AD8 viral stocks at a MOI of 0.9. PHA and unstimulated uninfected wells were treated with 250 μl C10 media. Plates were incubated at 37°C 5% CO2 for 48 h, whereupon multiplexed ELISA (culture supernatants) and flow cytometry (PBMCs) was performed for the day 5 time-point (post HIV infection).The cell culture and HIV infection protocol used in this study was adapted from previous studies , 39. BloPBMCs were centrifuged at 3,500 rpm for 5 min to pellet the cells and remove soluble HIV, and the cell culture supernatants were stored at −80°C for cytokine quantification. PBMCs were washed with sterile PBS supplemented with 2% FCS and then stained with 100 μl extracellular staining cocktail, fixed, and then stained with 100 μl intracellular staining cocktail. Data was acquired by flow cytometry on a BD LSR Fortessa . Data analysis was performed using FlowJo v10.4.1 software , according to the gating strategy . For theThe concentrations of 28 cytokines were assessed from cell culture supernatants using the Bio-Plex Pro Human Cytokine Group I 27-Plex Panel  and the Magnetic Luminex® Assay IL-1α Singleplex Kit (Research and Diagnostic (R&D) systems Inc., Minneapolis, Minnesota, USA) as per manufacturer's instructions. Data was acquired on the Bio-Plex® 200 system . Optimization of standard curves were performed automatically using the Bio-Plex manager software version 6.1 . Values with coefficients of variation <20% and with observed recoveries between 70 and 130% were considered reliable. Values that were below the detectable limit were assigned half of the lowest limit of detection value (LLOD), while values that were above the detectable limit were assigned double the highest limited of detection (HLOD) value.10 transformation and is displayed as mean concentration (Log10 pg/ml) ± standard deviation (SD). To understand the effect of various TLR agonists on cytokine expression, heat maps were generated by performing a single linkage hierarchical cluster analysis using R version 3.3.3 statistical software . Radial spider plots were created using Microsoft Excel© 2013 software .Statistical analyses and graphical representation of data was performed using the GraphPad Prism version 7.02 software for windows . For comparisons of cellular activation markers CD38, HLA-DR on CD4+ and CD8+ T cells, between stimulated conditions and the unstimulated control, a repeated measures two-way ANOVA with a Dunnett's multiple comparisons test was performed. Similarly, for CCR5 and cytokine comparisons, an ordinary one-way ANOVA with Dunnett's multiple comparison test was performed. Cellular activation results are displayed as mean percentage (%) ± standard deviation (SD) of CD4+ or CD8+ T cells. Cytokine data was normalized by Logp > 0.05) at day 3  or day 5 (post infection) (p < 0.05). Similarly, on day 5 and irrespective of infection status, PHA induced significantly elevated expressions of CD38+HLA-DR+ and CD38+HLA-DR-, but not CD38-HLA-DR+ on CD4+ T cells compared to the unstimulated infected control (p ≤ 0.0001)  . However 0.0001) . Represep < 0.05), while PHA significantly increased the CCR5 expression (16.6 ± 6.9% of CD4+ T cells) (p ≤ 0.0001) at day 3 (p = 0.0003), although both conditions had significantly higher CCR5 expression than the unstimulated but HIV-infected control (8.8 ± 2.9% of CD4+ T cells)  compared to the unstimulated control (6.9 ± 2.9% of CD4+ T cells) (at day 3 . Of noteT cells) . Represep < 0.01), this did not translate to a significant increase in any of the activated phenotypes (p < 0.05) on day 3 (p < 0.05), compared to unstimulated cells (p ≤ 0.0001) (p ≤ 0.0001) compared to the unstimulated infected control  . PHA, ir control . Represep < 0.05), R848 (8.1 ± 3.8% of CD8+ T cells) (p < 0.05) or Pam3CSK4 (6.7 ± 3.2% of CD8+ T cells) (p < 0.01) on day 3 (p < 0.01) on day 3. Only LPS (13.1 ± 7.6% of CD8+ T cells) maintained significantly lower CCR5 expression on CD8+ T cells than the unstimulated infected control (21.6 ± 7.8% of CD8+ T cells) on day 5 (p < 0.05) (CCR5 expression by CD8+ T cells was significantly lower than the unstimulated control (14.3 ± 7.8% of CD8+ T cells) with LPS (7.2 ± 3.8% of CD8+ T cells) (on day 3 . Convers < 0.05) . RepreseUnsupervised hierarchical clustering analysis and Radial spider plots were used to evaluate cytokine production by PBMCs in response to stimulation with various TLR agonists .p ≤ 0.0001 and p ≤ 0.0001, respectively) compared to the unstimulated control (p < 0.001), however these levels were generally lower than those observed with LPS or R848 , predicted >3-fold increased risk for HIV acquisition ( control . Pam3CSK or R848 .p ≤ 0.0001), R848 (p ≤ 0.0001), Pam3CSK4 (p ≤ 0.0001), and PHA conditions (p ≤ 0.0001) compared to the unstimulated HIV-infected control (p < 0.05) (p < 0.05)  , while I < 0.05) . Relevanp ≤ 0.0001), MIP-1α (p ≤ 0.0001), MIP-1β (p ≤ 0.0001), MCP-1 (p < 0.05), and RANTES (p < 0.05) compared to the unstimulated control (p < 0.01)  . R848 ap < 0.01) .p ≤ 0.0001), R848 (p ≤ 0.0001), Pam3CSK4 (p ≤ 0.0001) and PHA conditions (p < 0.001) compared to the unstimulated infected control (p < 0.01), R848 (p ≤ 0.0001), or Pam3CSK4 (p < 0.001) significantly increased MIP-1β compared to the unstimulated infected control (p < 0.01) (p ≤ 0.0001) (p < 0.05)  . Interes 0.0001) . Concent < 0.05) .p < 0.05), R848 (p < 0.05) and the PHA uninfected (p < 0.01) conditions compared to the unstimulated infected control , R848 (p < 0.01), and PHA stimulation (p < 0.001) compared to the unstimulated controls, while only IL-17 was significantly elevated with Pam3CSK4 (p ≤ 0.0001) (p < 0.05) (p ≤ 0.0001)  . Interes 0.0001) . PHA, bu < 0.05) . The ant 0.0001) .p ≤ 0.0001), R848 (p < 0.05), Pam3CSK4 (p < 0.001), and PHA-stimulated and uninfected (p < 0.001) and infected (p ≤ 0.0001) conditions compared to the unstimulated infected control (p < 0.001), Pam3CSK4 (p < 0.05), and PHA infected (p < 0.01) conditions compared to the unstimulated infected control , and R848 (TLR7/8) to a lesser extent, reduced HIV infection of CD4+ T cells compared to unstimulated cells (p < 0.001), with approximately 25% of CD4+ T cells infected  Biomedical Research Ethics Committee (BREC). All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the UKZN BREC (BE433/14).SA, J-AP, LL, LM, and DA acquired funding for the study and edited the manuscript. LL, LM, J-AP, AS, and DA assisted in study design and analysis of data. RC designed and ran experimental procedures, acquired and analyzed data, wrote and edited the manuscript. AS assisted in study design, provided laboratory space for experimental procedures and edited the manuscript.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.


By a combination of spectroscopic methods, the new compounds, designated dioxysceptrin (1) and ageleste C (2), were determined to be structural analogs of each other that differ at the imidazole moiety. Dioxysceptrin was also found to exist as a mixture of α-amido epimers. The sceptrin alkaloids exhibited weak cytotoxicity against cancer cells. Compounds 1 and 2 also moderately exhibited anti-angiogenic and isocitrate lyase-inhibitory activities, respectively.Two new sceptrin derivatives ( Phakellia flabellata and Agelas oroides, respectively, that were found almost 50 years ago ..1 and 2,1 exhibited the most potent anti-proliferative effects against six cancer cell lines , only 1 exhibited moderate anti-angiogenic activity comparable to sunitinib, a positive control. The anti-angiogenic activity of compound 1 was visualized and quantified , a key enzyme in microbial metabolism.Sceptrins and structurally-related alkaloids are known to exhibit a broad range of bioactivities, such as anticancer, antibacterial, antifungal, anti-inflammatory, and anti-biofilm activities ,3,4. In ll lines . For antantified a–c. In a6, with the solvent peaks (δH 2.50/δc 39.50) as internal standards, on a Bruker Avance 600 MHz spectrometer . High-resolution FABMS spectrometric data were obtained at the National Center for Inter-university Research Facilities (NCIRF), Seoul National University and acquired using a JEOL JMS 700 mass spectrometer with 6 keV-energy, emission current 5.0 mA, xenon as inert gas, and meta-nitrobenzyl alcohol (NBA) as the matrix. HPLC separations were performed on a SpectraSYSTEM p2000 equipped with a refractive index detector ) and a UV-Vis detector ). All solvents used were of spectroscopic grade or were distilled prior to use.Optical rotations were measured using a JASCO P-1020 polarimeter  with a 1 cm cell. CD spectra were obtained using an Applied Photophysics Chirascan Plus spectrometer . UV spectra were acquired using a Hitachi U-3010 spectrophotometer . IR spectra were recorded on a JASCO 4200 FT-IR spectrometer  using a ZnSe cell. NMR spectra were recorded in DMSO-dSpecimens of the Agelas kosrae sponge (Demospongiae: Agelasida: Agelasidae) were collected by hand using SCUBA offshore of Kosrae Island in the Federated States of Micronesia at a depth of 15 m on 23 October 2013. The sponge had an elongated repent form with several branches and had dimensions of 6 cm wide and up to 20 cm long. The texture was firm and compressible, and the color was purple on the surface and beige in the choanosome. The skeleton was composed of spicules cored primary fibres, echinated secondary fibres, and very rare achinated tertiary fibres with diameters of 100–200, 30–60, and 10–20 μm, respectively. The spicules, acanthostyles, (110–140 × 6–8 μm) and acanthoxeas (150–170 × 6–8 μm) were identical to those in the literature . A vouch2Cl2 (3 × 2 L). The combined extracts (31.5 g) were successively partitioned between H2O (2.6 g) and n-BuOH (28.6 g); the latter layer was repartitioned between H2O-MeOH  and n-hexane (21.3 g). Then, the H2O-MeOH layer was separated by C18 reversed-phase flash chromatography using sequential mixtures of MeOH and H2O as the eluents (six fractions in an H2O-MeOH gradient from 50:50 to 0:100) followed by acetone and finally EtOAc.Freshly collected specimens were immediately frozen and stored at −25 °C until use. Lyophilized specimens (121.4 g) were macerated and repeatedly extracted with MeOH (3 × 2 L) and CH1H NMR spectroscopy and cytotoxicity analyses, the fractions that eluted with 40:60 H2O-MeOH (0.84 g) and 30:70 H2O-MeOH (1.33 g) were chosen for further separation. The 40:60 H2O-MeOH fraction was separated by semipreparative reversed-phase HPLC , yielding, in order of elution, compounds 9 (tR = 30.7 min) and 7 (tR = 40.2 min). These compounds were further purified by analytical HPLC .Based on the results of the 2O-MeOH fraction was separated by semipreparative reversed-phase HPLC , yielding, in order of elution, compounds 1, 5, 6, 8, 10, 2, 3, and 4; tR = 16.5, 24.2, 28.0, 28.8, 38.5, 42.5, 48.7, and 49.1 min, respectively. Compounds 1, 5, 6, 8, and 2 were further purified by analytical HPLC under isocratic conditions . Compounds 10, 3 and 4 were also purified by analytical HPLC ; tR = 14.4, 24.4, and 26.9 min, respectively. The purified metabolites were isolated in the following amounts: 13.2, 4.2, 8.2, 7.6, 3.3, 2.5, 11.6, 9.1, 7.3, and 2.7 mg of 1–10, respectively.The 30:70 H1): yellow oil; max (log ε) 270 (3.09) nm; CD  Δε 205 (−1.40), 230 (+1.09), 244 (+0.55), 258 (+1.71), 279 (−4.78) nm; IR (ZnSe) νmax 3251, 1680, 1635, 1574, 1522, 1337 cm−1; 1H and 13C NMR data, m/z 651.0432 [M + H]+ .Dioxysceptrin (2): yellow oil; max (log ε) 269 (3.34) nm; CD  Δε 211 (+0.95), 233 (−0.99), 256 (+0.39), 279 (−3.57) nm; IR (ZnSe) νmax 3248, 1725, 1709, 1652, 1581, 1521 cm−1; 1H and 13C NMR data, m/z 544.9677 [M + H]+ .Ageleste C (6): yellow oil; max (log ε) 270 (3.15); CD  Δε 209 (+1.19), 230 (−1.84), 258 (+0.80), 280 (−1.68) nm.-Nakamuric acid methyl ester  shielding constant calculations without geometry optimization employing TmoleX Version 4.2.1 ) at the B3LYP/6-31G level in the gas phase. The CD spectra were simulated by overlapping each transition, where σ is the width of the band at 1/e height. ΔEi and Ri are the excitation energies and rotatory strengths, respectively, for transition i. In the current work, the value of σ was 0.10 eV.All conformational searches were performed using Macromodel ) software with “Mixed torsional/Low Mode sampling” in the MMFF force field. The searches were conducted in the gas phase with a 50 kJ/mol energy window limit and a maximum of 10,000 steps to thoroughly examine all low-energy conformers. The Polak-Ribiere conjugate gradient (PRCG) method was utilized for minimization processes with 10,000 maximum iterations and a 0.001 kJ (mol Å)2. Briefly, cells were seeded in 96-well plates with various doses of compounds and incubated for 72 h. The cells were stained as previously described [50 values were calculated through non-linear regression analysis using TableCurve 2D v5.01 .Anti-proliferative activity was evaluated using SRB staining assay in various cancer cell lines . Cells were purchased from the American Type Culture Collection . They were cultured in media supplemented with 10% fetal bovine serum (FBS) and antibiotics–antimycotics . All cells were maintained at 37 °C under a humidified atmosphere containing 5% CO2. After the tube formation, cells were photographed using an inverted microscope , then images were quantified with Angiogenesis Analyzer using Image J software. Tube formation activity was calculated using the following formula:  − Total segment # (VEGF−))/ − Total segment # (VEGF−)) × 100. The IC50 value was calculated through non-linear regression analysis using TableCurve 2D v5.01 . Cell viability with HUVEC cells were measured independently. First, HUVEC cells were seeded into a 96-well plate and the culture medium was replaced with a serum-free medium when it reached 60% confluency. After overnight starvation, cells were treated with samples and VEGF (50 ng/mL) in 2% FBS EBM-2 medium. Cells were further incubated for 24 h, and MTT assay was used to measure the cell viability. The formazan products were dissolved in dimethyl sulfoxide (DMSO). The absorbance was measured at 570 nm using VersaMax ELISA microplate reader .Anti-angiogenic activity was evaluated using human umbilical vein endothelial cells (HUVEC) using previously described experimental methods . HUVEC cDL-(+)-isocitrate, 3.75 mM MgCl2, 4.1 mM phenylhydrazine, and 2.5 μg/mL of recombinant ICL. The reaction was immediately initiated following the addition of a substrate with or without a prescribed concentration of the inhibitor dissolved in DMSO . Glyoxylate phenylhydrazone formation was spectrophotometrically assessed at 324 nm after incubation at 37 °C for 30 min. The percent inhibition of ICL enzyme activity for each compound was calculated relative to the inhibitor-free control and the IC50 values were calculated using nonlinear regression analysis (percent inhibition versus concentration). 3-Nitropropionic acid was used as a positive control. Protein concentrations were measured using the Bradford method with the Bio-Rad protein assay kit (Bio-Rad) and bovine serum albumin as the standard.A 1 mL aliquot of the reaction mixture contained 20 mM sodium phosphate buffer (pH 7.0), 1.27 mM threo-Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 19433 and Enterococcus faecium ATCC 19434) and Gram-negative bacteria  were used for antibacterial activity tests. Bacteria were grown overnight in Mueller Hinton (MH) broth at 37 °C, harvested by centrifugation and washed twice with sterile distilled water. Stock solutions of the compound were prepared in DMSO. Each stock solution was diluted with MH broth to give serial two-fold dilutions in the range of 128 to 0.06 μg/mL. The final DMSO concentration was maintained at 1% by adding DMSO to the MB broth. Aliquots (10 μL) of the broth containing approximately 5 × 105 colony-forming units (cfu)/mL of the bacteria were added to each well of a 96-well plate. The plates were incubated for 24 h at 37 °C. The minimum inhibitory concentration (MIC) values were determined as the lowest concentration of the test compound that inhibited bacterial growth. Ampicillin and tetracycline were used as reference compounds.Gram-positive bacteria  to give serial two-fold dilutions in the range of 128 to 0.06 μg/mL. The final DMSO concentration was maintained at 1% by adding DMSO to the broth. Aliquots (10 μL) of the RPMI 1640 broth containing approximately 104 cells/mL were mixed with the test compound solutions in each well of a 96-well plate. The plates were incubated for 24 h , 48 h (for A. fumigatus) and 96 h (for T. rubrum and T. mentagrophytes) at 37 °C. A culture with DMSO (1%) was used as a solvent control, and a culture supplemented with amphotericin B was used as a positive control.Potato dextrose agar (PDA) was used to cultivate S. aureus ATCC 25923 and Bacillus subtilis ATCC 6633) were used for disk diffusion assay. Bacteria were grown overnight in Mueller Hinton (MH) broth at 37 °C, harvested by centrifugation and washed twice with sterile distilled water. Stock solutions of the compound were prepared in DMSO. Bacterial cells (5 × 105 colony-forming units (cfu)/mL) were inoculated and 128 μg/disk of the compound was added to each agar plate. The plates were incubated for 24 h at 37 °C. Ampicillin was used as a reference compound.Gram-positive bacteria  and eight structurally-related known bromopyrrole-bearing alkaloids were isolated from the tropical sponge Agelas kosrae. The structure elucidation of compounds 1 and 2 were established by combined spectroscopic methods. Dioxysceptrin (1) was also found to exist as a mixture of α-amido epimers. Absolute configurations were determined by comparison of the experimental and calculated ECD data. The sceptrin alkaloids exhibited weak cytotoxicity against cancer cell-lines. Compounds 1 and 2 also moderately exhibited anti-angiogenic and isocitrate lyase-inhibitory activities, respectively.Two new sceptrin derivatives (


UNaV decreased from 150.3 ± 4.01 mEq/24-h to 122.8 ± 3.92 mEq/24-h (p < 0.001); UIE decreased from 186.1 ± 7.95 µg/24-h to 175.0 ± 7.74 µg/24-h (p = NS); both systolic and diastolic BP values decreased ; DDD decreased . UNaV and UIE were related both before  and after the diet . UNaV and UIE were significantly associated both before and after the diet (p < 0.0001 for both). After salt restriction UIE showed a non-significant decrease remaining in an adequate range. Our dietary suggestions were aimed at avoiding preserved foods, whereas the cautious use of table salt was permitted, an approach which seems safe in terms of iodine intake.A low-sodium diet is an essential part of the treatment of hypertension. However, some concerns have been raised with regard to the possible reduction of iodine intake during salt restriction. We obtained 24-h urine collections for the evaluation of iodine (UIE) and sodium excretion (UNaV) from 136 hypertensive patients, before and after 9 ± 1 weeks of a simple low-sodium diet. Body mass index (BMI), blood pressure (BP), and drug consumption (DDD) were recorded. Data are average ± SEM. Age was 63.6 ± 1.09 year. BMI was 25.86 ± 0.40 kg/m A low-salt diet constitutes a standard approach to the treatment of hypertensive patients ,2,3,4. SIn Italy iodine supplementation was first undertaken in 1921 and iodized salt was introduced by regulatory authorities in 1924 . Since tThe main concern raised by the reduction of sodium intake regards the possible deficiency of iodine intake during a low-sodium diet . In hypeAn additional problem arises from the type of population examined. As a general rule, when an iodine-sufficient population is studied, patients on a low-sodium diet show reduced UIC/UIE, but no substantial change in IS . When ioIn Italy, these concerns have already been raised . Iodine The aim of the present study was to evaluate the modification of UIE induced by a proposed low-sodium diet in hypertensive patients. Urinary iodine excretion and 24-h urinary sodium excretion (UNaV) were measured by standard methods before and after the administration of a dietary protocol.In a larger cohort of 291 hypertensive patients , we obtaThe low-sodium diet prescribed by a dietitian was based on simple recommendations printed on a single A4 sheet of paper . PatientTwenty-four hour urines were collected in accordance with a standard protocol: after the first morning void (to be discarded) patients were requested to record the time and to collect every subsequent void until the same time the following day, when they had to collect the content of the last void. All patients received written and verbal instructions, together with the appropriate urine containers.Blood pressure was measured by means of a semi-automated repeated-measures method  as previously described  both at UNaV was measured by an AutoAnalyzer , while urinary iodine was measured by means of a current commercial colorimetric method  all CVs are below 8%.All patients were on anti-hypertensive drug treatment. Patients on thyroid hormones, amiodarone or with a recent history of contrast-media exposure were excluded. Patients unable or unwilling to provide two (t0 and t2) 24-h urine collections , or with concomitant diseases such as congestive heart failure, atrial fibrillation, renal failure, diabetes, electrolyte abnormalities, secondary hypertension, or goiter were deemed ineligible.Finally, 136 patients, who had successfully provided 24-h urine collections twice in a two months period, were selected. Neither FFQ nor 24-h dietary recalls were used. The study population consisted of Caucasian Europeans, 98%, and Latin Americans, 2%. Of the 136 patients, 84 (61.76%) claimed to use iodized table salt routinely, in agreement with our national data . All subt-test, non-parametric analysis , repeated-measures ANOVA followed by multiple-comparison Newman-Keuls post-test, Kruskal-Wallis test (with Dunn’s multiple comparison test), one-way ANOVA followed by Bartlett’s test for equal variances, linear regression analysis, Fisher’s exact test and Chi-square test were carried out. The significance cut-off was set at p < 0.05.Statistical analysis was performed using a commercial software package: PRISM 7.0 . Paired Student’s Data are reported throughout as average ± SEM unless otherwise stated.Patients were 83 females and 53 males. Their mean age was 63.6 ± 1.09 year.2 before the dietary suggestions (visit t0) and 25.38 ± 0.37 kg/m2 afterwards (visit t2) .BMI was 25.86 ± 0.40 kg/mp < 0.001).UNaV decreased from 150.3 ± 4.01 mEq/24-h at visit t0 to 122.8 ± 3.92 mEq/24-h at visit t2   .t-test, p < 0.05).Drug consumption also decreased from visit t0 to visit t2  at t0, and 162.0 µg/24-h (lower and upper 95% CI 159.6 and 190.3) at t2.Median UIE values in females were 178.2 µg/24-h (lower and upper 95% CI 163.0 and 204.3) at t0, and 153.7 µg/24-h (lower and upper 95% CI 151.3 and 188.5) at t2.R square 0.006788) showed non-significant differences between UIE values in males vs. females both before and after the diet period (p = 0.6087). Bartlett’s test for equal variances gave non-significant results .Median UIE values in males were 188.0 µg/24-h (lower and upper 95% CI 164.9 and 215.2) at t0, and 170.0 µg/24-h (lower and upper 95% CI 155.9 and 210.5) at t2. One way ANOVA .UIE was below 100 µg/24-h in 28 patients before the suggested diet, and in 28 patients thereafter (Fisher’s exact test: p = NS). Furthermore, these data were reanalyzed with non-parametric tests , to avoid distributional assumptions . A possible UIE variability induced by the dietary suggestions was challenged with an F test to compare variances before and after the observation period. F test showed non-significant differences .UIE decreased from 186.1 ± 7.95 µg/24-h at visit t0, to 175.0 ± 7.74 µg/24-h at visit t2     and afte= 0.005) .p < 0.0001 for both).UNaV and UIE were significantly associated both before and after the dietary suggestions  and the final visit (time t2) was 9 ± 0.12 weeks:The proposed low-sodium diet induced a significant reduction in UNaV, BMI, BP, and DDD in our patients. Urinary iodine excretion showed a small, non-significant decrease.We did not monitor the actual diet in our patients, because of the inherent limits of FFQ and 24-h recall methods . InsteadIn our patients we did not evaluate urinary creatinine excretion, which is of limited value as a measure of the completeness of 24-h collection ,33, becaIn our patients, the decrease in UNaV after the dietary suggestions was paralleled by significant improvements in BMI and BP values, with an additional significant decrease in drug consumption.Our patients seemed to follow our suggestions, apparently reducing their sodium intake from (150.3 mEq × 23 =) 3.46 g/day, which is equivalent to 8.78 g/day of salt, to (122.8 mEq × 23 =) 2.82 g/day, which is equivalent to 7.17 g/day of salt. This small but significant decrease, albeit lower than that suggested in the Guidelines [This effective UNaV reduction did not appear to affect UIE, which showed a non-significant reduction from 186.1 to 175.0 µg/24-h. The approximate iodine intake thus decreased from (186.1/0.92 =) 202.3 μg/day, to (175.0/0.92 =) 190.2 μg/day ,36.The number of iodine-deficient patients (UIE below 100 µg/24-h) did not change from the basal observation to the final visit .Our data agree with previously published findings, which indicate that reducing sodium consumption in an iodine-sufficient population does not compromise iodine intake . In our Our instructions do not appear to induce a substantial nor significant decrease in iodine intake, and the number of iodine-deficient subjects did not change substantially, nor significantly . Differences between females and males were non-significant.In Italy, the primary source of salt is not processed or canned food (the use of which remains negligible), nor the amount of salt added at the table or during cooking; rather, it is constituted by three categories of food: bread, cheese, and cured meats . The aimThe fact that UNaV and UIE were significantly related and associated, both before and after the administration of the diet protocol, suggests a possible common source of both sodium and iodine; given that the three salty foods had been excluded from the dietary protocol, this common source was presumably table salt. Our results support this view: the non-significant decrease in UIE seen in our patients seems to fit in with the current low use of iodized salt (3–8%) by the Italian food industry , with thIn an area of iodine sufficiency, albeit in a mild iodine-deficient country, where table salt is the main source of iodine, a low-sodium approach which primarily limits the salty foods  seems safe in terms of iodine intake, and does not worsen the IS.