Pediococcus pentosaceus was cultivated in MRS medium supplemented or not with polydextrose under different conditions in order to evaluate its effect on cell growth, lactic acid and bacteriocin-like inhibitory substance (BLIS) production. Independent variables were pH , rotational speed , polydextrose concentration  and temperature , while cell concentration and productivity after 24 h, maximum specific growth rate, specific rate of substrate (glucose) consumption, volumetric and specific lactic acid productivities, yields of biomass and lactic acid on consumed substrate were the dependent. The maximum cell concentration (10.24 ± 0.16 gX L−1) and productivity (0.42 ± 0.01 gX L−1 h−1) were achieved at pH 6.0, 35 °C, 150 rpm using 1.5% polydextrose, while the maximum specific growth rate (0.99 ± 0.01 h−1) and yield of biomass (2.96 ± 0.34 gX gS−1) were achieved at the same pH and polydextrose concentration, but at 25 °C and 50 rpm. The specific substrate consumption rate (0.09 ± 0.02 gS gX−1 h−1) and the volumetric lactic acid productivity (0.44 ± 0.02 gP L−1 h−1) were maximized at pH 6.0, 35 °C, 50 rpm and 0.5% polydextrose. BLIS produced in this last run displayed the highest antibacterial activity against Escherichia coli, while the same activity was displayed against Enterococcus faecium using 1.5% polydextrose. These results appear to be quite promising in view of possible production of this BLIS as an antibacterial agent in the food industry. Because of its highly branched structure, it is fermented slowly by a mix of bacteria in the distal portion of colon in humans and animals . PropertRegarding the substances released by polydextrose fermentation and their beneficial effects on health, Hernot et al.  reportedPediococcus stand out. Some LAB strains are capable of producing antimicrobial compounds, such as organic acids, hydrogen peroxide, and bacteriocins or Bacteriocin-Like Inhibitory Substance (BLIS), which can inhibit the growth of spoilage and pathogenic bacteria [There are many lactic-acid bacteria (LAB) that have been described as good bacteriocin producers ,15, amonbacteria ,17. BLISbacteria ,19. Bactbacteria ,21.Pediococcus pentosaceus is a gram-positive, facultative anaerobe, catalase- and oxidase-negative bacterium, which is morphologically classified as a coccus commonly arranged in tetrads. Many strains of this species, which was successful in the fermentation of different matrices including goat milk [Listeria monocytogenes, and their possible harmful effects to food consumers [P. pentosaceus SM1, isolated from dry sausage, was found to coincide with P. pentosaceus 1934, the producer of a thermoresistant Class IIa bacteriocin with a molecular mass of 5.37 kD that proved to be active against Micrococcus luteus CECT 241, Pediococcus acidilactici ATCC 25740, P. pentosaceus ATCC 33316, Lactococcus lactis 11454, Lactobacillus curvatus ATCC 51436, Lactobacillus sakei CECT 906T, Lactobacillus plantarum NCAIM B 01133, NCAIM B 1074, CECT 220 and ATH. Despite its relatively low activity (70 AU mL−1), this bacteriocin, which was optimally produced under aerobic conditions, was stable for 1 h at 100–121 °C and for one year at −8 °C and −2 °C [P. pentosaceus ST44M was isolated from Marula fruit (Sclerocarya birrea) and characterized by PCR analysis, 16SrDNA sequencing and typical carbohydrate reactions. The produced ST44M bacteriocin, with a molecular mass of about 6.5 kDa, was attributed to the Class IIa, was shown to be stable for 2 h at 100 °C and in a wide range of pH (2–12), and displayed antimicrobial activity against either gram-positive bacteria such as Listeria ivanovii subsp. ivanovii ATCC 19119 and Enterococcus faecium HKLHS or gram-negative bacteria . Its production was almost independent of temperature in the range 26–37 °C and appeared to be linked to the primary metabolism [P. pentosaceus (R38 and Cb4) isolated from vegetables were reported to produce thermostable bacteriocins strictly related to pediocin PA-1 that exerted antimicrobial activity against both gram-positive (Listeria sp.) and gram-negative (Aeromonas sp. and Yersinia sp.) indicator strains, the former being active also against Staphylococcus aureus [P. pentosaceus ATCC 43200, which was isolated by Piva and Headon [P. pentosaceus FBB61, was proven to be a very good, commercially available pediocin or BLIS producer [oat milk , have beoat milk , which aoat milk  and whosonsumers . P. pentnd −2 °C . P. penttabolism . Two strd Headon  from cucP. pentosaceus ATCC 43200 has thoroughly been investigated in this study at different concentrations, aiming to improve BLIS production for possible use as a food biopreservative.Despite the large use of prebiotics , there iP. pentosaceus ATCC 43200 was grown in de Man, Rogosa, and Sharpe (MRS) broth , some samples supplemented with polydextrose. Temperature, pH, polydextrose concentration and rotational speed were selected as the independent variables and set according to the 24-1 Plackett-Burman fractional factorial design shown in 600nm) as described later. When the OD600nm reached 0.8–0.9, which corresponds to 107–108 CFU mL−1, the inoculum suspension was transferred to 450 mL of the same MRS broth in 2 L Erlenmeyer flasks. Bacterial growth was followed for 24 h by triplicate sampling every 3 h.To prepare the inoculum, aliquots of a cell suspension stored at −70 °C in 20% glycerol were added to 250 mL Erlenmeyer flasks containing 50 mL of MRS broth and shaken for 12 h at 100 rpm and 30 °C. After this period, 2 mL of cell suspension were taken to determine biomass concentration by optical density measurements at 600 nm  broth and MacConkey broth , respectively. After that, 100 μL of suspensions of both strains were added to tubes containing 5.0 mL of the above broths and allowed to grow in a BOD incubator, model TE424 , for 12–14 h at 30 °C. Ten microliters of suspension was periodically drawn to test bacteriocin antimicrobial activity.4-1 Plackett–Burman fractional factorial design was used to study the effects of four independent variables, namely pH, polydextrose concentration, rotational speed, and temperature, on P. pentosaceus ATCC 43200 cultures. For this purpose, cell concentration and productivity after 24 h, maximum specific growth rate, specific rate of substrate (glucose) consumption, volumetric and specific lactic acid productivities, and yields of biomass and lactic acid on consumed substrate were selected as the responses. Such a design was composed of Controls A and B without polydextrose, 8 factorial runs, and 4 central point runs carried out in triplicate under the conditions defined in A 2P. pentosaceus ATCC 43200 was followed by optical density measurements at 600 nm (OD600nm) every 3 h for a total fermentation period of 24 h. The calibration curve y = 1.3677 x − 0.0008 (R2 = 0.9998) was constructed plotting data of cell mass concentration versus OD600nm of cell suspensions at different dilutions , and the results were expressed in g dry weight per liter (gDW L−1).Cell growth of The progressive pH decrease during fermentation was measured by a pH meter, model 400M1 .−1 prepared with high-purity water . For this purpose, fermented broth samples were centrifuged at 5000× g for 20 min at 4 °C  and then filtered through membranes with pore diameters of 45 µm . The CFS was analyzed by an HPLC system, model Prominence , constituted by SIL-20ACHT auto-injector, UV SPD-20A light source, RI-10A refractive index detector, CBM-20A system controller, two LC20AD pumps, CTO20AC column oven, Aminex HPX-87H  ion exclusion column and DGU-A20 degasser. The equipment was programmed to analyze 30 µL samples in the wavelength range from 190 to 230 nm, employing 5.0 mM sulfuric acid as a mobile liquid phase at 0.6 mL min−1 flowrate.Glucose and lactic acid concentrations were determined in triplicate samples of cell-free supernatants (CFS) by High-Performance Liquid Chromatography (HPLC), using standard solutions at concentrations in range to 0.1–2.0 g LE. faecium 101 and E. coli ATCC 25922 as the indicator strains. Samples of CFS prepared by centrifugation at 5000× g for 10 min at 4 °C and filtration through a 45-μm porous membrane had their pH adjusted to 6.5 by the addition of 1.0 N NaOH and then were heated at 80 °C for 3 min to avoid bacteriocin inactivation by proteases. Quantitative analyses were carried out by the spot-on-lawn assay using suspensions of E. faecium 101 and E. coli ATCC 25922 at concentrations of 3 × 106 CFU/mL and 6 × 106 CFU/mL, respectively. Briefly, 10 μL of indicator strain suspension were spread onto 10 mL of BHI Agar (for E. faecium) or MacConkey Agar (for E. coli) previously supplemented with 1.0% agar. After medium solidification, 10 μL of each double dilution of every CFS were placed on the medium surface. Plates were prepared in duplicate and were incubated for 16–18 h at 37 °C. The antimicrobial activity of each CFS was expressed as arbitrary units (AU) per mL by the equation [D is the dilution factor, n the first dilution exhibiting a negligible inhibition zone, and P the volume of sample deposited onto the agar surface.Halo formation was considered to determine BLIS activity by the diffusion agar method against equation :(1)Antimµmax) and the generation time (gt) were calculated by the equations:X0 and fX are the biomass concentrations and the beginning and the end of the exponential phase, and t0 and ft the corresponding times.The maximum specific growth rate (YX/S) and lactic acid (YP/S) on consumed substrate and of lactic acid on biomass (YP/X) after 24 h were calculated by the equations:P is the lactic acid concentration after 24 h, iS and S are the initial substrate (dextrose) concentration and substrate concentration after 24 h, while iX and X are the corresponding values of biomass concentration.The yields of biomass (QX) and lactic acid (QP) productivity after 24 h were defined as:t is the time of 24 h.Volumetric biomass (qS) and specific lactic acid productivity (qP) were calculated by dividing the corresponding volumetric values by the biomass concentration detected at the end of each run.Specific substrate consumption rate  was performed on the results of cell growth, pH, and lactic acid production after 24 h, as well as those of the BLIS concentration at the beginning and the end of logarithmic growth phase and 24 h. Response Surface Methodology (RSM) and Multiple Regression Analysis (MRA) were used to explain the responses obtained by PCA as functions of the selected independent variables .4-1 Plackett–Burman fractional factorial design, carried out with 1.0% polydextrose addition, P. pentosaceus ATCC 43200 reached a biomass concentration (4.75 ± 0.17 gDW L−1) after 12 h about 25% higher than in Control A performed at the same starting pH (5.0) but only 8% higher than in the Control B (pH 6.0), after which it stopped growing. This result provides a first indication not only of the stimulating effect of polydextrose on P. pentosaceus growth during cultivation in MRS medium, but also the prominent role played by the medium acidity. However, it can be seen from the kinetic growth parameters listed in μmax) followed an opposite trend, being in the central point runs (0.87 ± 0.01 h−1) about 7 and 2% lower than in Controls A and B, respectively.Considering that this kinetic parameter is characteristic of the exponential phase of growth, the slower cell growth observed in central point runs may be due to the cell energy spent in this phase to cleave such a polysaccharide to dextrose, in order to make its transport through the cell membrane possible ,37.X = 10.24 ± 0.16 gDW L−1) and volumetric cell productivity (QX = 0.42 ± 0.01 g L−1 h−1) were obtained in run 8 carried out at highest levels of the independent variables  including polydextrose concentration (1.5%). Compared to Control B and central point runs (runs 9–12), these results represent 1.34- and 1.15-fold gains in terms of biomass concentration and 1.33- and 1.21-fold gains in terms of cell productivity.Extending the comparison to all the runs of the experimental design, one can see in P. pentosaceus growth in the pH range 5.0–8.0 [P. pentosaceus Mees 1934 cultivated in MRS medium at pH 6.0 grew up to 6.5 g L−1, while at pH 6.5 it was unable to exceed 2.6 g L−1 [P. pentosaceus CFR SIII isolated from cucumber was able to grow up in MRS at pH 6.5 and 37 °C for 16 h producing a heat-stable, anti-listerial and cell lytic bacteriocin [P. pentosaceus ALP57 was able to grow effectively for 25 h on modified APT broth even at pH 4.1 [These results corroborate what is described in the literature for  5.0–8.0 ,39,40. T.6 g L−1 . P. pentteriocin . Howevert pH 4.1 .P. pentosaceus are susceptible to changes in temperature because they happen simultaneously. According to Papagianni and Anastasiadou [P. pentosaceus Mees 1934 and P. acidilactici NRRL5627 was investigated by Anastasiadou et al. [The growth profile and bacteriocin production by tasiadou , the ranu et al.  and Anasu et al. , who proP. pentosaceus ATCC 43200 when it was cultivated anaerobically in MRS supplemented with 1.5% peptone in bioreactor for 24 h at 30 °C and agitation of 200 rpm. Under such optimal conditions, the cell mass concentration (3.41 gDW L−1) was 66% higher, the generation time (1.28 h) 38% shorter and the BLIS activity against different indicator strains significantly higher than in MRS medium without any supplement taken as a control; it was in the range of 11.0–19.5 mm and the exponential phase started 4 h before. Production of BLIS by this Pediococcus strain was also influenced by the initial pH of the MRS medium as well as the addition of sugars (sucrose) and prebiotic (inulin). The CFS of the fermented control medium at pH 5.0 displayed antimicrobial activity against E. faecium 101 5.3% higher than that at pH 6.0, and even 20% higher than those of all supplemented media, regardless of the concentration of supplements. BLIS production was favored either at pH 5.0 or in the absence of any additional supplements, which were able, instead, to stimulate growth and lactate production by P. pentosaceus ATCC 43200 [Azevedo et al.  reportedP. pentosaceus ATCC 43200 was characterized as a homofermentative strain able to survive the anaerobic environment [As is known, glucose is often the preferable carbon source of acid lactic bacteria when they are submitted to fermentation conditions, under which pyruvate is reduced to lactic acid by lactate dehydrogenase ,44. Partironment ,44.Polydextrose is a branched polymer, consisting of glucose monomers linked mainly via α- and β-1-6 glycosidic bonds, which is associated with the regulation of ATP-dependent transporter activities and participates in phosphoenolpyruvate production in the glycolytic pathway . Lahtine−1) found in run 2, performed in MRS broth supplemented with 0.5% polydextrose, which corresponded to 68.74% of the initial glucose and the highest specific rate of its consumption (qS = 0.09 ± 0.02 gS gX−1 h−1) with the exception of Control B (−1) 47.47 and 25.32% higher than those obtained in Control A (6.12 ± 0.33 g L−1) and the central point runs (8.70 ± 0.50 g L−1) (YX/S = 0.46 ± 0.08 gX gS−1) (YP/S = 0.71 ± 0.04 gP gS−1)   . As the gX gS−1)  was 35.2gP gS−1) , it is e 0.01 h) , was theqS values were obtained when the broth was supplemented with higher levels of polydextrose, which appears to be a rule in fermentation processes when complex molecules are partially cleaved and utilized as carbon sources. Further analyzing the results of QP = 0.36 ± 0.02 g L−1 h−1) and yield of lactic acid on biomass (YP/X = 1.87 ± 0.14 gP gX−1) were obtained in the central point runs, while the highest YP/S (1.35 ± 0.17 gP gS−1) was in run 6; these were carried out using the intermediate and lowest concentrations of polydextrose, respectively, which confirms preference of this bacterial strain for a directly assimilable carbon source such as glucose from the beginning of growth.Lower P. pentosaceus ATCC 43200 CFS aliquots of fermented broths were withdrawn during the exponential phase of growth and tested for antimicrobial activity against two indicator strains, the one representative of gram-negative bacteria (E. coli ATCC 2592) and the other of gram-positive ones (E. faecium 101).E. faecium 101 was as high as 6400 AU mL−1 when tests were made applying samples from Control A, central point runs and run 5. This value was 4 to 8 times higher than those (800–1600 AU mL−1) obtained using fermented broths from other runs. Since both Control A and central point runs were performed under similar conditions except for the percentage of polydextrose supplemented, this result demonstrates that the presence of this polymer in the medium did not significantly influence BLIS production.As shown in P. pentosaceus ATCC 43200 synthesized BLIS during its growth as a product of its primary metabolism, thereby confirming the results reported in the literature by Piva and Headon [P. pentosaceus BCC 3772 [P. pentosaceus 05-10 [Moreover, the antimicrobial activity of CFS at the start of fermentation suggests that d Headon  and Azevd Headon , and forBCC 3772  and P. pus 05-10 . Nonethe−1 against Listeria monocytogenes and 3200 AU mL−1 against Enterococcus faecalis ATCC 29212 at pH = 6. Papagianni and Sergeledis [P. pentosaceus Mees 1934 (170 AU mL−1) by reducing the pH of MRS broth from 6.8 to 6.5. Finally, Engelhardt et al. [−1 by P. acidilactici HA 6111-2 in acidic MRS (pH 3.5). Some authors observed that pediocin release from the cell to the broth occurred very well as soon as the broth underwent acidification. Abrams et al.  reportedrgeledis  describet et al.  reportedE. faecium 101 (6400 AU mL−1) was detected in acidic (pH 5.0) media ; however, overly acidic conditions (pH 4.0) remarkably reduced this activity, likely due to possible partial inactivation of bacteriocin or BLIS or even interference with its mechanism of action. In this respect, it is worth remembering that pore formation happens quickly in the host cell as soon as the identified pediocin or BLIS is connected to cell receptors placed on the cell wall surface. As a harmful consequence, ion transport is highly affected, inducing ion concentration instability between the intra- and intercellular environments [The results obtained in the present work partially agree with the above findings, since the highest antimicrobial activity against ronments ,55,56. Bronments . ObviousE. coli ATCC 25922 as a representative of gram-negative bacteria. It can be seen that maximum BLIS activity (6400 AU mL−1) occurred under less acidic conditions than against E. faecium 101, which leaves us leaning towards interference on the mechanism of action. Gram-negative bacteria are in fact scarcely susceptible to bacteriocin action because in order for the bacteriocin to penetrate them, it must also cross the external membrane [P. pentosaceus KTU05-10 on agar plates containing E. coli ATCC 25922 and E. coli 110, observed a maximum bacteriocin concentration (6400 AU mL−1) coincident with the maximum one observed in run 2 in this study.membrane ,59. To omembrane  structurmembrane , who anaThese results as a whole show how much the conditions of bacteriocins or BLIS productions affect their concentration, activity and even targets.Statistical analyses were carried out on the results pertaining to cell growth, pH decrease, substrate consumption and concentrations of products at the end of cultures. As a first step, Principal Component Analysis (PCA) was performed aiming to take advantage of the correlation among responses. Such a statistical tool is increasingly used also in biotechnology owing to several advantages, among which the reduction of multidimensional data sets to a lower number of dimensions for further analysis stands out . Data vaMicrobial behavior could be understood through the coefficients of responses estimated by the software Minitab 17, for which we considered linear combinations of experimental data of cell, substrate (glucose), lactate and BLIS concentrations as well as pH. The software was able to estimate absolute values for each coefficient, which expressed how participative each result was. Values of all estimated coefficients that were part of the previously mentioned components are listed in P. pentosaceus ATCC 43200. As for the second component (PC2), initial biological results obtained between 3 and 4 h were the most significant; therefore, we assumed that it represented the lag phase of growth and was associated with initial BLIS production. Furthermore, the third component (PC3) was composed of maximum coefficients of BLIS activity when this peptide tested against E. faecium 101 (0.326) and E. coli ATCC 25922 (0.414).Examining the first component (PC1), there were relevant values of coefficients in particular intervals of time, namely cell growth (9–18 h), substrate consumption (3–21 h), lactic acid production (6–12) and pH decrease (9–18 h). Gathering this information, we noticed that PC1 expressed exponential growth of Response Surface Methodology (RSM) and Multiple Regression Analysis (MRA) were used to explain the responses obtained from PCA as function of pH, polydextrose concentration, rotational speed, and temperature. As shown in p values were <0.05. pH and polydextrose concentration had statistically significant effects (p = 0.000) on both cell growth and BLIS production, which were evaluated according to their linear effects. Although three different rotational speeds  were tested, this variable was not statistically significant (PC1: p = 0.460 and PC2: p = 0.620).For each component, interactions between the variables and their significance for biological responses were also evaluated. Significance was assumed when the p = 0.000). As can be seen in −1. Even though runs 2, 6 and 8 resulted in PC1 equivalent to run 5, run 8 showed the maximum cell concentration (Xmax = 10.24 ± 0.16 g L−1).However, when agitation was combined with pH, their interaction strongly affected cell growth (p = 0.01) on both cell growth and BLIS production, while the most noticeable quadratic effect was that of pH (p = 0.00). The importance of this effect is confirmed in runs 1, 3, 5 and 7 whose initial pH was 4.0. Despite the combinations between pH and rotational speed or temperature, cell growth was impaired  by acidification of the culture medium at the start of fermentations, and their PC1 were smaller than −5. 3D. Response surface plots were then generated aiming to find the optimal conditions able to ensure the highest values of PC3, i.e., to maximize BLIS production. E. coli ATCC 25922 (6400 AU mL−1) and consequently allowed a PC3 equivalent to 3.880.In addition, as illustrated in E. faecium (3200 AU mL−1) was noticed. On the other hand, the low value of PC2 in run 4 is consistent with the slight antimicrobial activity against the same strain (800 AU mL−1). The effect of each variable/factor  on the responses  are presented in the Pareto charts and main effects plots provided in Therefore, we can infer that these conditions may be the optimal ones for BLIS production. Furthermore, the initial BLIS production explained by PC2 was the highest in central runs, when strong antimicrobial activity against E. coli ATCC 25922 (6400 AU mL−1) and consequently allowed a PC3 equivalent to 3.880. Therefore, we can infer that these conditions may be the optimal ones for BLIS production. Following these steps, 3D Response surface plots were generated, aiming to find the optimal conditions able to ensure the highest values of PC3, i.e., to maximize BLIS production. P. pentosaceus ATCC 43200 fermentation in MRS broth has been investigated under different operating conditions according to a Plackett–Burman design, paying particular attention to cell growth and BLIS production by this strain. As for cell growth, a polydextrose concentration of 1.5% (run 8) almost tripled the maximum cell concentration (Xmax = 10.24 ± 0.16 g L−1) compared to Control A at pH 5.0 (3.78 ± 0.05 g L−1) with a consequent increase in cell productivity (QX = 0.42 ± 0.01 g L−1 h−1), although the highest maximum specific growth rate (µmax = 0.94 ± 0.01) was obtained in the absence of polydextrose (Control A). Instead, no direct influence of the polydextrose concentration on BLIS production was found. In fact, the BLIS concentration was almost the same in the fermented broths from the Control (MRS) and the central point runs (MRS + 1% polydextrose), when E. faecium 101 was used as a target microorganism for its quantification. Unexpectedly, relevant results were obtained in terms of antibacterial activity against the gram-negative strain E. coli ATCC 25922 of the fermented broth (CFS) from run 2 (6400 AU mL−1), which suggests that more in-depth studies in this regard should be carried out in the future. To complete this study, a statistical analysis of the results was finally proposed using the so-called Principal Component Analysis. Through three models, we were able to confirm that interactions of (i) pH and rotational speed or (ii) pH and temperature are more important for cell growth and BLIS production than those involving polydextrose concentration. In conclusion, the analysis of the response surface suggested the addition of polydextrose in a concentration of 0.5% (run 2) aiming to optimize the growth of P. pentosaceus and the simultaneous production of BLIS. Since the microbial activity of BLIS can be greatly influenced by its degree of purity, further studies will deal with BLIS purification.The effect of polydextrose concentration on


Generalised additive models revealed that stranding event was a stronger predictor for δ13C and δ15N values than body length, sex, or reproductive status, indicating that spatiotemporal differences explained isotopic variation of G. m. edwardii in New Zealand waters better than ontogenetic factors.Isotopic niche analyses can elucidate a species’ foraging ecology. Using isotopic values of Globicephala melas edwardii) strandings on the Aotearoa New Zealand coast, their trophic niche remains poorly understood. To assess the isotopic niche of G. m. edwardii within New Zealand, ontogenetic  and spatiotemporal  variation were investigated. Stable isotopes of carbon (δ13C) and nitrogen (δ15N) were examined from skin samples of 125 G. m. edwardii  collected at mass-stranding events at Onetahua Farewell Spit in 2009 (n = 20), 2011 (n = 20), 2014 (n = 27) and 2017 (n = 20) and at Rakiura Stewart Island in 2010 (n = 19) and 2011 (n = 19). Variations in δ34S values were examined for a subset of 36 individuals. General additive models revealed that stranding event was the strongest predictor for δ13C and δ15N values, whilst sex was the strongest predictor of δ34S isotopic values. Although similar within years, δ13C values were lower in 2014 and 2017 compared to all other years. Furthermore, δ15N values were higher within Farewell Spit 2017 compared to any other stranding event. This suggests that the individuals stranded in Farewell Spit in 2017 may have been feeding at a higher trophic level, or that the nitrogen baseline may have been higher in 2017 than in other years. Spatiotemporal differences explained isotopic variation of G. m. edwardii in New Zealand waters better than ontogenetic factors.The quantification of a species’ trophic niche is important to understand the species ecology and its interactions with the ecosystem it resides in. Despite the high frequency of long-finned pilot whale ( Stable isotope analysis has steadily grown as an ecological tool over recent years , with thδ34S) combined with carbon (δ13C) and nitrogen (δ15N) isotopes, are now increasingly being used to provide clarity around prey source pathways, e.g., estuarine or marine [Multiple isotopes have been used in foraging research including carbon ,12, nitrr marine . The comr marine ,21, inclr marine ,23,24, er marine ,26, the r marine . Hence, r marine .Orcinus orca [Tursiops truncatus [Globicephala melas [Trophic niche partitioning between species is a common strategy to reduce resource competition ,30. Isotnus orca , bottlenruncatus  and longla melas .G. m. melas isotopic values have been noted [G. m. edwardii is the most frequently stranded cetacean by number and several locations have been identified as local stranding hotspots [G. m. edwardii from three stranding events in New Zealand described six cephalopod species present in their stomachs [G. m. edwardii from two stranding hotspots in New Zealand. Specifically, we investigated (1) the isotopic niche of G. m. edwardii in New Zealand using carbon, nitrogen, and sulphur isotopes, (2) ontogenetic variation in isotope values by sex, body length, age, maturity status and reproductive group and (3) spatiotemporal overlap in isotopic niche. Whilst both spatial and seasonal differences in en noted ,47,48, den noted . In New hotspots . Stomachstomachs ,52,53. Wstomachs ,55, it dstomachs . FurtherG. m. edwardii in New Zealand waters, archived skin samples (n = 125) were analysed from individuals collected from stranding events between 2009 and 2017  and Rakiura Stewart Island , mature females (n = 5), immature males (n = 5) and immature females (n = 5) within each stranding event. In one stranding event (FWS2014), more mature females of known reproductive group were available, and these were therefore included in analyses to increase comparative statistical power of mature female reproductive groups  were analysed to compare ontogenetic and spatiotemporal variation. Additionally, a subset of 36  samples from sexually mature individuals with the highest, lowest, and median carbon and nitrogen isotope values recorded per stranding event were analysed for sulphur isotope values. Immature individuals excluded from analyses of sulphur isotopes to avoid confounding the data with individuals that were not fully weaned.Carbon and nitrogen stable isotopes from skin samples , Gaithersburg, MD, USA) and δ15N relative to Pee Dee Beleminte (PDB) standard followed by correction for O17. International laboratory reference materials from NIST were run at the start and end of eve ry batch of analyses for data normalisation [δ13C and δ15N values, whilst precision was measured to better than 0.24‰ for δ13C and 0.22‰ for δ15N values. Stable isotope ratios were expressed as delta values (δ) in per mil units (‰), which represent the ratios of heavy to light isotopes within a sample (Rsample), relative to the ratio in an international standard (Rstandard) as:Carbon and nitrogen isotope analysis was carried out at the Environmental and Ecological Stable Isotope Analytical Facility at the National Institute for Water and Atmospheric Research (NIWA), Wellington. Around 1.0 mg of each homogenised skin sample was weighed into tin capsules using a six decimal place (g) microbalance. Tin capsules were formed into balls containing the sample and were analysed by a FLASH 2000 elemental analyser with MAS 200 R autosampler linked to a DELTA V Plus continuous flow isotope ratio mass spectrometer . Stable isotope values were calculated using ISODAT (Thermo Fisher Scientific) software; lisation . A workiẟ13C (0.08‰), ẟ15N (0.04‰) and ẟ34S (0.16‰) was assessed from replicates positioned every ten samples. A subset of 36 skin samples from mature individuals was processed for sulphur isotope analysis at IsoTrace Limited, Dunedin. Samples were analysed using the Carlo Erba NC 2500 elemental analyser coupled to a Europa Hydra isotope ratio mass spectrometer. Stable isotope values were normalised against international standards of Vienna PDB, AIR and Canyon Diablo Troilite for carbon, nitrogen, and sulphur, respectively. Two international reference materials comprising USGS40 mixed with IAEA-S1 , and USGS-41 mixed with IAEA-S2  used for data normalisation in a three-point system. Replicate analysis of the keratin internal working laboratory standard was used to determine machine drift, and precision of 13C relative to 12C compared to proteins. The lipid content of ecological samples therefore affects δ13C values [δ13C values [G. m. edwardii skin samples  [δ13C and δ15N values), stranding location, stranding event and stranding year. Body length was fitted as a continuous variable, whereas sex, maturity status, stranding location, stranding event and stranding year were fitted as factors. As body length and age were highly correlated , and age was not available for all individuals, body length (n = 125) was included in GAM models as a proxy rather than age itself (n = 108). Models were built with Gaussian distribution with gamma set to 1.4 to prevent overfitting [Following testing assumptions of normality using Shapiro–Wilk tests, Kruskal–Wallis tests were used in the R package “rstatix”  to compas, e.g., ,80. Speas (GAMs)  using ths (GAMs) . Predictrfitting  with allrfitting ) was usirfitting  and “ggprfitting  with ell3) using δ13C, δ15N and δ34S data using the R packages “scatterplot3d” [40, e.g., [40 of another group. Data were presented as a pairwise grid of one-dimensional isotopic density distributions, two-dimensional pairwise isotopic scatter plots and two-dimensional NR40 ellipses of five random NR40 estimates. Overlap probability was calculated at the 95% level using a Bayesian approach with 10,000 iterations and reported as mean posterior overlap, e.g., [Niche regions (NR) were presented in three-dimensions . Data w0, e.g., , replaciG. m. edwardii stranded and triple isotope niche size was examined through Pearson’s correlation analysis both with and without FWS2009 data included. The FWS2009 stranding event appeared anomalous as it had a much larger niche size for the number of animals stranded compared to all other events, and did not fit the trend of the other stranding events. Finally, isotopic range of δ13C using the highest and lowest values were calculated using the formula:δ15N and δ34S were calculated in the same way at the level of (1) the entire dataset, and (2) each stranding event.The relationship between number of ple size . IsotopiAll data analysis was completed in R version 4.0.5 .δ13C values from 71 (57%) samples, whilst 54 samples (43%) were not lipid-corrected . Overall, neither δ15N values  nor δ34S values  were normally distributed.Lipid corrections were performed on orrected . Followiδ15N value was 12.59 ± 0.72‰ . No significant correlations were found between δ13C values and body length  nor age , respectively. Futher, no significant differences were found in the δ13C values between males  and females  and mature  individuals  or among reproductive groups  nor age , respectively. No differences in the δ15N values between males  and females , between immature  and mature individuals  or among reproductive groups  were detected . Sulphur isotope values did not differ significantly between sex  nor body length .The mean n = 87) had significantly lower δ13C and higher δ15N values  compared to those stranded at SI  were larger for females at both FWS  and SI . The TA and SEAc values were larger at FWS than SI for both males and females, respectively. The TA was largest for pregnant females at FWS , and smallest for mature males at SI , with Wilcoxon tests describing four pairs as not significantly different: FWS2014, FWS2017 (p = 0.40); and FWS2009, SI2010 (p = 1); FWS2009, SI2011 (p = 0.25) and SI2010, SI2011 (p = 0.25). Mean δ13C values were lowest in FWS2017 stranded individuals (n = 20), whereas the highest mean δ13C values were observed in those stranded at FWS2009 (n = 20). Nitrogen isotope values differed among stranding events , with higher δ15N values recorded in individuals from FWS2017 (n = 20) than any other stranding event. Nitrogen isotope values were also lower at the FWS2014 stranding event (n = 27) than any other FWS stranding event. Differences in p = 0.10) nor stranding location  were calculated by ontogenetic variation and stranding event. Pairwise comparisons showed the NR40 overlaps of individuals from differing ontogenetic groups  than either pregnant/lactating females (33.56 ± 11.24‰3) or males (20.72 ± 7.18‰3). Mean niche size was similar across several stranding events; FWS2011 (6.62 ± 3.60‰3), FWS2014 (4.32 ± 2.27‰3), SI2011 (4.11 ± 2.21‰3) and SI2010 (3.78 ± 2.04‰3). The combined niche width of individuals stranded at FWS2009 (17.62 ± 9.42‰3) and FWS2017 (15.52 ± 8.25‰3) were much larger than those of all other stranding events  of an individual from SI being found in the NR40 of an individual stranded at FWS. Several pairs were considered to have low probability of NR40 overlap (<10%), with individuals from FWS2017 seemingly the least likely to be detected within the NR40 of any other stranding event  and those that were temporally close  was detected. However, when the FWS2009 stranding event was removed from the dataset, a significant positive correlation was revealed between the number of animals involved in the stranding event and the niche width . Finally, isotopic range was found to be similar between δ13C (3.27‰), δ15N (4.75‰) and δ34S (4.30‰) values for the entire pooled dataset  along with the largest range of δ34S values (3.27‰). In contrast, the largest range of δ13C values were recorded at the SI2011 stranding event (1.90‰) and the smallest range of δ34S values (1.15‰) were recorded at FWS2014. Finally, the largest range of δ15N values were recorded at the FWS2017 (3.85‰) stranding event, whereas the smallest range was at the FWS2011 stranding (1.20‰). No significant correlation between stranding group size and niche size  were lower than mean values reported for other cetacea in New Zealand waters around the same time period, e.g., teuthophagus common dolphins Delphinus delphis  and mal n = 37; ). Howeve, n = 8; ) and sou n = 18; ). Lower  griseus . This co griseus ,97,98. Iglobally ,100,101.ulations ,102,103.G. m. edwardii δ34S values from this study were similar to those reported in G. m. melas in the Mediterranean [δ34S (n = 36) values indicated a large contribution to diet from marine sulphate, indicating marine foraging pathways [δ13C values with high δ34S values observed in this study has previously been described as typical of oceanic feeding behaviour [G. melas is primarily an oceanic species [Sulphur isotopes can provide useful information on foraging prey source pathways ,21, G. merranean . High δ3pathways ,104,105.ehaviour , corrobo species ,108. G. m. melas in the Strait of Gibraltar [T. truncatus [G. melas previously. Furthermore, higher cadmium levels have been reported in female G. m. edwardii from New Zealand waters than in males [δ13C and δ15N values and were less likely to be found in the triple isotope niche of males (56%) than the other way around  and latitude [δ13C values compared to FWS due to the more southerly location [δ15N values were consistently lower in SI than FWS. The lack of significant differences in sulphur isotope values suggests that these carbon and nitrogen isotopic variances are likely due to variation in primary productivity and baseline isotope values between the two locations rather than differences in diet or food web pathways. Future studies would benefit from baseline isotopic information obtained from either: (1) sampling suspended particulate organic matter in surface waters or a sessile primary consumer; or (2) employing compound specific isotope analysis to tease out confounding baseline versus trophic level drivers of elevated ẟ15N values [Spatial differences in isotopic composition within a population are well recorded in cetacea, including G. melas ,100. Forlatitude . It was location , howeverN values ,129,130.ẟ13C and ẟ15N, indicating that stranding event was an important driver of variation for carbon and nitrogen isotopic values. Individuals involved in the SI2010 mass-stranding had the smallest niche size of all the stranding events, which indicates little inter-individual difference in prey and foraging locations for animals involved in this stranding event. However, SI2010 was also the event with the smallest number of overall individuals stranded  and D.  delphis  across tmisphere ,48, datamisphere , preventG. m. edwardii strand all around New Zealand, only two stranding hotspot locations [Resource partitioning of socially and spatially distinct groups has been noted in other cetacea ,143. Altocations  were expG. m. melas populations [G. melas populations, including both shallow and deep foraging dives [G. macrorhynchus in the northeastern Atlantic revealed that individuals may be able to adapt foraging states and behaviour per dive in response to immediate physiological and environmental constraints [G. m. edwardii utilise in New Zealand waters due to a lack of tagging, video, or distribution data for this species.Population homogeneity has been recorded in northern hemisphere ulations , suggestulations  or coopeulations . Multiplng dives ,147 and ng dives ,149 and ng dives  in captistraints . HoweverG. m. edwardii in New Zealand waters. Overall, spatiotemporal variation appeared to have a greater effect on isotopic values than ontogenetic variation, with significant differences in δ13C and δ15N values detected between stranding location and event. Whilst δ34S values did not directly relate to ontogenetic or spatiotemporal factors, incorporating sulphur isotope data improved isotopic niche calculations and provided insight into drivers of other isotopic differences. In particular, δ34S values determined possible drivers of isotopic niche differences between stranding events, which were not easily identified using just δ13C and δ15N values. Finally, our study showed the benefits of long-term tissue archiving when supported by robust life history datasets. Further sampling of G. m. edwardii and their associated prey from additional locations over multiple seasons would improve understanding of spatial and seasonal niche changes for G. m. edwardii. In addition, satellite tagging of G. m. edwardii individuals would provide missing information about their movements, foraging ranges, and habitats. This study was the first to investigate isotopic variation of


Plasmodium falciparum  malaria. A longitudinal cohort study performed in the Southern part of Benin, identified a group of infants who were able to control asymptomatic malaria infections (CAIG).Immunoglobulin G (IgG) antibodies are thought to play important roles in the protection against IgG antibodies against distinct merozoite antigens were quantified in plasma from Beninese infants. Functionality of these antibodies was assessed by the merozoite-phagocytosis assay using THP-1 cells and primary neutrophils as effector cells. Gm allotypes were determined by a serological method of haemagglutination inhibition.Purified IgG from infants in CAIG promoted higher levels of merozoite-phagocytosis than did IgG from children who were unable to control asymptomatic infections . High level of merozoite-phagocytosis activity was significantly associated with high levels of IgG against AMA1  and GLURP-R2 . Moreover, infants of the G3m5,6,10,11,13,14,24 phenotype showed higher merozoite-phagocytosis activity  than those presenting other G3m phenotypes.The results of the present study confirm the importance of antibodies to merozoite surface antigens in the control of asymptomatic malaria infection in Beninese infants. The study also demonstrated that G3m phenotypes impact the functional activity of IgG. This last point could have a considerable impact in the research of candidate vaccines against malaria parasites or other pathogens.The online version contains supplementary material available at 10.1186/s12936-022-04361-w. Plasmodium genus. Of these, Plasmodium falciparum causes the most severe forms of malaria and is the primary cause of malaria related mortality and morbidity in children under 5 years of age in sub-Saharan Africa [P.falciparum malaria after repeated infections [Malaria is caused by parasites of the n Africa . Immune fections , 3. Howefections , 5.P. falciparum parasitaemia below the detection threshold of thick blood smear (CAIG) as determined by monthly thick blood smears (TBS) [To study the development of immunity against clinical malaria during infancy, samples and data were collected from children residing in Tori Bossito in Benin from birth to 18 months of age. The analyses of clinical and parasitological data obtained during the follow-up allowed us to identify a group of infants who were able to clear asymptomatic infections or at least to reduce the rs (TBS) . These crs (TBS) .P. falciparum malaria infections while other correlated these IgG to malaria risk or exposition [P. falciparum-infected red blood cells (iRBC) in vivo, free circulating merozoites and occasionally trophozoites [It is generally accepted that antibodies of the cytophilic subclasses (IgG1 and IgG3) may be particularly important in protection against clinical malaria –12. Accoposition , 13–15. position . In FcgRposition , 17, 18.position . Neutrophozoites –23.http://www.imgt.org/). Gm allotypes have been shown as implicated in malaria susceptibility [Gm allotypes are allelic variations in the CH1, CH2 and CH3 IgG heavy chain, that induce alterations in the amino acid sequences which may affect their binding to FcgRs . Gm allotibility , 26, 27,Here, samples and data were used from a well-established birth-cohort from Benin, to investigate the functional activity of serum IgG antibodies. Infants were typed with respect to their Gm allotypes and specific antibodies  against a panel of merozoite-antigens were quantified by ELISA. The functional activity of IgG was investigated in the merozoite-phagocytosis assay. IgG levels and specific G3m phenotypes were strong predictors of the merozoite-phagocytosis activity by monocytes and neutrophils, respectively.The study was conducted in the district of Tori Bossito in southwest Benin between July 2007 and January 2010, where 567 children were followed-up from birth to 18 months of age. This follow-up consisted in an epidemiological, parasitological and immunological follow-up detailed elsewhere –28. HealP. falciparum vaccine antigen candidates AMA1, MSP1, MSP3, MSP2-3D7, MSP2-FC27, GLURP-R0 and GLURP-R2 were quantified at 6, 9, 12, 15 and 18 months by ELISA [TBS were also performed monthly to detect asymptomatic malaria infections. In addition, mothers were invited to bring their infants to the health centre, at any time, in case of fever (suspected by the mother) or clinical signs, whether or not related to malaria, and the same protocol was applied. Venous blood samples were collected quarterly for hematological and immunological measurements. Venous blood was centrifuged for plasma isolation and genomic DNA extraction. IgG1 and IgG3 responses against P. falciparum parasites or at least maintain P. falciparum at very low parasitaemia, below the detection threshold of thick blood smear [P. falciparum but not able to control asymptomatic malaria infection (IG group) and a group of 23 children for which P. falciparum has not been detected by TBS examination during the survey (NPfDG group). These two groups were composed of all other infants for whom sufficient plasma volume was available.The control of asymptomatic malaria infection was defined as follow: the clearance of blood od smear . CAIG gr™ columns containing protein G coupled to Sepharose were used to purify IgG from 60 μl of plasma diluted 1/10 in phosphate buffered saline (PBS), as per manufacturer's instructions (Sigma-Aldrich). IgG concentration was measured using a NanoDrop®ND-1000 spectrophotometer, and samples were stored at −80 °C. Purified IgG samples were diluted in PBS to obtain a final concentration of 50 ng/mL before performing OP assays. Pooled IgG from malaria-exposed hyperimmune Liberian adults or malaria-non immune Danish blood donors served as internal controls [Ab SpinTrapcontrols .P. falciparum line NF54 was cultured and merozoite isolation was performed as described previously [l-glutamine, 0.02 g/l hypoxanthine, and 25 μg/ml gentamicin) at 37 °C in a humidified 5% CO2, 2% O2 and 93% N2 atmosphere. The parasitaemia was monitored by examination of Giemsa-stained thin blood smears, and parasite cultures were synchronized with 5% sorbitol treatment for 10 min. Synchronized trophozoite-stage parasites were harvested using a magnetic separation column (Vario MACS) and subsequently treated with 10 μM of epoxysuccinyl-l-leucylamido (4-guanidino) butane (E64) for 6–8 h to allow schizonts to mature without rupture. Segmented schizonts were centrifuged, resuspended in 4–6 ml RPMI 1640 medium, and then filtered through a 1.2 μm/32 mm syringe filter to obtain merozoites. The filtrate was passed over an LS MACS Column twice to remove the haemozoin. Free merozoites were stained with 10 μg/ml of ethidium bromide (EtBr) for 30 min before used in OP assay.eviously . Briefly4Cl, 10 mM KHCO3 and 0.1 mM EDTA) and incubated for 10 min.Primary neutrophils were purified from freshly drawn blood from a healthy Danish donor as described previously . Brieflyl-glutamine and 25 μg/ml gentamicin) and distributed in 96-well, U-bottom plates (1 × 105 cells/150 µl/well). The human monocytic cell line THP-1 was maintained as described previously [5 cells/150 µl/well).The cells were centrifuged and further purification of neutrophils was carried out with the EasySep Human Neutrophil Isolation Kit (StemCell Technologies) according to the manufacturer’s protocol. For OP assay, neutrophils were resuspended in cell medium , 4 nM eviously . Likewis2 humidified incubator. To stop phagocytosis, plates were centrifuged for 5 min at 400 g at 4 °C, followed by 2 washes with cold fluorescence-activated cell sorting (FACS) buffer (PBS + 0.5% BSA + 2 mM EDTA). Then, cells were fixed with 200 μl of FACS fixative  and kept at 4 °C until analysed in a CytoFLEX S flow cytometer (Beckman Coulter). Viable neutrophils or THP-1 cells were gated by forward scatter and side scatter properties, and EtBr-positive events were enumerated using primary neutrophils or THP-1 cells alone [The OP assay was performed as described in detail . Brieflyls alone . The perThe determination of Gm allotypes was performed before our study and included in our statistical analyses. Detailed description on the determination of IgG Gm allotypes of the Tori Bossito cohort is available elsewhere . BrieflyConsidering G3m allotypes, no child presented G3m16 or G3m21, both located on the CH2 domain of the IgG heavy chain, and absent in populations from sub-Saharan Africa. Similarly, due to the uncertain location of the Gm28 allotype on either IgG1 or IgG3 sub-classes among sub-Saharan Africans , this alIgG quantification was performed before our study and included in the statistical analyses.The Enzyme Linked Immunosorbent Assay (ELISA) was used to assess antibody levels against seven merozoite antigens. Details about the recombinant proteins and the procedure are described elsewhere , 29. BriPfDG groups using Chi-square test for categorical variables and Mann–Whitney U-test and Kruskal–Wallis test for quantitative variables. Kruskal–Wallis test was used to determine if the distribution of OP values was different between groups while the Chi-square test compared the proportion of infants with low/high OP  between groups.The statistical analysis was carried out using the Stata software version 13. Comparisons of demographic and clinical characteristics were investigated between the CAIG, IG and NPfDG), antibody levels against P. falciparum merozoite antigens and G3m phenotypes carriages were studied by multivariate regressions. Multivariate regressions studied the correlation between OP values, groups, antibody levels and G3m phenotypes using control variables. In this study, only control variables with P < 0.20 among demographic and clinical characteristics were specifically used. The correlation between OP values and groups  was studied using a multivariate ordered logit regression model; NPfDG, IG, and CAIG was coded 0, 1 and 2, respectively, according to OP values observation. If the coefficient and P value was significant and positive, the OP values increased with groups in the order 0, 1 and 2 otherwise the OP values did not increase with groups. The association between OP values, IgG levels and G3m phenotypes were studied using a generalized linear multivariate regression model where only control variables with P < 0.20 among demographic and clinical characteristics were specifically used. In these models, validity assumptions  have been checked and also the deviance and Pearson were shown as regards the model fitting.Finally, the correlation between OP values, groups  with the possibility to withdraw at any time.PfDG) and 80 children tested parasite-positive at one or more of the monthly visits. Of the 80 parasite-positive children, 27 were able to clear asymptomatic malarial infections or to reduce P. falciparum parasitaemia under detection threshold of thick blood smear (CAIG) while 53 had P. falciparum infection with clinical symptoms (IG) .Infants from CAIG had more asymptomatic infections P < 0.001) when compared to infants from IG while there was no difference regarding symptomatic infections between the two groups  (Table The distribution of bed net use and gender was similar between the three groups (P = 0.429). In contrast, the age of the mothers P = 0.001) and the number of antenatal visits (P = 0.005) was highest in N and the Merozoite-phagocytosis assays are typically performed with primary cells or with the THP1 cell line –35. To cPfDG groups , respectively  against AMA1, MSP2-3D7, MSP2-FC27, MSP1, MSP3, GLURP-R0, and GLURP-R2 were quantified by ELISA  and MSP1 IgG1  while levels of AMA1 IgG3 showed a modest association . Second, we investigated the association between merozoite-phagocytosis by neutrophils and specific IgG levels  . However, a significant low level of IgG3 against MSP2-3D7 was found in infants carrying G3m5,10,11,13,14  (Table PfDG groups. OP was previously correlated to malaria protection and judged as a reliable vaccine evaluation criterion through analysis of antibody responses [The results of the present study showed a higher ability of antibodies from infants included in the CAIG group to stimulate phagocytosis of monocyte cell line THP-1 or primary neutrophils compared to infants belonging to IG and Nesponses , 19, 36,PfDG groups (P ≤ 0.001, Additional file P. falciparum infections in Papua New Guinea [Infants from the CAIG presented higher IgG concentrations against AMA1, MSP1 and GLURP-R2 compared to infants belonging to IG and Nw Guinea . IgG1 tow Guinea  and suggin vitro OP activity observed in CAIG group could reflect the antibody involvement in the control of parasite growth. However, it is difficult to affirm that OP activity observed in this group is responsible for parasitemia control observed during the follow-up. Unfortunately, certain constraints such as the presence of maternal antibodies until 12 month of age and the quantity of available plasma at 15 month of age did not allow us to perform a prospective study.The pattern of malaria infections in CAIG group during the survey was very different from the one of IG group. In addition to have the capacity to control their parasitaemia, individuals included in CAIG group presented more asymptomatic infections than IG group Fig. . InfantsP. falciparum malaria after repeated infections. The correlation of high concentrations of IgG subclasses against AMA1 and MSP1 to high levels of OP may reflect the repeated exposure of these children to malaria which can lead to a better antibodies’ memory responses and phagocytosis capacity. Moreover, in this cohort, it has been shown that the levels of IgG against P. falciparum merozoite antigens reflected malaria exposure [PfDG group had lower levels of phagocytosis compared to CAIG and IG groups. Note that a slightly higher bed net possession was observed in NPfDG group, however other factors might be involved in lower exposition to P. falciparum infections. However, it is possible that a proportion of infants in NPfDG group could also be protected from malaria infections by other mechanisms.Immune epidemiological studies have shown that children living in malaria endemic areas may develop naturally acquired immunity against exposure . Thus, t. [. [The finding that the levels of total IgG to GLURP-R2 were associated with a high OP and not significant when considering specific IgG1 or IgG3 may suggest a potential implication of IgG2 and/or IgG4. Indeed, the total IgG level to GLURP was associated with a reduced risk of malaria by Nebie et al.  or Kana . [.  while an. [Indeed, IgG3 and IgG4 against GLURP were associated with a reduced risk of clinical malaria by Nebie et al.  while on. –42. In a. . IgG2 anP. falciparum merozoites. The effector functions of IgG3 are numerous among which activation of complement pathways, neutralization, antigen recognition and cell activation via FcgR receptors [As regards the influence of G3m phenotypes on OP, our results showed a high level of OP associated with the carriages of G3m5,6,10,11,13,14,15 and G3m5,6,10,11,13,14,24. No work previously investigated associations between Gm phenotypes and opsonizing antibodies to eceptors , 44. Amoeceptors , 45. The. demonstrated a protective effect of the Gm5,6,13,14; 1,17 phenotype carriage in Benin [P. vivax PvMSP1-19 and PvAMA-1 antigens in Brazilian Amazon endemic area [Facer  was the in Benin  while thmic area . Moreovemic area . G3m5,6,mic area , 48 or qThis work showed that G3m5,6,10,11,13,14,24 and G3m5,6,10,11,13,14,15 phenotypes were associated with high OP using primary neutrophils while only G3m5,6,10,11,13,14,24 phenotype was associated with high OP in THP-1 cell lines with a decreased significance. Other factors such as FcgR polymorphisms may also impact OP activities. The genotypes of primary neutrophils and THP-1 cells used in this study were respectively FcgRIIA 131 RR/FcgRIIIB NA2NA2 and FcgRIIA 131 RH/FcgRIIIB NA1NA2. It is worth noting that FcgRIIA 131 RR/FcgRIIIB NA2NA2 was previously associated with malaria protection and FcgRIIA 131 RH/FcgRIIIB NA1NA2 with a trend of protection .However, this aspect deserves further studies, performed on diverse populations in high number in order to explore this potential role of Gm phenotypes in the functionality of IgG, which could have a considerable impact in the research of vaccine candidates against malaria.This study shows that the group of infants able to control infection had probably a better functionality of IgG through a high OP compared to a control group. It showed also that the inherent polymorphism of the IgG heavy chain represented by the Gm phenotypes play an important role in IgG functionality, opening up avenues for the efficacy of antibodies in the search for anti-malarial vaccines.Additional file 1: Table S1. IgG levels in the study group.


The adsorbent features were characterized by Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscope (FESEM), and energy dispersive X-ray analysis (EDX). Various effective parameters on the sorption and desorption cycle, such as sorption time, the mass of adsorbent, pH, salt addition, and material ratio, were investigated and optimized. The data were evaluated using isotherm models, and experimental data were well-fitted to both Langmuir (R2 = 0.975) and Freundlich (R2 = 0.985) models. Moreover, kinetic of reaction was agreement with pseudo-second-order (R2 = 0.999) as compared pseudo-first-order (R2 = 0.760). The maximum adsorption capacity for tetracycline and cefixime was achieved at 116.27 and 105.26 mg·g−1, respectively. Hence, the prepared adsorbent can be used as an alternative material for enhanced determination of pharmaceutical substances in biological fluids.In this study, a magnetic solid-phase extraction method was developed based on multi-wall carbon nanotubes decorated by magnetic nanoparticles (Fe Recently, the consumption of antibiotics as medicine has increased significantly, and their usage has grown globally . CefiximRecently, traditional strategies and recent technology were combined to develop high-performance methods for efficient removal of antibiotics from water , such as2·g−1 [Amongst the aforementioned methods, the techniques based on green and synthetic nanoparticles are the most common approach because the nanoparticles have some significant merits, such as being affordable and having low toxicity, easy operation, high surface area to volume ratio, and high dispersibility ,10,11,122·g−1 , high su2·g−1  as well 2·g−1 . Further2·g−1 ,22.Cadmium sulfide (CdS) as a soft nanoparticle (NPs) can increase the possible interactions between adsorbents and antibiotics due to an enhancement in active sites for adsorption . The low3O4) were doped on MWCNTs and functionalized by CdS nanoparticles (Fe3O4@MWCNTs-CdS) for adsorption of cefixime and tetracycline, the selected antibiotics, in wastewater samples Furthermore, MNPs help to an easy collection of MWCNTs from a large volume of aqueous solution. MWCNTs gain high extraction efficiency through π–π interactions with selected antibiotics and also avoid the CdS NPs aggregation. The presence of CdS improved the adsorption efficacy by introducing various complexation, electrostatic interactions, and H-bonding. Hence, the newly synthesized Fe3O4@MWCNTs-CdS nanocomposite provided excellent adsorption efficiency for selected antibiotics from wastewater samples.In this study, MNPs (Fe2·4H2O), Iron(III) chloride hexahydrate (FeCl3·H2O), sodium hydroxide (NaOH), polyethylene glycol (PEG), cadmium acetate dehydrate (Cd(CH3COO)2·2H2O), methanol (CH3OH), sodium thiosulfate pentahydrate (Na2S2O3·5H2O), hydrochloride acid , nitric acid , and sulfuric acid  were of analytical grade and supplied by Merck Chemicals . The pharmaceutical analytical standards of tetracycline and cefixime were purchased from Solarbio Science & Technology Co., Ltd. .Multi-walled carbon nanotubes (MWCNTs)  were purchased from Chengdu Organic Chemicals Co. Ltd. , Iron(II) chloride tetrahydrate . UV–visible absorption spectra were recorded on RAYLEIGH UV-2601  double-beam UV–visible spectrophotometer fitted with a tungsten lamp as the source, volume 3.5 mL quartz cuvette cell . Various pH values were measured by WTW Inolab 720 pH meter .Morphological and compositional information of the solid product was studied by field-emission scanning electron microscopy and energy-dispersive X-ray spectroscopy (FESEM/EDX) from TESCAN MIRA3 . Fourier transform infrared spectrometry (FT-IR) spectra were obtained with KBr pellets in the range 450–4000 cm3O4) were pre-treated by dispersion in strongly acidic conditions (3:1 molar ratio) that contained HNO3 (65%) and H2SO4 (96%) for 48 h under magnetic stirring at room temperature. Finally, activated MWCNTs were collected and washed several times with acetone and excess distilled water and dried at 85 °C for 24 h [Pristine MWCNTs NPs before magnetization (Fefor 24 h .2·4H2O (0.005 mol) and 0.5 g of FeCl3·6H2O (0.001 mol) were added to the solution and stirred. The solution was heated until 50°C, and an aqueous sodium hydroxide solution (2 M) was added gradually at pH 10. Eventually, the mixture was stirred for 2 h, washed several times with distilled water, and dried at 80°C for 24 h.Activated MWCNTs were magnetized as follows: 0.5 g of pre-treated MWCNTs was dispersed in 50 mL distilled water under magnetic stirring. After that, 1.0 g of FeCl3O4@MWCNTs. Then, 5 mmol Cd(CH3COO)2·2H2O, 5 mmol Na2S2O3·5H2O, and 10 mL of polyethylene glycol (PEG) were mixed under stirring and heating at 60 °C. Then, 1 g of Fe3O4@MWCNT was added to the above solution and stirred vigorously for 2 h, and this solution was kept in the oven at 90 °C for 24 h. Thereafter, the obtained Fe3O4@MWCNTs-CdS was easily separated by an external magnet and washed with excess distilled water, methanol, and ethanol. Then, the separated adsorbent dried in the oven at 85°C for 24 h. In the next step, the CdS nanoparticles (yellow product) were synthesized with the same procedure without the addition of Fe3O4@MWCNTs into the PEG solution. The characterization and surface morphology of dried CdS nanoparticles and Fe3O4@MWCNTs-CdS nanocomposite were studied by using FTIR and FESEM/EDS.To further modification of the adsorbent, the cadmium sulfide (CdS) nanoparticles were immobilized on the surface of Fe−1. All samples were stored at 4 °C in the refrigerator.Wastewater from the drug company was analyzed as a model of real sample analysis. Wastewater was collected from the vicinity of a pharmaceutical company located in Tehran, Iran. The simulated samples were spiked by the addition of an adequate amount of antibiotics standard solution to give a final concentration of 0.1–100 mg·L3O4@MWCNT-CdS was added to the aqueous samples containing selected antibiotics with different initial concentrations (0.5–10 mg·L−1). Then, the mixture was shaken on a plate shaker (350 rpm) for 10–90 min in the different solutions’ pH range of 3–12. Then, the adsorbent was separated from the solution by an external magnetic field that was applied by putting a magnet on the outer tube’s wall. Residual solutions were analyzed by spectrophotometry to measure the residual concentration of antibiotics in the solution and removal percentage.The experimental adsorption procedure includes subsequent steps: first, 40 mg Fe3O4@MWCNT, and newly fabricated Fe3O4@MWCNTs-CdS nanocomposite are given in −1 that is attributed to the stretching of O-H. Peaks at 2924 cm−1 and 2849 cm−1 are ascribed to C-H stretching. The vibrations at 1714 cm−1 and 1378 cm−1 are assigned to C=O and C-OH, respectively. In −1 refers to Fe-O stretching, which confirmed the magnetization of MWCNT [−1, and the peaks in the range of 500–600 cm−1 correspond to metal–sulfur bonding. Hence, the peaks at 641 cm−1 are ascribed to the vibration of the metal sulfide (Cd-S) bands as reported for quantum dots materials [3O4@MWCNTs-CdS. In addition to all previous peaks, some additional peaks at 1632 cm−1, 1016 cm−1, and 641 cm−1 are related to the CdS nanoparticles, which confirms the successful coating of CdS nanoparticles onto the surface of Fe3O4@MWCNT.The FT-IR spectra of the activated MWCNTs, Feof MWCNT . In Figuaterials . Figure 3O4@MWCNTs.3O4@MWCNTs-CdS are illustrated in 3O4@MWCNTs-CdS nanocomposite confirmed the presence of main elements C, O, Fe, Cd, and S in the backbone of the adsorbent.The elemental composition of the prepared materials was analyzed with the energy-dispersive X-ray spectroscopy (EDX) technique. The EDX spectra for CdS nanoparticles and FeThe total removal rate of TC and SF by using Fe CNT, 5% Fe CNT-CdS, 10% Fe CNT- CdS, 20% Fe CNT-CdS, and CdS are demonstrated in A real sample was obtained from pharmaceutical company wastewater. The experiment was conducted for un-spiked and spiked (10 mg·−1) samples. The cefixime and tetracycline were not detected in the un-spiked wastewater, while detection of TC and SF in the spiked sample was conducted successfully, with a recovery percentage of 91.38 % and 87.44 %, respectively.LPZC). The pHPZC for MWCNTs adsorbent and IEPs for tetracycline were reported in pH 2–4 and pH~6.3, respectively [3+ at pH < 5, TCH2± at pH 5–7, and TCH−/TC2− at pH > 7 [a 2.5) [pH is an important parameter affecting both analyte and adsorbent surfaces. As illustrated in ectively ,36,37. Ft pH > 7 ,39. Thus [a 2.5) , and due [a 2.5) . FinallyDifferent mass of adsorbents in the range of 5–60 mg was carried out for MSPE performance due to the crucial importance of adsorbent amount. The results given in w/v) into the sample solution (w/v) does not affect the extraction of antibiotics. By increasing salt concentration up to 0.5% (w/v), the extraction recovery decreased slightly due to the mass transfer of analytes to the surface of the adsorbent or interference with salt ions. In this regard, salt addition is not necessary for our experiments.Salt augmentation to sample solution can influence extraction efficiency. In this study, the effect of salt concentration was investigated by the addition of different amounts of NaCl salt in the concentration range of 0–10%  are the Elovich model constant. The values of these parameters can be derived from the slope and intercept that are displayed in 2 = 0.999 TC, R2 = 0.998 SF). In addition, the Elovich model was conducted, and the low values of R2 (<0.889) demonstrate that the electron-sharing process does not limit the kinetic. In this respect, the adsorption mechanism was conducted through chemisorption.For the evaluation of adsorption, kinetic pseudo-first-order, pseudo-second-order, and Elovich models, as expressed in Equations (1), (2) and (3), respectively, were studied.In order to perform the isotherm models, the experimental process is conducted by variation of initial concentrations of TC and SF at constant time. Then, the equilibrium isotherm is plotted in eC is the equilibrium concentration of TC and SF antibiotics. eQ (mg·g−1) is the experimental equilibrium adsorption capacity, mQ is the maximum adsorption capacity (mg·g−1), 1/n is the empirical parameter related to the energetic heterogeneity (average energy of sites), and .L.K and .F.K (L·mg−1) are the Langmuir and Freundlich equilibrium adsorption constants, respectively. adK is constant, and Qs is the theoretical adsorption capacity of D-R model (mg·g−1), which are related to multilayer adsorption with chemical and physical adsorption onto the heterogeneous surface. The values of the parameters are calculated under linear plots  confirms that the adsorption pattern is following a multilayer process.In these equations, ar plots B–D and lar plots  perform .D. = Qe/CeK (thermodynamic constant), ΔH , and ΔS (entropy) can be expressed by Equations (6) and (7). The obtained calculations from the experimental (Thermodynamic parameters such as ΔG (free energy change), rimental  determinThe stability of the adsorbent was evaluated under a continuous adsorption-desorption reusability process. After each adsorption process, the adsorbent was separated with an external magnet and washed with 6 mL methanol three times. Then, adsorbent was dried, and we proceeded to investigate their reusability for five cycles. The adsorbent was provided appropriate stability since the removal efficiency (first cycle > 0.91 %) was not changed significantly after five adsorption-desorption cycles (first cycle > 0.84 %).The two selected antibiotic molecules (TC and SF) possess three functional groups, including hydroxide (-OH), amine (-NH), and carbonyl (COO-). Furthermore, Fe-CNT-CdS adsorbent possesses various active sites on the surface, including -OH and COO-, sulfur, cadmium, and iron oxide. The proposed functional groups of TC and SF and active adsorbent sites  can attract each other. Thus, the attraction is involved with the electrostatic interaction. Hydrogen binding and π–π interaction occur between the antibiotic’s molecules and magnetic adsorbent.3O4@MWCNTs-CdS) via a simple procedure. This nanocomposite was used as an adsorbent to remove cefixime and tetracycline antibiotics from drug company wastewater and biological urine fluid. Isotherm models, including Langmuir and Freundlich models, were investigated. The experimental data were in agreement with the Langmuir model, which suggests a monolayer of tetracycline and cefixime onto the adsorbent and provides adsorption capacity 116.27 mg·g−1 and 105.26 mg·g−1, which indicates high potential adsorbent in comparison with other published works in the treatment of TC and SF The kinetic mechanism was examined using pseudo-first-order and pseudo-second-order models. The experimental data in the current study were fitted with a pseudo-second-order model (R2 = 0.999). The proposed method is environmentally friendly due to the lack of toxic organic solvents.In this study, CdS NPs were decorated on magnetic multi-walled carbon nanotubes as novel nanocomposite (Fe


Background: Icaritin is a natural product with a wide range of anti-tumor effects. However, its anti-tumor mechanism has not been thoroughly studied. This study examined the inhibitory effect of icaritin on nasopharyngeal cancer and its underlying mechanism using network pharmacology along with in vivo and in vitro experiments.Methods: MTT and clone formation assays were used to detect the effects of icaritin on the viability and proliferation of nasopharyngeal carcinoma cells, followed by the construction of a HONE1 xenograft tumor model to evaluate the anti-tumor efficacy of icaritin in vivo. A public database was used to predict prospective targets, built a protein-protein interaction (PPI) network, and analyze gene enrichment and biological processes. Based on network pharmacological data, cell cycle-related proteins were identified using western blotting. Besides, cell cycle distribution, apoptosis, and intracellular reactive oxygen species (ROS) generation were identified using flow cytometry. In addition, SA-β-Gal staining was performed to detect cellular senescence, and western blotting was performed to detect the expression of P53, P21, and other proteins to verify key signaling pathways.Results: Icaritin effectively inhibited the viability and proliferation of nasopharyngeal carcinoma cell lines and showed good anti-tumor activity against HONE1 nasopharyngeal carcinoma cells in vivo. Key protein targets, including AKT1, HSP90AA1, CDK4, CCND1, and EGFR, were screened using PPI network topology analysis. GO and KEGG analysis revealed that the cell cycle, p53 signaling, and cell senescence pathways may be the main regulatory pathways. Flow cytometry and western blot experiments showed that icaritin caused S-phase arrest and promoted an increase in ROS. SA-β-Gal staining showed that icaritin significantly induced cellular senescence, and western blotting showed that the expression of senescence-related proteins p53 and P21 increased significantly. Moreover, inhibition of ROS levels by N-Acetylcysteine (NAC) enhanced cell viability, reversed cellular senescence and reduced cellular senescence-associated protein expression.Conclusion: The results of network pharmacological analysis and in vivo and in vitro experiments showed that icaritin effectively inhibited the growth of nasopharyngeal carcinoma cells, promoted ROS production, induced cellular senescence, and inhibited tumor cells, which are related to the regulation of P53/P21 signal pathway. A common malignant tumor in otolaryngology, nasopharyngeal carcinoma develops from the nasopharynx’s epithelial cells . Its incEpimedium sagittatum (Sieb. et Zucc.) Maxim. or Epimedium brevicornum Maxim. of the family Berberidaceae, which tonifies the liver and kidney, reinforces the muscles and bones, and dispel wind and dampness . Fetal bovine serum (FBS) was purchased from Gibco . 3--2, 5-diphenyltetrazolium bromide (MTT), crystal violet, a cell-based ROS assay kit, N-acetylcysteine (NAC), and a senescence-associated β-galactosidase  kit were obtained from Beyotime Biotechnology . Annexin V-FITC/PI and propidium iodide (PI) were purchased from KeyGEN BioTECH . The primary antibody against γ-H2AX was obtained from Cell Signaling Technology . Primary antibodies against ki67, GAPDH, PARP1, cyclin D1, CDK2, CDK4, Bax, Bcl-2, P53, P21, p-EGFR, EGFR, p-AKT, AKT, and HIF1α were obtained from Proteintech . Antibody against horseradish peroxidase (HRP)-conjugated secondary antibodies (goat-anti-mouse or goat-anti-rabbit) were obtained from ZSGB-BIO .2 atmosphere.The human nasopharyngeal carcinoma cell lines HONE1 and HNE1 were purchased from Zhongqiaoxinzhou Biotech . HONE1 and HNE1 cells were cultured in DMEM and RPMI-1640 media containing 1% penicillin-streptomycin and 10% FBS, respectively. Both cell lines were cultured at 37°C in a 5% COHONE1 and HNE1 cells were seeded into 96 well plates at the density of 3,000 cells per well, and different concentrations of icaritin were added to the cells after 24 h culture. After 72 h, 20 μl MTT solution (5.0 mg/ml) was added and incubated at 37°C for 4 h, then added 150 μl of DMSO. The optical density was measured at 570 nm using a microplate reader .HONE1 and HNE1 cells were seeded into 6 well plates, 5,000 cells per well. After 24 h incubation, the cells were treated with icaritin  for 48 h. The fresh culture medium was replaced and cultured for 2 weeks and stained with 0.1% crystal violet solution for 10 min. The colonies were photographed using a digital camera .https://pubchem.ncbi.nlm.nih.gov/) and imported into SwissTargetPrediction (http://www.swisstargetprediction.ch/), species selection of Homo sapiens, and prediction of icaritin targets. The HERB database was searched for icaritin targets and duplicate targets were merged and removed.SMILES of icaritin were obtained by searching Pubchem (https://www.disgenet.org/) and TTD databases (http://db.idrblab.net/ttd/) were searched with “nasopharyngeal carcinoma” as the keyword. Then, the targets were merged and duplicates removed to obtain candidate targets for nasopharyngeal carcinoma.The DISGENET (http://www.uniprot.org/) to unify the target names. The targets were imported into VENNY2.1 (https://bioinfogp.cnb.csic.es/tools/venny/) to obtain the intersection targets of the compound and disease. The intersecting targets were imported into STRING (https://cn.string-db.org/cgi/input.pl), “Homo sapiens” was selected as the species, and medium confidence >0.4 was selected as the minimum interaction threshold, unlinked targets were hidden, and other parameters were kept at default settings. The tsv file was saved after updating and imported into Cytoscape 3.7.1. Then, network analysis was performed, and the clusters with high correlation were calculated using the MCODE plugin.The icaritin and nasopharyngeal carcinoma targets were imported into UniProtKB (https://metascape.org/), and the species was set as “Homo Sapiens”. The three modules of GO: molecular function, biological process, and cell composition were selected for GO enrichment analysis, and KEGG was selected for pathway analysis. The KEGG pathways of p < 0.01 were considered significant, and the results of enrichment analysis were visualized using the microbiology online mapping platform (http://www.bioinformatics.com.cn/).Icaritin-nasopharyngeal carcinoma intersection targets were imported into the Metascape database .HONE1 and HNE1 cells were seeded into 60 mm cell culture dishes at a density of 3 × 105 cells. After 24 h incubation, the cells were treated with 10, 20, or 40 μM icaritin for 48 h. The cells were then collected and stained with Annexin V-FITC/PI working solution for 15 min in the dark. The apoptotic cell ratio was determined using a FACScan flow cytometer.HONE1 and HNE1 cells were seeded into 60 mm cell culture dishes at a density of 3 × 10HONE1 and HNE1 cells were collected after treatment with different concentrations of icaritin for 24 h or 48 h or preated with NAC (5 mM) for 1 h before administration. After lysis, the supernatant was collected and the concentration was quantified by BCA Protein Assay Kit. After SDS-PAGE protein electrophoresis, transferring to PVDF membranes, and blocking with 5% non-fat milk, the membrane was incubated with primary antibodies at 4°C for 12 h, and then incubated with the corresponding secondary antibody at room temperature for 2 h. The protein bands were detected using an ECL kit, and the band density was analyzed by Scion Image software.2 atmosphere. Then the cells were collected and analyzed using FACScan flow cytometer. The average DCF fluorescence intensity was used to express the intracellular ROS level in each group.After treatment with icaritin  for 24 h, the cells were incubated with 10 μM dichlorofluorescin diacetate (DCFH-DA) at 37°C for 20 min in a 5% CO4 cells per well. After 24 h incubation, the cells were treated with 10, 20, or 40 μM icaritin for 24 h. The cells were then stained with SA-β-Gal solution overnight. Six separate fields were counted under a microscope in each group.HONE1 and HNE1 cells were seeded in 6 well plates, 5 × 106) were injected subcutaneously into the left flanks of the mice. Tumor volume (mm3) = 1/2 × length × width2. When the tumor volume reached 100 mm3, the mice were administered 50 or 100 mg/kg of oral icaritin daily. Tumor volume and body weight were recorded every 2 days. After 14 days, the tumor tissues were harvested and fixed in formalin for immunohistochemistry.BALB/c Nude mice (6-weeks-old) were purchased from HFK Bioscience Co., Ltd. . The Institutional Animal Care and Use Committee at the Institute of Radiation Medicine approved all animal experiments in this study. HONE1 cells . Statistical comparisons were performed using the Student’s t-test and one-way ANOVA. To explore the effect of icaritin on nasopharyngeal carcinoma, two nasopharyngeal carcinoma cell lines, HONE1 and HNE1 were chosen as the study’s two nasopharyngeal cancer cell lines. According to MTT data, icaritin significantly and dose-dependently reduced the viability of HONE1 and HNE1 cells . The celin vivo. The results indicated that the tumor volumes in the low (50 mg/kg) and high (100 mg/kg) dose icaritin treatment groups were significantly smaller than those in the blank group  and KEGG pathway were selected in ascending order of the lization . The reslization . The patlization . Combinilization .Based on the results of the network pharmacology analysis, we examined the changes in PI3K/AKT, EGFR and HIF-1 signaling pathways, and the results showed that these pathway-related proteins were not significantly altered, and we speculate that icaritin may exert anti-tumor effects through other pathways . The cloin vivo SA-β-Gal staining results showed that senescent cells were significantly increased in the tumor tissues of the iacritin-treated group, and a significant increase in the expression of senescence-related proteins such as P53 in the tumor tissues, consistent with the in vitro cellular results , molecular chaperones (HSP90AA1 and HSP90AB1), tyrosine kinase family members (EGFR and ERBB2), P53/P21 and PI3K/AKT. Based on the results of the network pharmacological analysis, we examined the expression of p-AKT and p-EGFR, and it turned out that these proteins were not significantly altered, and we hypothesized that icaritin exerted its antitumor effects through other pathways. in vitro and in vivo showed that icaritin significantly induced cellular senescence. We then examined the key signaling pathway of senescence regulation, P53/P21, and found a significant dose-dependent increase in the expression levels of both P53 and P21 proteins in vitro and in vivo. In addition, the P21 protein plays a role as an inhibitor of cell cycle progression, as P21 binds to the cell cycle protein kinase complex and inhibits its kinase activity, which is also closely related to the cycle regulation of icaritin (Cellular senescence, induced when cells undergo irreversible cycle arrest, is one of the main ways to treat tumor . Networkicaritin .Senescence is mainly associated with G0/G1 arrest, however, our experimental results showed that icaritin-induced nasopharyngeal carcinoma cell lines were blocked in S phase and positive for SA-β-Gal staining, which is a very interesting phenomenon and warrants further exploration of the mechanism of icaritin-induced senescence. Studies have shown that senescence can be triggered by multiple stimuli, such as oxygen reactive substances and DNA damage . We specin vitro and in vivo investigations. Combining with network pharmacological analysis and cellular validation, we found that icaritin exerts anti-tumor effects by promoting ROS production, damaging DNA, and inducing S-phase arrest to inhibit proliferation of nasopharyngeal carcinoma cells. The results provide a new direction for exploring the mechanism of icaritin and its use in treatment of nasopharyngeal carcinoma.Icaritin has been shown to have effective anti-tumor properties in both


In this work, we evaluated the possible anti-inflammatory effects of MOMAST® GR25 in both acute and chronic inflammatory models. MOMAST® GR25 decreased the levels of prostaglandin (PG) E2 and 8-iso-PGF2α in isolated rat colon, liver, and heart specimens stimulated with lipopolysaccharide (LPS). In vivo, compared to controls, rats treated with MOMAST® GR25 (100 mg/kg to 1 g/kg) showed a significant reduction in both licking/biting time in the formalin test. In a rat model of osteoarthritis by monoiodoacetate (MIA) injection, MOMAST® GR25 showed pain-relieving properties when acutely administered, reducing mechanical hyperalgesia and spontaneous pain. Moreover, a repeated daily treatment with MOMAST® GR25 (300 mg/kg) fully counteracted osteoarticular pain without the development of tolerance to the antinociceptive effect. Taken together, our present findings showed that MOMAST® GR25 could represent a potential strategy for the treatment of inflammation and pain.MOMAST Olea europaea L. (family Oleaceae) is a typical plant of the Mediterranean area known for its recognized pharmacological properties [Olea europaea have been attributed to the presence of phenolic components, which showed antioxidant, anti-inflammatory, immunomodulatory, anti-atherogenic, hepato-protective, anticancer, antimicrobial, hypolipidemic, and hypoglycemic activities [operties . The bentivities . In parttivities ,4,5.Hydroxytyrosol (HT) and the secoiridoid oleuropein (OE) are two abundant phenolic compounds in virgin oil, olives, and waste water from olive oil production ,7,8. ParInterestingly, verbascoside is a phenylpropanoid glycoside found in a variety of plant species ,13, incl® GR25  is a polyphenolic granular complex rich in HT and other polyphenols from olive  . MOMAST® GR25 is characterized by the presence of HT, tyrosol, verbascoside, OE, and other polyphenols, whose identification was performed according to the laboratory organization and technical conditions of analysis requirements [MOMASTirements  , two polyphenolic liquid complexes from olive  pressing juice, exerted protective effects, by decreasing lactate dehydrogenase (LDH), prostaglandin (PG)E2, and 8-iso-PGF2α, levels, as well as tumor necrosis factor α (TNFα), cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) gene expression, in rat isolated tissues, after treatment with lipopolysaccharide (LPS) [A previous study from ours showed that MOMASTde (LPS) .® GR25 on isolated rat colon, liver, and heart specimens treated with LPS, a validated ex vivo model of inflammation, comparing it to HT. Moreover, we studied the acute and chronic effects of MOMAST® GR25 in a formalin test and in a rat model of monoiodoacetate (MIA)-induced osteoarthritis.In the present study, we evaluated the possible anti-inflammatory effects of MOMASTAdult Sprague-Dawley male rats (200–250 g) were housed in Plexiglass cages (40 cm × 25 cm × 15 cm), in colony rooms , on a 12 h/12 h light/dark cycle (light phase: 07:00–19:00 h), with free access to tap water and food, 24 h/day throughout the study, with no fasting periods. Rats were fed with a standard laboratory diet. The detailed Materials and Methods are provided in Colon, liver, and heart specimens were obtained from rats treated with the vehicle from our previous experiments, approved by Local Ethical Committee (University “G. d’Annunzio” of Chieti-Pescara) and Italian Health Ministry .All animal manipulations were carried out according to the Directive 2010/63/EU of the European parliament and of the European Union council (22 September 2010) on the protection of animals used for scientific purposes and with IASP. The ethical policy of the University of Florence complies with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health . Formal approval to conduct the manipulations and experiments described was obtained from the Italian Ministry of Health (No. 171/2018-PR) and from the Animal Subjects Review Board of the University of Florence. 2 inhalation (100% CO2 at a flow rate of 20% of the chamber volume per min), and tissue specimens were immediately collected and maintained in a humidified incubator with 5% CO2 at 37 °C for 4 h, in RPMI buffer with added bacterial LPS (10 μg/mL) (incubation period).Rats were sacrificed by CO® GR25 (5 and 25 µg/mL) and HT (5 and 25 µg/mL), which were used as the reference standard. Tissue supernatants were collected, and the PGE2 and 8-iso-PGF2α levels (ng/mg wet tissue) were measured by radioimmunoassay (RIA), as previously reported [During the incubation period, tissues were treated with scalar concentrations of MOMASTreported .® GR25 was orally administered, suspended in a 1% carboxymethylcellulose sodium salt (CMC) solution, at doses of 100 mg/kg, 300 mg/kg and 1g/kg. A standardization of testing conditions was carefully taken [After 2-week acclimation, MOMASTly taken .Formalin solution was injected, subcutaneously, under the plantar surface of a hind paw, and pain-related behaviors were scored during two successive phases, as previously reported .A MIA  injection was used to induce unilateral osteoarthritis into the tibio-tarsal joint ,23. A moThe evaluation of mechanical hyperalgesia was performed by an analgesimeter , as previously described by Leighton and collaborators (1988) and Bird and collaborators (2016) ,25.The evaluation of spontaneous pain was performed by an incapacitance apparatus  in order to record variations in the postural equilibrium ,27. An uStatistical analysis was performed using GraphPad Prism version 5.01 for Windows . Means ± SEM were measured for each experimental group and statistical analysis of data was performed by one-way analysis of variance (ANOVA), followed by Newman–Keuls comparison multiple test or by Bonferroni test .® GR25 (5 and 25 µg/mL) supplementation on PGE2 and iso-PGF2α levels in colon, liver, and heart treated with LPS. In this context, PGE2 and 8-iso-PGF2α levels, in the supernatants of tissues, were analyzed by RIA, following the treatment of tissue specimens with LPS+MOMAST® GR25 (5 and 25 µg/mL), LPS+HT (5 and 25 µg/mL), LPS, or vehicle. LPS treatment significantly increased PGE2 and 8-iso-PGF2α levels, with respect to vehicle, in all tested tissues. Compared to LPS, both MOMAST® GR25 (5 and 25 µg/mL) and HT (5 and 25 µg/mL) decreased PGE2 and 8-iso-PGF2α levels induced by LPS, in colon, liver and heart specimens  was more effective in inhibiting LPS-induced 8-iso-PGF2α in all tissues analyzed, with respect to HT-treated groups (® GR25 (5 and 25 µg/mL) was able to significantly reduce PGE2 levels as compared to HT  was orally administered 20 min before the test. Control animals received vehicle (1% CMC). After intraplantar injection of formalin, rats treated with MOMAST® GR25  showed short nociceptive behavioral responses (first and second phase) compared to vehicle-treated group  significantly decreased nociceptive behavioral response compared to the vehicle-treated group  when the neuropathic component is predominant. The animals were tested before (0 min) and after the administration of MOMAST® GR25. The effect of MOMAST® GR25 on the animal’s pain threshold was measured using a painful mechanical stimulus (Paw Pressure test), while the incapacitance test was used to measure spontaneous pain by evaluating the difference between the weight burdened on the contralateral paw vs the ipsilateral one. In the presence of monolateral pain, the animal tends to load more weight on the contralateral paw than on the ipsilateral leg injected with algogen MIA agent.To confirm the potential anti-inflammatory effect of MOMAST® GR25, at doses of 100 mg/kg, 300 mg/kg, and 1 g/kg, 14 days after damage induced by MIA on the ipsilateral paw. The weight borne by these animals on the ipsilateral paw at the paw pressure test is significantly reduced compared to control animals (vehicle + vehicle) . In this pain model, the highest tested dose (1 g/kg) of MOMAST® GR25 completely reversed the hyperalgesia induced by MIA at 30 min after administration, reaching a value of 65.8 ± 0.8 g, and the anti-hyperalgesic effect was still active even at 15 min (52.5 ± 1.4 g) and remained up to 45 min (57.5 ± 1.4 g). The intermediate dose of 300 mg/kg increased the threshold of pain in mice from 15 min from administration up to 45 min. The anti-hyperalgesic effect generated is lower than that achieved with the dose of 1 g/kg but statistically significant with respect to the control. The dose of 100 mg/kg showed a mild effect, increasing the pain threshold only 30 min after treatment (44.2 ± 0.8 g). No significant change was found on the contralateral paw after administration of MOMAST® GR25, thus excluding any central analgesic effect (c effect .® GR25 (1 g/kg) was able to reduce the weight difference burdened by the animal on the hind legs 15 min after administration (22.8 ± 1.6 g), the efficacy increased at 30 min, reaching the value of 7.1 ± 1.8 g, remained significant even at 45 min (17.6 ± 1.1 g), and then ended at 60 min (40.3 ± 2.0 g). The intermediate dose of 300 mg/kg partially reduced the weight difference between the two legs at 30 min after treatment, while the lowest dose in this test was ineffective . Using this experimental protocol, the protective effect of MOMAST® GR25 on MIA-induced osteoarthritic damage was evaluated. Treatment was administered daily and started in parallel with the induction of the damage (day 1). Pain measurements were conducted 7 and 14 days after the beginning of the experiment, both 24 h after the last administration and 30 min after the last daily treatment.The same behavioral measures (paw pressure and incapacitance tests) were performed following repeated treatments with MOMAST® GR25 prevented the development of joint pain when it was measured 24 h after the last dose; the measurement made 30 min following the new daily administration showed no significant changes in the algic threshold value recorded. The anti-hyperalgesic effect did not show tolerance since similar results were obtained 14 days after the start of treatment. At this time, MOMAST® GR25 was able to prevent the onset of mechanical hyperalgesia measured with the paw pressure test. Similar results were found in the incapacitance test  showed that OE significantly slowed down the progression of osteoarthritis lesions in animals [® GR25, acutely administered before the behavioral tests 14 days after damage, determined pain relief and a reduction in postural unbalance in a dose-dependent manner (100 mg/kg–1 g/kg) for at least 45 min. The therapeutic effect was accompanied by the protective one when the treatment with MOMAST® GR25 (300 mg/kg) started the same day of MIA injection (day 1) and continued, daily, for two weeks. Chronic treatment with MOMAST® GR25 fully prevented the development of mechanical hyperalgesia and spontaneous pain at both 7 and 14 days. Interestingly, the repeated administration of MOMAST® GR25 did not induce tolerance to the anti-nociceptive effects, in contrast to other drugs, such as morphine, that shows tolerance to the analgesic effects both in naïve [® GR25 did not significantly modify the pain threshold of the contralateral (control) paw, thus excluding central analgesic properties and highlighting a pure anti-hypersensitive profile. Many of the behavioral effects are in line with the antioxidant property of MOMAST® GR25 previously described. Mounting evidence suggests that the production of ROS is upregulated during persistent inflammatory and neuropathic pain [® GR25 in osteoarthritis pathology since it has been able to exert direct effects on the antioxidant system by stimulating glutathione peroxidase, SOD, and glutathione reductase activities and also maintaining higher reduced glutathione (GSH) levels in cells [® GR25 could also be attributed to presence of verbascoside. It possesses substantial beneficial activities and exerts anti-inflammatory, analgesic, and antioxidative properties in in vitro and in vivo studies [Our study demonstrates that MOMAST effects ,31. In t effects . Oxidati effects ,34. Inte effects . Actualldiabetes ,37,38. Arageenan . In addirageenan , as wellembranes . On the embranes ,43. MIA embranes ,45; its embranes ,46,47, iembranes . All theembranes . Indeed,embranes . Our fin animals . In part animals . In thisin naïve ,51 and iin naïve , which rin naïve . Furtherhic pain . To supphic pain . Olive ohic pain . In thisin cells ,58. In a studies ,60,61. I studies . Doses f studies . The exp studies . General studies ,66 exist® GR25 could reduce production of these pro-inflammatory markers in serum. Another limitation of our study is that we have not evaluated the effects of the MOMAST® GR25 administration on systemic cytokine levels, which could contribute to further deepening the potential mechanisms involved in the protective effects induced by this polyphenolic complex. In conclusion, taken together, our present findings showed that MOMAST® GR25 could represent a potential strategy for the treatment of inflammatory reactions and pain.Kim et al. 2016) showed that serum PGE2 levels were significantly higher in the vehicle-treated MIA group compared with the control  group, in rats, suggesting a possible link between behavioral measures and PGE2 levels . Accordi16 showed