A simple Δ15N-based isotope mass balance (IMB) model has been proposed to provide estimates of efflux/influx (E/I) ratios across root plasma membranes, the proportion of inorganic nitrogen assimilation in roots (Proot) and translocation of inorganic nitrogen to shoots (Ti/Tt) under steady-state conditions. We used the IMB model to investigate whether direct selection for yield in canola (Brassica napus L.) has resulted in indirect selection in traits related to nitrogen use. We selected 23 canola lines developed from 1942 to 2017, including open-pollinated (OP) lines developed prior to 2005 as well as more recent commercial hybrids (CH), and in three separate experiments grew them under hydroponic conditions in a greenhouse with either 0.5 mM ammonium, 0.5 mM nitrate, or 5 mM nitrate. Across all lines, E/I, rootP and Ti/Tt averaged 0.09±0.03, 0.82±0.05 and 0.23±0.06 in the low nitrate experiment, and 0.31±0.06, 0.71±0.07 and 0.42±0.12 in the high nitrate experiment, respectively. In contrast, in the ammonium experiment average E/I was 0.40±0.05 while Ti/Tt averaged 0.07±0.04 and rootP averaged 0.97±0.02. Although there were few consistent differences between OP and CH under nitrate nutrition, commercial hybrids were collectively better able to utilize ammonium as their sole nitrogen source, demonstrating significantly greater overall biomass and a lower rootP and a higher Ti/Tt, suggesting a somewhat greater flux of ammonium to the shoot. Average root and whole-plant Δ15N were also slightly higher in CH lines, suggesting a small increase in E/I. An increased ability to tolerate and/or utilize ammonium in modern canola hybrids may have arisen under intensive mono-cropping.Nitrogen isotope discrimination (Δ Brassica napus L.), and has become one of the world’s most important oil crops (Nitrogen (N) is the primary limiting nutrient for most plants growing in natural and agricultural ecosystems  and nitrmissions  and groumissions . Canola il crops . Like otil crops . Though il crops . Better E/I) describes the bi-directional movement of inorganic N between root and rhizosphere and relates to the N-uptake efficiency; i.e., a low E/I indicates a high uptake efficiency because there is less leakage of inorganic N back to the rooting medium.Both nitrate and ammonium have two major transport systems responsible for N uptake: high-affinity (HATS) and low-affinity (LATS) transport systems that are most effective at low or high N concentrations, respectively . Nitrate14N and 15N), with 14N being the predominant form . However, the stable isotopes of N may show differences in rates of chemical reaction, or in physical processes such as diffusion, that result in small changes in 15N/14N ratios between N pools. Changes in the relative abundance of 15N and 14N, called isotope fractionation, can provide integrated or tracer information about N fluxes within/through ecosystems  E/I at the root, (2) leaf vs. root partitioning of N assimilation, and (3) fluxes of inorganic and organic N to the shoot. E/I in balsam poplar . The highly correlated E/I from the two methods suggested the IMB model can be used for estimating N-uptake efficiency (1 – E/I), which is difficult to measure directly. In the second approach, the IMB model was tested by measuring the δ15N of inorganic and organic N forms in xylem sap in black cottonwood (Populus trichocarpa Torr. & Gray) to compare the calculated proportion of inorganic nitrogen assimilated in roots (rootP) and translocation of inorganic nitrogen to shoots (Ti/Tt) to direct measurements. Salix eriocephala Michx.) under In a series of articles, utrition , and in utrition  and hearr NO3− .13C) of plant tissue can provide information on intrinsic water-use efficiency of C3 plants  lines developed prior to 2005 as well as more recent commercial hybrids (CH), and in three separate experiments grew them under hydroponic conditions in a greenhouse with either 0.5 mM ammonium, 0.5 mM nitrate or 5 mM nitrate. We hypothesized that direct selection for yield in canola may have resulted in the indirect selection of N-uptake efficiency, NUE and WUE.The experiment used 23 historical canola lines developed in Canada from 1942 to 2017, including open-pollinated lines developed prior to 2005 and more recent commercial hybrids Table 1.-2s-1) on an 18/6 h day/night photoperiod. Temperatures in the greenhouse were maintained between 20-24°C. To prevent overheating of the hydroponic solution, a 10 m cooling coil of ¼ inch stainless steel pipe was submersed in the main reservoir. Cold tap water flowed through the coil under the control of a thermostated solenoid valve .The hydroponic system Figure 1th strength Johnson’s solution  supplemented with either 0.5mM or 5mM nitrate (as Ca(NO3)2; δ15N= 3.5‰) for the low nitrate or the high nitrate experiment, respectively, and 0.5mM ammonium (as (NH4)2SO4; δ15N= 0.2‰) for the ammonium experiment. The solution was monitored daily for oxygen levels, pH and temperature. Powdered calcium carbonate (CaCO3) was added to buffer pH in the range of 6-7.5. Media 15N of the hydroponics solution. However, based on the total nitrogen content and isotopic composition of the harvested plants, 10.22, 21.26 and 2.36% of the supplied N was consumed in the ammonium, low nitrate and high nitrate experiments, which would have resulted in isotopic enrichments of 0.5, 0.4 and 0.1‰, respectively. Accordingly, source δ15N values were adjusted by these amounts for purposes of calculating discrimination.The hydroponics solution was a modified 1/10d method  and phend method . The sol13C and δ15N using a Vario EL Cube Elemental Analyzer  interfaced to an Isoprime Isotope Ratio Mass Spectrometer  in the Stable Isotope Lab, Agriculture and Agri-Food Canada Lethbridge Research and Development Centre, Lethbridge, Alberta, Canada.Canola plants were harvested into leaves, stems, and roots after 45 days of growth. Once freeze-dried, these parts were weighed and then pulverized using a Wiley mill  followed by a Geno/Grinder . Sub-samples (3 mg) were packed into tin capsules and analyzed for %C, %N, δ15N) and isotope discrimination (Δ) were expressed as:Nitrogen isotopic composition (δRsample and Rstandard are the 15N/14N ratios of the sample and the arbitrary standard (air N2), respectively. Plants were divided into three major parts , and the whole-plant isotope discrimination was calculated as the weighted sum of these parts:where, 15Ni is the discrimination relative to the external media N source and if is equal to the fraction of tissue nitrogen contributing to overall plant nitrogen content. The percent ratio of inorganic nitrogen (Ti) relative to total nitrogen (Tt) translocated to the leaves (Ti/Tt), as predicted by the IMB model  or GS (17‰), as appropriate for either nitrate or ammonium assimilation. Assuming leaves and roots are the major sites of nitrogen assimilation, the amount of stem N  that originated from the leaves (stem-leaff) was approximated by:where, Δand the proportion of total plant nitrogen assigned to the leaf pool was:15Nstem frequently exceeded Δ15Nroot in the ammonium experiment, in which case all stem N was assumed to originate from the roots and stem–leaff was assigned a value of zero. The proportion of total plant nitrogen assimilated in the roots (rootP) was given by:ΔE/I) was then calculated as:and efflux over influx  as a fixed factor and historical lines nested within the group effect. Pearson correlation coefficients (r) were used to examine relationships between all physiological variables determined among the 23 canola lines.All statistical analyses used R version 3.5.1 . One-wayPlants reached greater size in the nitrate experiments than in the ammonium experiment, but given that these were all separate experiments, we are unable to test for differences in performance between N sources or concentrations. There were no obvious signs of N limitation or toxicity in all three experiments. Foliar nitrogen averaged (±SD) 4.48±0.69% in the 0.5mM (low) nitrate experiment, 4.90±0.94% in the 5 mM (high) nitrate experiment, and 5.64±1.27% in the 0.5 mM ammonium experiment, which is within the expected 3-6% sufficiency range for herbaceous plants .15N in the ammonium experiment. Across all 23 canola lines, the leaf, stem and root Δ15N averaged (±SD) 6.1±0.8‰, 8.2±0.9‰ and 7.1±1.0‰, respectively (P< 0.001) Group means, ranges and statistical differences are summarized in 15N, whole-plant Δ15N and whole-plant δ13C. The difference in the whole-plant Δ15N can largely be ascribed to the difference in root Δ15N. The CH group had 76% higher biomass than the OP group, while the root:shoot ratios were similar. The CH group also had significantly higher average root and whole-plant Δ15N than the OP group, by 0.6‰ and 0.3‰, respectively. For whole-plant δ13C, the CH group was 0.5‰ higher than the OP group. Although the differences were small, the IMB model estimates suggested significantly higher E/I and Ti/Tt, and lower Proot in the CH group as compared to the OP group. There was no statistical difference between OP and CH lines in whole-plant C/N, being 9.95±2.03 for the OP group and 10.40±2.44 for the CH group, respectively.Plants showed considerable discrimination against 15N was 0.7±0.8‰, 1.7±0.8‰ and 5.7±1.3‰, whereas whole-plant Δ15N and δ13C were 1.7±0.6‰ and -30.8±0.7‰, respectively. Estimated E/I, rootP and Ti/Tt averaged 0.09±0.03, 0.82±0.05 and 0.23±0.06, respectively.Unlike the ammonium experiment, there were no statistically significant differences between the OP and the CH groups in either the low nitrate experiment Table 3 15N were 2.0±2.4‰, 6.0±1.2‰ and 11.3±0.9‰, and the whole-plant Δ15N and δ13C were 4.8±1.3‰ and -31.5±0.6‰, respectively. The average E/I was 0.31±0.06, while rootP was 0.71±0.07 and Ti/Tt was 0.42±0.12.In the high nitrate experiment, whole-plant biomass averaged 11.92±6.65 g and the mean root:shoot ratio was 0.16±0.13. Mean values for the leaf, stem and root ΔrootP and Ti/Tt were consistently and very strongly negatively related to each other in all three experiments (P< 0.001), as would be expected based on Equation 7. Otherwise, the strength and direction of trait-to-trait correlations varied considerably between experiments.Genetic correlations between growth and isotope-based physiological traits in each experiment are shown in Ti/Tt (P< 0.05), and negatively to rootP (P< 0.05). Root:shoot ratio was almost but not quite significantly negatively correlated to whole-plant δ13C (P = 0.058). rootP was negatively correlated with biomass (P< 0.05). Whole-plant δ13C was significantly and positively correlated with the whole-plant C/N ratio (P< 0.05).In the ammonium experiment, whole-plant biomass was positively correlated to 13C (P< 0.01) but not quite E/I (P = 0.061), and negatively correlated to root:shoot ratio (P< 0.05). Similar to the ammonium experiment, E/I was not correlated with either Ti/Tt or rootP, however rootP was also not significantly correlated with biomass. In sharp contrast to the ammonium experiment, whole-plant δ13C was positively related to E/I (P< 0.01) and negatively related to whole-plant C/N, but not quite significantly so (P = 0.063).In the low nitrate experiment, biomass was positively correlated to whole-plant δ13C (P< 0.05). In contrast to both the ammonium and the low nitrate experiments, root:shoot ratio was significantly positively correlated to rootP and E/I (P< 0.001). Further, rootP was significantly and strongly correlated to E/I (P< 0.001). In concordance with the ammonium experiment, but not the low nitrate experiment, whole-plant δ13C was positively related to whole-plant C/N (P< 0.001). By contrast, in concordance with the low nitrate experiment, but not the ammonium experiment, whole-plant δ13C was positively related to E/I (P< 0.001).In the high nitrate experiment, biomass was positively correlated only to whole-plant δ14N) over heavier N (15N) during assimilation, organic N in plants is generally depleted in 14N while remaining unassimilated inorganic N is enriched  and lowest in the low nitrate experiment (1.7‰), with the high nitrate experiment in the middle (4.8‰). The leaf Δ15N, which represented up to 70% of plant N in this study and is often used as a proxy for whole-plant Δ15N, also followed the same order.There are three major competing fates for inorganic N taken up by roots, including (1) assimilation in the root cytosol, (2) loading of unassimilated N into the xylem for root-to-shoot translocation, or (3) efflux back to the rooting medium . Since tenriched . In this15N by NR is thought to exceed discrimination by GS , lodgepole pine (Pinus contorta Dougl.) and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco), and in every case found E/I was increased at the higher concentration.Measuring g plants . Due to ectrodes  or by tha tracer . These m15N values, on average, were both close to zero, while stem Δ15N and root Δ15N were not. We found similar patterns in nitrate-grown black cottonwood and heart-leaved willow  and by combining it with the organ level data on N partitioning (Equation 7). In the nitrate experiments, leaf Δd willow . Leaf Δ1le e.g., . The oveE/I and other flux parameters using the CATE or microelectrode methods has been limited to a few genotypes  was supported by the group differences we found in the ammonium experiment. In this experiment, the CH group had better growth than the OP group, as well as higher Δ15N at the root, stem, leaf and whole-plant levels. The commercial hybrids also had a greater mean leaf-root Δ15N difference. These differences indicate that the commercial hybrids, which are more recent than the open-pollinated lines, tend to have higher E/I and Ti/Tt, and a lower rootP. The higher Ti/Tt and lower rootP suggest that hybrid lines have a slightly greater capacity for ammonium translocation in the xylem and proportionally greater ammonium assimilation in the leaves. The greater relative growth of these lines when provisioned solely with ammonium also suggests increased resistance to ammonium toxicity. In contrast, the slightly higher E/I suggests more futile cycling at the soil-root interface and a modest reduction in uptake efficiency. This result is surprising, as we might initially expect a greater growth demand to result in relatively less N leakage, not more. Overall, the slightly higher efflux implies an even faster rate of ammonium uptake by the CH group. By harvest, we calculate that gross N influx and efflux averaged 244±18 and 97±14 mg N per plant for the OP group, and 430±38 and 182±33 mg N per plant for the CH group, respectively.Another profound advantage of the IMB model is its utility for screening large numbers of plants. The assessment of genotypic variation in es e.g.,  or to spes e.g., . In the 15N relative to root Δ15N observed in the ammonium experiment underscores an important limitation of the current IMB model in assuming that the root inorganic N content is negligible. Ti/Tt with consequent effects on the estimation of rootP and, to a lesser extent, E/I. They also noted that the fraction of inorganic N in roots typically varies from 1 to 10% of the total N . We estimated the genetic correlations between key physiological traits and biomass across all 23 canola lines. The trait-to-trait correlations varied considerably between experiments , but the calculation of E/I is independent of root mass. Increased root-to-shoot ratio was also correlated with increased E/I in heart-leaved willow  or in whole-plant δeriments Figure 3d willow  and in bd willow . A highed willow .This paper represents the first use of the IMB model to assess time-integrated nitrogen-use traits in a commercial crop. Relative to older open-pollinated lines, modern canola hybrids appear to be collectively able to better utilize ammonium as their sole nitrogen source. Selection for yield in canola may have resulted in this change. In western Canada, urea is the predominant source of nitrogen used to fertilize canola . Given tThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.YH performed hydroponics experiments, analyzed data, and drafted the manuscript. RDG provided scientific insight into data analysis and edited the manuscript. RYS conceived of the study, obtained funding, and edited the manuscript. All authors contributed to the article and approved the submitted version.This work was funded by the Canola Agri-Science Cluster (2018-23), a partnership between Agriculture and Agri-Food Canada (AAFC) and the canola industry  under the Canadian Agricultural Partnership to RYS. Research for RDG was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant GR004049. YH received stipends through the Research Affiliate Program of AAFC.B. napus breeder, AAFC-Saskatoon) and the canola industry partners for providing the seed material, Brett Hill (AAFC-Lethbridge) for assistance with stable isotope analysis, and Melina Biron (UBC-Vancouver) for advice on hydroponic system design.We would like to thank Sally Vail (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The intercompartmental clearance (Q) and the volume of the peripheral compartment (pV) were 2.85 liters/h and 6.01 liters, respectively. In the final model, we found that albumin (ALB) affected the CL of caspofungin with an adjustment factor of −1.01, and no other covariates were identified. In this study, HTx was not found to affect the PK of caspofungin. Based on the simulations, the dose of caspofungin should be proportionately increased in patients with decreased ALB levels.The effect of heart transplantation (HTx) on the pharmacokinetics (PK) of caspofungin is not well-characterized. The aim of this study was to investigate the population PK of caspofungin in HTx and non-HTx patients and to identify covariates that may affect the PK of caspofungin. Seven successive blood samples were collected before administration and at 1, 2, 6, 10, 16, and 24 h after the administration of caspofungin for at least 3 days. This study recruited 27 HTx recipients and 31 non-HTx patients with 414 plasma concentrations in total. A nonlinear mixed-effects model was used to describe the population PK of caspofungin. The PK of caspofungin was best described by a two-compartment model. The clearance (CL) and volume of the central compartment ( Invasiv2–an cells .After HTx, patients need to be monitored in the surgical intensive care unit (SICU). Renal complications  and low-16–20–25–A total of 58 patients and 414 plasma concentrations were included in the study. We divided these patients into two groups. A total of 27 patients  who received HTx were recruited in this study as the HTx group, and 31  critically ill patients who had not received HTx were enrolled as the control group. The demographic and clinical characteristics of the study population are summarized in After analysis of one-, two, and three-compartment models, the time course of plasma caspofungin concentrations was found to be best described by a two-compartment model with an additive residual error of 0.213 mg/liter and a proportional error of 13.4%. The population PK parameter estimates with 95% confidence intervals (CI) based on bootstraps are listed in Goodness-of-fit (GOF) plots are displayed in cV), interventricular space (Q), and peripheral compartment distribution volume (pV) of the HTx group were 3.14 liters (1.35 to 10.02 liters), 2.85 liters/h, and 4.98 liters (1.52 to 8.59 liters ), respectively. The cV, Q, and pV of the control group were 5.27 liters (0.81 to 14.03 liters), 2.85 liters/h, and 6.31 liters (4.07 to 11.73 liters), respectively. The median (range) area under the curve (AUC) values in the HTx group and control group were 126.86 mg·h/liter (26.78 to 292.27 mg·h/liter) and 105.64 mg·h/liter (52.21 to 217.99 mg·h/liter), respectively. The results indicated a significant difference between the two groups with respect to CL and AUC (P < 0.05) (The median (range) CL in the HTx group and control group were 0.35 liter/h (0.18 to 0.98 liter/h) and 0.45 liter/h (0.22 to 0.84 liter/h), respectively. The median (range) central compartment distribution volume ( < 0.05) .Candida spp. when MIC was 0.06 mg/liter or less. However, the common maintenance dose of 50 mg is not suitable for all situations. When patients infected by Candida glabrata and MIC 0.25 mg/liter or less, the recommended maintenance dose is 50–100 mg . For Candida albicans, the recommended maintenance dose is 50 to 200 mg when MIC is 0.25 mg/liter or less. For patients infected by Candida parapsilosis (MIC of 0.125 mg/liter or less), the recommended maintenance dose is 50 to 150 mg. For patients infected by C. parapsilosis (MIC was 0.25 mg/liter) and ALB of 30 g/liter or more, the recommended maintenance dose is 200 mg. More recommended dose regimens can be found in The probability of target attainment (PTA) values based on different dose regimens and minimum inhibitory concentrations (MICs) with ALB levels of 37 g/liter are shown in To the best of our knowledge, this is the first population PK analysis of caspofungin for HTx recipients. Our results suggest that HTx may not significantly affect the PK of caspofungin. However, we observed a significant difference in CL between the two groups. The possible reason for this difference was ALB levels. The ALB level in the HTx group was 40.3 g/liter (29.7 to 49.3 g/liter), while the ALB level in the control group was 33.7 g/liter (26.41 to 48.40 g/liter). Our finding suggests that ALB may associate with the CL of caspofungin.V of caspofungin , C. glabrata (MIC of 0.125 mg/liter or less), and C. parapsilosis (MIC of 0.03 mg/liter or less), the recommended maintenance dose of 50 mg can obtain a high PTA. This is consistent with previous studies  patients aged 18 years or older who received caspofungin in SICU and accepted cardiac surgery and (ii) acceptability of multiple blood sampling. Patients who were allergic to caspofungin, those receiving drugs that can affect the PK of caspofungin, and those who could not receive multiple blood sampling were excluded. Patients who underwent HTx in SICU were recruited as the HTx group. Patients who never received HTx but underwent cardiac surgery and being in SICU were recruited in the control group. This study was approved by the Ethics Committee of Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences H).The clinical data, including age, weight, height, sex, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), total protein (TP), albumin (ALB), serum creatinine (sCr), platelet count (PLT), procalcitonin (PCT), and creatinine clearance (CrCL) were evaluated on the day of sample collection. Other potential covariates included CRRT, ECMO, and baseline sequential organ failure assessment (SOFA) scores. The components of the SOFA scores were collected from medical records.g for 10 min at 4°C and stored at −80°C before analysis.Caspofungin  was administered by 1-h intravenous infusion at a dose of 50 mg every 24 h after a loading dose of 70 mg. Blood samples were collected before administration and at 1, 2, 6, 10, 16, and 24 h after the administration of caspofungin for at least 3 days. Plasma was separated by centrifugation of blood samples at 2,000 × m/z 547.3 → 538.2 for caspofungin and m/z 549.3 → 540.3 for caspofungin-d4 (IS). The linear range of the method was 0.4 to 25 mg/liter. The intraday and interday precision were 1.56 to 3.32% and 3.40 to 7.48%, respectively, and the deviation of accuracy was 0.96 to 2.21%.All plasma samples were detected by the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. First, 200 μL of acetonitrile-dissolved extraction working solution was added to 100 μL of plasma sample and mixed by vortexing at 3,000 rpm for 1 min. After centrifugation at 14,000 rpm for 15 min at 4°C, 2 μL supernatant was injected for LC-MS/MS analysis. The chromatographic column was an Agilent poroshell 120 SB-C18  column. The mobile phase was acetonitrile with 0.1% formic acid (A) and water with 0.1% formic acid (B). In the initial 0 to 0.5 min, the mobile phase was composed of 95% B and 5% A, and then within 0.5 to 1.5 min, the mobile phase B decreased linearly from 95% to 5% and remained unchanged for 1.5 to 2 min. Finally, it returned to 95% B and 5% A, and the entire analysis time was 5 min. The flow rate was 0.5 mL/min, and the column temperature was 35°C. A positive ion mode multiple reaction monitoring detection mode through electrospray ionization was performed with the transitions of We analyzed population PK data using the nonlinear mixed-effects modeling software NONMEM 7.3 . We employed one-, two-, and three-compartment pharmacokinetic models to fit the data and selected the most suitable basic structural model. Further, all potential covariates for caspofungin PK parameters reported in previous studies were analyzed. The covariates included demographic characteristics, renal function indices, liver function indices, HTx status, ECMO, and CRRT. Different covariate types have different variable models. Discrete covariates (such as sex) were modeled as follows.For continuous covariates (such as weight), an exponential model was selected with average covariates values and an adjusting factor.ijC is the individual value. tv,jC is the population typical value. cov in j is an impact factor. covave is the average value of a continuous covariate. Interindividual variability is shown as jη, which is normally distributed with mean 0 and variance ω2. Residual variability was modeled using a mixture of additive and proportional error models.In the above equations, 2. For the forward stepwise covariate evaluation, it was considered significant when the OFV was reduced by at least 3.84 ; for the backward covariate elimination, it was considered significant when the OFV was increased by at least 10.82 (α = 0.001).The first-order conditional interaction estimation method with interaction was used for the analysis. The asymptotic distribution of the change in the OFV is χIn our study, GOF plots, bootstrap, visual predictive check (VPC), and NPDE were used for model validation. VPC and bootstrap were conducted on Pearl-speaks-NONMEM version 4.8.0 . The GOF plots and NPDE statistics were generated using R and the add-on package NPDE version 2.0 . BootstrALB-CL, and CL interindividual variations and their calculated standard deviations from bootstraps  at various dosage regimens . The median ALB level of the research population was 37 g/liter. We generated 1,000 virtual patients whose pharmacokinetic parameters were randomly assigned according to the distributions with the median of CL, θotstraps  for the otstraps . The AUCectively . The tes2 test, Fisher’s exact test, and t test were used to evaluate the differences between the HTx group and control group.Statistical analysis was performed using SPSS version 23.0 . Population PK analyses were performed with the software NONMEM . Simulation was performed using Matlab R2017b . A Fortran compiler was adopted, and the runs were executed on Pirana (version 2.9.0). Categorical data are expressed as frequency (percentages) and continuous data are presented as medians (range). χThe raw data analyzed during the current study are available from the authors on reasonable request.


We report on the electrochemical determination of one the most effective and widely used chemotherapeutic, anti-inflammatory, and immunomodulator agents, methotrexate (MTX), using low-cost, green, and facile one-pot prepared graphitic carbon nitride (g-CN ) nanosheets. The g-CN nanosheets have been characterized utilizing Fourier transform infrared spectroscopy, X-ray diffraction(XRD), scanning electron microscopy(SEM), and density functional theory (DFT). In comparison to the bare carbon paste electrode (CPE), the g-CN -modified electrode showed a spectacular enhancement in the electrochemical oxidation and detection abilities of MTX. The proposed material exhibits very low limits of detection (12.45 nM) and quantification (41.5 nM), while possessing a wide linear range of 0.22–1.11 μM and 1.11–27.03 μM under optimized conditions at pH 7.0. Due to the ease of preparation of g-CN, it can be adopted for the cost-effective detection of MTX in industrial and clinical analyses. N- methyl] (methyl)amino}benzoyl)-L-glutamic acid) is an antineoplastic [Methotrexate (MTX)  as an electrode modifier. In addition to being a low-cost material, g-CN can be synthesized via environmentally friendly routes at large scales ,38. More2SO4) and dimethylformamide (DMF) were purchased from Sigma-Aldrich . Hydrochloric acid (HCl) was purchased from Honeywell . Carbon black and graphite sheets with 0.3 mm thickness were obtained from Xinruida, . All the materials used were reagents of analytical grade without any further purification. Deionized water (DI) was prepared by an ultrapure water system using Millipore Direct-Q3 with UV from Merck .Urea was purchased from Alfa Aesar  and was purified extra through recrystallization in absolute ethyl alcohol. Ethylenediaminetetraacetic acid disodium and polyvinylidene difluoride powder (PVDF) were also obtained from Alfa Aesar. Sulfuric acid was created. The paste was then carefully packed into the electrode’s cavity. Before use, the CPE’s surface was polished on a fresh piece of paper. Prior to scanning, the CPE was submerged in the supporting electrolyte. The paste was emptied, renewed, and polished following each scan. By combining 1.0 g of graphite with 15 mg of g-CN and homogenising the mixture for two minutes with a spatula, the modified electrode was created. To create the paste, 0.27 mL of paraffin was also added and mixed for 45 min. The paste was packed and regenerated as previously mentioned. The characterization and computational procedures are included in the Plasma. Fresh plasma samples were provided for the tests by a healthy person. By adding 10 mL of the supernatant to 9 mL of pH 7 Britton–Robinson buffer (BRB), MTX stock solution concentration was obtained. The solution was then, without further pretreatment, transferred to an electrochemical cell for analysis.®.Methotrexate Five Methotrexate® 2.5 mg tablets were weighed and ground to prepare a stock solution containing 1 mM of MTX. To 25.0 mL of ethanol, the calculated quantity of the powder containing a specified amount of drug is added. Hence, 25.0 mL of ethanol was added to the flask and mixed with a vortex mortar to dissolve the added amount. After filtering the solution through 0.45-m filter paper, the 1 mM of MTX stock solution was prepared for further analysis.Urine. Fresh urine samples were taken from a healthy person prior to the experiments. The desired concentration was achieved by adding various concentrations of MTX stock solution to 10 L of supernatant that had been spiked to 9 mL of pH 7 BRB. Without applying any additional pretreatment, the solution was transferred to an electrochemical cell for analysis.2-bond, replaced one carbon atom in the aromatic ring. As a result of this alteration, the electronic characteristics of the graphite layer are improved, and the electrochemical oxidation of the carbon surface is prevented. While the other, less distinct peaks at 399.1, 400.9, and 404.1 eV, respectively, are for tertiary nitrogen (N single bond C), amino nitrogen (N single bond H), and nitrogen-oxide groups. It is noteworthy that the estimated nitrogen content of the g-CN powder surpasses 12%. The HR-XPS O 1s spectrum in The morphology of the prepared g-CN nanosheets was investigated using Field emission scanning electron microscopy (FESEM) as shown in 2/g. In addition, the Barrett-Joyner-Halenda (BJH) model was used to determine the total pore volume and average pore diameter, as shown in 3/g and 1.868, respectively. The nitrogen adsorption/desorption isotherm of the synthesized g-CN nanosheets is shown in −5 Ha, 0.002 Ha/Å and 0.005 Å for energy, force, and displacement tolerance, respectively, through a Monkhorst−Pack k-point sampling of (2 × 2 × 2) [adsE, g-CNE, MTXE, and totalE are the energy of the adsorbed molecule, the total energy of the surface isolated, the total energy of the single adsorbate, and the total energy of the adsorbate on the adsorbent, respectively.The structures of the MTX and g-CN bulk  were lef× 2 × 2) . As MTX × 2 × 2) , as listBefore setting the possible scenarios for the adsorption of MTX on g-CN, the possibility of adsorbing MTX via its central atoms was excluded due to the steric hindrance of its neighboring groups . The totThe mechanism of adsorbing the amino group through its Nitrogen atom on the g-CN surface was due to the difference in the electronegativity between the two atoms, which caused the development of partial negative charges (∂−) on the Nitrogen of the amino group and partial positive charges (∂+) on the carbon atom of the g-CN, leading to the attraction between them. However, the electronegativity difference between Hydrogen and Nitrogen of the amino group was found to be 0.84 eV , which i−3 M K3Fe(CN)6 in 0.10 M KCl and recording the current vs. voltage peak at different scan rates, the electroactive surface area of the prepared g-N/CPE sensor platform was calculated from the cyclic voltammograms using the Randles–Ševčík equation for a quasi-reversible reaction, Equation (2) [A is the electrode electrochemical surface area, C is the concentration of the redox probe, D is the diffusion coefficient, pI is the peak current, and n is the number of electrons involved in the electrochemical anodic oxidation. K3Fe(CN)6 has a D value of 7.6 × 10−6 cm2 s−1 [2 and 0.0506 cm2, respectively, using the slopes of the pI vs. 1/2 plot and Equation (2).For the purpose of sensing MTX in Britton–Robinson (BR) buffer at 7.0 pH using 0.10 mM of MTX, square wave voltammetry (SWV) was used to examine the electrochemical behavior of bare against g-CN nanosheets-modified CPE platforms. In the absence of MTX, CPE there are no discernible anodic or cathodic peaks, indicating electrochemical inactivity of the sensor platform within the working-potential window. The anodic oxidation peak current of MTX on bare CPE was 13.61 μA at 0.78 V, which was dramatically increased upon the use of g-CN, reaching 24.96 μA at 0.78 V and demonstrating the simple oxidation of MTX. This means that the sensitive lower potential detection of MTX can be accomplished using g-CN /CPE. The highest electrochemical activity toward MTX, good conductivity, and a high rate of electron transfer have all been demonstrated for 15 mg of g-CN /CPE, as shown in tion (2) :(2)IP=2. cm2 s−1 . The ele−3 M K3Fe(CN)6 in 0.1 M KCl was utilized to examine the reaction kinetics, mass transport, and charge-transfer coefficient at the electrode surface using electrochemical impedance spectroscopy (EIS), as shown in CT) at the surface of the electrode from the diameter of the quasi-circle in the high-frequency window. A typical Warburg-type equivalent circuit model can be obtained from the Nyquist plots. As a result, when using the proposed g-CN to modify the CPE, the charge transfer is superior using the bare CPE. When the bare CPE is modified with g-CN, the RCT drops dramatically from 4400 to 2616.2 Ω, which can be attributed to the large surface area and interactive nature of g-CN that facilitates electron transfer. Additionally, −3 M K3Fe(CN)6 in 0.1 M KCl. The g-CN/CPE electrode has an anodic peak current value that is nearly 1.5 times greater than that of bare CPE. Additionally, compared to the bare CPE, the use of the g-CN/CPE significantly reduced the peak separation (Ep—Ep) from 0.34 to 0.21 V, indicating enhanced electron transfer. Therefore, g-CN has good conductivity, a high rate of electron transfer, and good catalytic activity toward the electrochemical oxidation of MTX.A 1:1 solution of 5.0 × 10p) is shifted into more positive values, indicating an irreversible electrochemical behavior of MTX. A linear relationship is observed, and the measured current is less than 0.5: pI (μA) = 0.4941 mVs−1 + 0.5006 µA, R2 = 0.9996, which indicates a diffusion-controlled process. Furthermore, the linear relationship between the peak current (pI) versus the square root of the scan rate (ν1/2) in the range of 0.010–0.250 V s−1. pI (μA) = 0.4941 mVs−1 + 0.5006 µA, R2 = 0.9996 also supports the idea that the anodic oxidation process is diffusion-controlled.The effect of scan rate on the electrochemical anodic oxidation of MTX on g-CN /CPE is investigated using cyclic voltammetry, as shown in −1, MTX shows two linear behaviors based on its concentration in the BRB. In the range of 2.22 × 10−7 to 1.11 × 10−6 M, the regression equation is pI (μA) = 1.9713 C μM + 2.0488 μA, R2 = 0.9971, while in the range of 1.11 × 10−6 to 27.03 × 10−6 M, the regression equation is pI (μA) = 0.4872 C μM + 3.6038 μA, R2 = 0.9983. The main cause for the decrease in the slope of the second linear range at higher concentrations is the increase in the required energy for anodic stripping in addition to the Ohmic drop at such high levels of MTX. Additionally, this platform’s limit of detection (LOD) was calculated using the formula LOD = 3 SD/x, where SD is the standard deviation of the peak currents of MTX oxidation for n trials (n = 5) and x is the slope of the calibration curve. LOD was found to be 12.449 nM. Using the formula LOQ = 10 SD/x and the limit of quantification (LOQ) was determined to be 41.496 nM.For the validation of the proposed sensing protocol, the approved International Conference on Harmonization (ICH) guidelines for validating analytical methods were adopted in our study ,52. Squa−1, MTX shows two linear behaviors based on its concentration in the BRB. In the range of 0.22 × 10−6 to 1.11 × 10−6 M, the regression equation is Ip (μA) = 1.9840 C μM + 2.0266 μA, R2 = 0.9924, while in the range of 1.11 × 10−6 to 27.03 × 10−6 M, the regression equation is pI (μA) = 0.5061 C μM + 3.6293 μA, R2 = 0.9990, as shown in Utilizing SWV at a scan rate of 0.1 V s−1, MTX shows two linear behaviors based on its concentration in the BRB. In the range of 0.11 × 10−6 to 1.11 × 10−6 M, the regression equation is Ip (μA) = 2.0735 C μM + 2.0071 μA, R2 = 0.9907, while in the range of 1.11 × 10−6 to 13.16 × 10−6 M, the regression equation is pI (μA) = 0.4872 C μM + 3.6038 μA, R2 = 0.9983, as shown in Utilizing SWV at a scan rate of 0.1 V s−1, MTX shows two linear behaviors based on its concentration in the BRB. In the range of 0.22 × 10−6 to 1.11 × 10−6 M, the regression equation is pI (μA) = 1.9422 C μM + 2.0606 μA, R2 = 0.9995, while in the range of 1.11 × 10−6 to 13.16 × 10−6 M, the regression equation is pI (μA) = 0.4969 C μM + 3.5521 μA, R2 = 0.9988.At a scan rate of 0.1 V s−1   and 0.1 −1 see .Due to the importance of monitoring MTX, it was extensively studied in the literature as shown in In conclusion, this study demonstrates the preparation of g-CN for the detection and determination of MTX in buffer, spiked plasma, urine samples, and dosage form. The g-CN /CPE sensor platform showed the highest cost-efficiency among other published counterparts. In addition, DFT calculations helped to elucidate the oxidation mechanism. The proposed material showed limits of detection and quantification of 12.45 and 41.5 nM, respectively, while possessing a wide linear range of 0.22–1.11 μM and 1.11–27.03 μM under optimized conditions at pH 7.0, revealing the spectacular potential of the investigated sensor for use in a plethora of electrochemical applications.


Astragalus propinquus Schischkin (RAP) plant, as a traditional Chinese medicine, has been widely used to treat stroke, cerebral ischemia, qi deficiency, and hypertension. Buyang Huanwu decoction is traditionally used to treat stroke in China for more than 200 years and has a significant effect on cerebral ischemia, and RAP is monarch medicine of Buyang Huanwu decoction. Therefore, this study was designed to observe the regulatory effect of RAP on transmembrane iron transporters and ferroptosis-related factors in cerebral ischemia-reperfusion injury (CIRI) in rats.  The dried roots of the − (System Xc−) light chain subunit (XCT) and heavy chain subunit (SLC3A2), glutathione peroxidase 4 (GPX4), nuclear factor erythroid 2-related factor (NRF2), heme oxygenase-1 (HO-1), and iron-responsive element-binding protein 2 (IREB2) in the brain tissues of rats was assessed by Western blotting. RAP decreased the infarct size and neuronal injury after CIRI in rats. Similarly, RAP treatment regulated the expression of iron transporters. As such, RAP was able to reduce the expression of Fn, FHC, FLC, Tf, TfR, DMT1, and TRPC6 and increase the expression of FPN1 through a Tf/TfR-independent pathway after CIRI in rats.  Middle cerebral artery occlusion (MCAO) was used to block blood flow in the blood supply area of the middle cerebral artery in seventy male SD rats to induce focal CIRI to establish a rat model of CIRI. RAP was administered to explore the regulatory effect of RAP on iron transmembrane transport under the condition of CIRI. The infarct size was measured using 2,3,5-triphenyl-tetrazolium chloride (TTC) staining, the pathological structure of brain tissue was observed by HE staining, and neuronal injury was evaluated by Nissl staining after treatment. Then, changes in the iron transporters ferritin (Fn), ferritin heavy chain (FHC), ferritin light chain (FLC), transferrin (Tf), transferrin receptor (TfR), divalent metal transporter 1 (DMT1), L-type calcium channel (LTCC), transient receptor potential canonical 6 (TRPC6), and ferroportin 1 (FPN1) were observed by immunohistochemistry staining (IHC) and Western blotting. The expression of key factors of ferroptosis, including the membrane sodium-dependent cystine/glutamate antiporter System Xc RAP stimulation inhibited ferroptosis by regulating the expression of the key ferroptosis factors XCT, SLC3A2, GPX4, NRF2, HO-1, and IREB2. In conclusion, RAP regulates transmembrane iron transport and ferroptosis to improve CIRI. Ischemic stroke is a destructive brain injury and one of the leading causes of death and physical disability worldwide , 2. RepeIron overload occurs resulting in DNA damage, lipid peroxidation, neuronal apoptosis, and mitochondrial autophagy in CIRI . Under n−, which consists of XCT and SLC3A2 and is mainly responsible for intracellular cystine uptake [Ferroptosis plays an important role in the occurrence and development of ischemic stroke by affecting iron metabolism and lipid peroxidation , 14. Thee uptake , 16. Thee uptake . Recent e uptake , 19.Thrombolytic and neuroprotective therapies are recommended for patients suffering from ischemic stroke. Thrombolytic therapy is one of the most effective treatment measures, but it has strict time window restrictions and side effects. Neuroprotective agents are considered to be the most promising drugs, and the purpose of their treatment is to reduce brain tissue injury and inhibit nerve cell death after cerebral ischemia reperfusion. Edaravone is an antioxidant free radical scavenger, but it has a single therapeutic target and also causes various adverse reactions .Astragalus propinquus Schischkin (RAP) is a traditional Chinese medicine (TCM), also known as Huang Qi or Radix Astragali [https://www.theplantlist.org. RAP was first recorded in the earliest Chinese pharmacy monograph “Shen Nong's Herbs” with more than 2000 years history [Astragalus propinquus Schischkin (Huangqi) 120 g, Angelica sinensis (Oliv.) Diels (Danggui) 6 g, Paeonia lactiflora Pall (Chishao) 4.5 g, Ligusticum striatum DC. (Chuanxiong) 3 g, Lumbricus (Dilong) 3 g, Prunus persica (L.) Batsch (Taoren) 3 g, and Carthamus tinctorius L. (Honghua) 3 g, among which RAP is monarch medicine, with a dosage of up to 120 g [The dried roots of plant stragali , 22. Its history . Accordi history . In TCM, history –27. Buya history . It has  history , 30. BHDto 120 g . In thisAstragalus polysaccharides, saponins, flavonoids, and amino acids. The 2020 edition of “Chinese Pharmacopoeia” takes the content of calycosin and astragaloside as the quality control standard for RAP [stragalus propinquus Schischkin dried roots [Astragalus propinquus Schischkin. Compared with granule, it has the characteristics of high absorption and availability and rapid curative effect [Modern pharmacological studies show that RAP possesses a variety of chemical components, mainly including  for RAP . Currented roots . RAP inje effect .Astragalus polysaccharide upregulates hepcidin and reduces iron overload in mice [Astragalus polysaccharide treatment significantly reduces apoptosis, regulates oxidative stress, and upregulates GSH peroxidase activity [Experimental studies confirm that RAP and Astragaloside IV can alleviate brain tissues in ischemia-reperfusion injured rats . Astraga in mice . Moreoveactivity . Calycosactivity , 41. TheIn the present study, we first determined the neuroprotective effect of RAP in CIRI model rats. Then, we clarified the mechanism of RAP in CIRI iron injury and identified the molecular targets of RAP and its monomer calycosin in transmembrane iron transport and ferroptosis. In addition, we compared the difference in the efficacy of granule and injection of RAP.All experimental procedures were approved by the Ethics Committee of Hebei University of Chinese Medicine . In this study, 70 SD healthy male rats weighing 120–150 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. . All rats were housed with free access to food and water at a constant temperature of 22 ± 2°C under a 12 h light/dark cycle with a relative humidity of 40–60%. All animals were treated as humanely as possible to alleviate the pain suffered during the experiment.The MCAO model was established according to methods outlined by Longa . The ratAll rats were randomly divided into 7 groups: the sham group, model group, RAP granule group, RAP injection group, calycosin group, edaravone group, and deferoxamine group. The CIRI model was established in all groups except for the sham group. The sham group and model group received a single intraperitoneal injection of 0.9% NaCl; the RAP granule group received a stomach feeding of 2.5 g/kg/d RAP granule ; the RAP injection  group, calycosin  group, edaravone  group and deferoxamine  group were intraperitoneally injected with RAP (2 mL/kg/d), calycosin (10 mg/kg/d), edaravone (6.3 mg/kg/d), and deferoxamine (70 mg/kg/d), respectively. These administrations were given 7 days prior to the operation and 3 days following reperfusion, as shown in μm). The column temperature was 30°C, the nebulizer temperature was 40°C, the nitrogen flow rate was 1.6 mL/min, and the injection volume was 10 μL. The mobile phase was eluted with acetonitrile (A) and 0.2% acetic acid solution (B) in the gradient mode. The proportion of acetonitrile varied from 20% to 40% within 30 min  at a flow rate of 0.9 mL/min.The RAP granule/RAP injection was evaluated by high performance liquid chromatography (HPLC) analysis. Briefly, HPLC-ELSD was performed using Agilent-1260  equipped with Thermo BDS Hypersil C-18  was used to measure the pixel area of each tissue in each picture and the corresponding infarcted pixel area, using pixels as the standard unit. Then, the proportion of tissue infarcted area (%) was calculated with the following equation:Paraffin slices were dewaxed in water and then xylene twice, 5 min each time, rehydrated in an alcohol gradient of 100%, 95%, and 80%, 5 min each time, and then washed with tap water. The slices were immersed in hematoxylin for 3 minutes, followed by washing with tap water 2-3 times, and then in hydrochloric acid alcohol differentiation solution, followed by washing with tap water. The slices were incubated in blue solution for 5–15 s, followed by washing with tap water, and then stained with eosin for 10 s, followed by washing with tap water. The slices were then incubated in 80% alcohol for 5 min, 90% alcohol for 5 min, 100% alcohol for 5 min, and then xylene. The slices were sealed with neutral gum, after which they were observed by optical microscopy, and the images were collected.The sections were immersed in xylene two times for 15 min each, placed into a series of ethanol concentrations starting from 50% to 100% for 5 min each and then rinsed with distilled water 3 times. Subsequently, the sections underwent Nissl staining according to standard protocols. Then, the pathological changes in the brain tissue were observed under a microscope.2O2 at 37°C for 10 min and washed 5 times with PBS. Then, the sections were heated in a microwave oven in sodium citrate buffer for 20 min for antigen retrieval and washed 5 times with PBS. Then, the slices were incubated with primary antibodies overnight at 4°C, using the following primary antibodies: anti-Fn ; anti-Tf ; anti-TfR ; anti-DMT1 ; anti-LTCC ; anti-TRPC6 ; and anti-FPN1 . The next day, the sections were incubated with a secondary antibody at 37°C for 1 hour, DAB for development of color, and hematoxylin for counterstaining after washing with running water, and then washed back to blue with running water. Finally, the stained sections were observed under a light microscope.In the present study, IHC and Western blotting were carried out as described in our previous study , 44. ForFor Western blotting, the brain tissues were homogenized by using RIPA lysis buffer to extract total protein. The proteins were separated by SDS-PAGE and then transferred from the gel to nitrocellulose membranes. The membranes were blocked with 5% skim milk and then incubated overnight at 4°C with the following primary antibodies: anti-FHC ; anti-FLC ; anti-Tf ; anti-TfR ; anti-DMT1 ; anti-TRPC6 ; and anti-FPN1 . The membranes were then washed 3 times with TBST and incubated with the secondary antibody for 1 hour. Protein band densities were scanned and analyzed by using a computerized image analysis system and ImageJ.The brain tissues were homogenized by using RIPA lysis buffer to extract total protein. The proteins were separated by SDS-PAGE and then transferred from the gel to nitrocellulose membranes. The membranes were blocked with 5% skimmed milk and then incubated overnight at 4°C with the following primary antibodies: anti-XCT ; anti-SLC3A2 ; anti-GPX4 ; anti-NRF2 ; anti-IREB2 , and anti-HO-1 . The membranes were then washed 3 times with TBST and incubated with the secondary antibody for 1 hour. Protein band densities were scanned and analyzed by using a computerized image analysis system and ImageJ.P < 0.05 and at P < 0.01.Statistical analysis of the data was performed using SPSS (IBM SPSS Statistics v 26.0.0). All data satisfying normality are expressed as the mean ± SEM. The differences among the multiple groups were evaluated by one-way ANOVA, followed by LSD for comparison of two groups. The results were considered statistically significant at The RAP granule and RAP injection were evaluated by high performance liquid chromatography (HPLC) analysis to identify the presence of Calycosin-7-O-glucoside and Astragaloside A according to their retention times Figures .P < 0.01). Compared with the model group, the area of cerebral infarction in the RAP granule group, RAP injection group, and edaravone group decreased, especially in the RAP granule group (P < 0.01). These results show that RAP can alleviate the brain injury caused by ischemia.To explore the effect of RAP on CIRI, the cerebral infarction area in rats after MCAO and after administration of various agents was observed. As shown in Figures Under a pathological microscope, the hippocampal CA3 region of rats in the sham group was characterized by an intact cellular structure and orderly tissue arrangement. In the model group, the arrangement of the brain tissue was disordered, the structure of the neurons was seriously damaged, and some nerve cells were necrotic. The RAP granule group, RAP injection group and calycosin group showed effective reductions in neuronal injury, and the tissue arrangement in these groups was significantly better than that in the model group .P < 0.01). However, after MCAO, the arrangement of cells in the CA3 region of the hippocampus was loose, the cell bodies of some residual neurons shrank, the nuclei were pyknotic, and the Nissl bodies in the cytoplasm were decreased. After drug treatment, the neuronal injury was improved, the number of Nissl bodies in the cytoplasm was increased, and the differences were most significant in the RAP granule group and the RAP injection group  before and after administration of the various agents.(1) Effect of RAP on Fn, FHC, and FLC Protein Expression. According to the results of the IHC, the expression level of Fn protein in the model group was significantly higher than that in the sham group (P < 0.01). Compared with the model group, the expression of Fn protein decreased in the RAP granule group, RAP injection group, and calycosin group, especially in the RAP granule group . Compared with the model group, the expression level of FHC protein in the RAP granule group, RAP injection group, and calycosin group decreased, especially in the RAP injection group (P < 0.01). As shown in P < 0.01). Compared with the model group, the expression level of FLC protein in the RAP granule group, RAP injection group, and calycosin group decreased, especially in the RAP granule group (P < 0.01).(2) Effect of RAP on LTCC Protein Expression. As shown in Figures P < 0.01), and compared with the model group, the expression level of LTCC protein in the RAP granule group, RAP injection group, and calycosin group decreased, especially in the RAP injection group (P < 0.05).(3) Effect of RAP on Tf Protein Expression. Compared with the sham group, the expression level of Tf protein in the model group increased (P < 0.01), and compared with the model group, the expression level of Tf protein in the RAP granule group, RAP injection group, and calycosin group decreased, especially in the RAP granule group . Compared with the model group, the expression level of Tf protein in the RAP granule group (0.565 ± 0.08), RAP injection group (0.554 ± 0.07), and calycosin group (0.673 ± 0.06) decreased, especially in the RAP injection group  Effect of RAP on TfR Protein Expression. As shown in P < 0.01), the TfR protein expression level in the model group was increased. Compared with the model group, the TfR protein expression level of the RAP granule group, RAP injection group, and calycosin group decreased, with the RAP granule group decrease being the most significant (P < 0.01). Western blot results showed that the expression level of TfR protein in the model group (0.838 ± 0.08) was significantly higher than that in the sham group (0.569 ± 0.05) (P < 0.01). Compared with the model group, the expression level of TfR protein in the RAP granule group (0.450 ± 0.06), RAP injection group (0.469 ± 0.06), and calycosin group (0.558 ± 0.08) decreased, especially in the RAP granule group  Effect of RAP on DMT1 Protein Expression. As shown in P < 0.01). Compared with the model group, the protein expression level of DMT1 in the RAP granule group, RAP injection group, and calycosin group was decreased, and the difference was statistically significant in the RAP injection group (P < 0.01). Western blot results showed that compared with the sham group (0.612 ± 0.07), the expression level of DMT1 protein in the model group (0.809 ± 0.05) increased (P < 0.05). Compared with the model group, the expression level of DMT1 protein in the RAP granule group (0.594 ± 0.05), RAP injection group (0.635 ± 0.06), and calycosin group (0.633 ± 0.05) decreased, especially in the RAP granule group  Effect of RAP on TRPC6 Protein Expression. As shown in P < 0.01), and compared with the model group, the expression level of TRPC6 protein in the RAP granule group, RAP injection group, and calycosin group decreased, especially in the RAP granule group (P < 0.01). Western blot results showed that compared with the sham group (0.580 ± 0.08), the expression level of TRPC6 protein in the model group (0.867 ± 0.08) increased (P < 0.05). Compared with the model group, the expression level of TRPC6 protein in the RAP granule group (0.494 ± 0.06), RAP injection group (0.555 ± 0.04), and calycosin group (0.577 ± 0.06) decreased, especially in the RAP granule group  Effect of RAP on FPN1 Protein Expression. As shown in P < 0.01), and compared with the model group, the expression level of FPN1 protein in the RAP granule group, RAP injection group, and calycosin group increased, especially in the RAP granule group (P < 0.05). Western blot results showed that compared with the sham group (0.750 ± 0.07), the expression level of FPN1 protein in the model group (0.447 ± 0.08) decreased (P < 0.05). Compared with the model group, the expression level of FPN1 protein in the RAP granule group (0.779 ± 0.08), RAP injection group (0.784 ± 0.11), and calycosin group (0.924 ± 0.12) increased, especially in the calycosin group  Effect of RAP on Expression of the XCT Protein. As shown in P < 0.01), and compared with the model group, the expression level of XCT protein in the RAP granule group (0.767 ± 0.07), RAP injection group (0.853 ± 0.03), and calycosin group (0.857 ± 0.04) increased, especially in the calycosin group (P < 0.01).(2) Effect of RAP on Expression of the SLC3A2 Protein. As shown in P < 0.01), and compared with the model group, the expression level of SLC3A2 protein in the RAP granule group (0.773 ± 0.07), RAP injection group (0.847 ± 0.04), and calycosin group (0.883 ± 0.09) increased, especially in the calycosin group (P < 0.01).(3) Effect of RAP on Expression of the GPX4 Protein. As shown in P < 0.05), and compared with the model group, the expression level of GPX4 protein in the RAP granule group (0.719 ± 0.06), RAP injection group (0.667 ± 0.06), and calycosin group (0.784 ± 0.05) increased, especially in the calycosin group (P < 0.01).(4) Effect of RAP on Expression of the NRF2 Protein. As shown in P < 0.05), and compared with the model group, the expression level of NRF2 protein in the RAP granule group (0.686 ± 0.04), RAP injection group (0.702 ± 0.11), and calycosin group (0.653 ± 0.09) increased, especially in the RAP injection group (P < 0.01).(5) Effect of RAP on Expression of the HO-1 Protein. As shown in P < 0.05), and compared with the model group, the expression level of HO-1 protein in the RAP granule group (0.587 ± 0.07), RAP injection group (0.543 ± 0.06), and calycosin group (0.696 ± 0.06) increased, especially in the calycosin group (P < 0.01).(6) Effect of RAP on Expression of the IREB2 Protein. As shown in P < 0.01). Compared with the model group, the protein expression level of IREB2 in the RAP granule group (0.554 ± 0.08), RAP injection group (0.619 ± 0.08), and calycosin group (0.537 ± 0.07) decreased, especially in the calycosin group (P < 0.05).Ischemic brain tissue injury is further aggravated after the restoration of blood perfusion, otherwise known as CIRI, and its pathophysiological process is a cascade reaction of multiple linked factors and pathways. The recovery of neural function is helpful for alleviating CIRI . PreviouAstragalus polysaccharides, astragalosides, and Astragalus flavonoids, which have antioxidant, anti-inflammatory, and antiapoptotic effects and can treat ischemic cardio-cerebrovascular diseases. Two dosage forms, RAP granules and RAP injection, are widely used to treat ischemic cerebrovascular diseases in the clinic [Traditional Chinese medicine, with its ability to affect multiple targets and pathways, has prominent advantages in the prevention and treatment of CIRI. RAP is a commonly used Chinese herbal medicine for the treatment of cardio-cerebrovascular diseases. Modern pharmacological studies have shown that RAP contains a variety of active ingredients, such as e clinic , 51. As e clinic –54. In ae clinic .Brain iron homeostasis is critical to the normal physiological function of neurons. CIRI can lead to iron deposition in neurons, which catalyzes the production of a large number of free radicals, leads to a series of brain injuries , and finally induces cell death –58. StudIn the iron transmembrane transport system, the Tf/TfR-dependent pathway plays an important role and is closely related to maintaining iron homeostasis in the brain. Tf is the main iron transporter and requires receptor-mediated endocytosis to cross the blood–brain barrier –64. TfR 3+ is reduced to Fe2+ in the endosome and transported to the cytoplasm through DMT1. In the iron overloaded state, supersaturation of transferrin exists in the form of nontransferrin bound iron (NTBI), and the uptake of NTBI needs to be mediated by NTBI transporters such as DMT1, LTCC, and TRPC6 [In addition, iron transport across cell membranes may involve in Tf/TfR-independent pathways, such as DMT1, LTCC, TRPC6, and FPN1. In the transferrin cycle, Tf binds to TfR1 on the cell surface, followed by endocytosis and acidification, resulting in iron release from transferrin. The free Fend TRPC6 . Excessind TRPC6 . In a stnd TRPC6 . Iron tond TRPC6 . The imbnd TRPC6 , 71, 72.Moreover, one transporter, FPN1, capable of transferring iron out of cells, has been found in the brain thus far , 12. In −, composed of XCT and SLC3A2, is critical in ferroptosis [Ferroptosis is a new type of iron-dependent programmed cell death that is characterized by iron-dependent lipid peroxidation . Recent roptosis , 74–76. roptosis . In this− , enhance the activity of GPX4, inhibit lipid peroxidation, downregulate the expression of IREB2, regulate iron metabolism, and inhibit the occurrence of ferroptosis. Previous studies have shown that the NRF2/HO-1 pathway plays an important role in CIRI and ferroptosis [In addition, iron metabolism is required for the accumulation of lipid peroxides and the execution of ferroptosis . One sturoptosis , 81. NRFroptosis , 82, 83.roptosis . GPX4 isroptosis . In thisroptosis . Based oGenerally, brain iron transmembrane transporters are involved in the process of CIRI. RAP downregulated Tf, TfR, Fn, FHC, FLC, DMT1, LTCC, and TRPC6, upregulated FPN1, regulated the expression of iron transporters, and played a role in the prevention and treatment of CIRI. We also found that the key factors of ferroptosis were involved in ischemic stroke. Calycosin inhibited the occurrence of ferroptosis by regulating the expression of XCT, SLC3A2, GPX4, NRF2, HO-1, and IREB2.


Brachionus calyciflorus changed in response to CPZ and gradually increasing temperatures. The results showed that the concentration range of rotifer DAC was 1.06~2.51 ng/g. At 18, 25 and 32 °C, the 24 h 50LC was 1.795, 1.242 and 0.833 mg/L, respectively. Compared to the control, exposure to CPZ significantly decreased life expectancy at hatching, the net reproduction rate, generation time, population growth rate and dopamine concentration of B. calyciflorus in all three temperatures (p < 0.05). The toxicity of CPZ to rotifers was increased by high temperature. These findings indicated that CPZ is highly toxic to rotifers, displaying high ecological risks to aquatic ecosystems.Global warming and environmental pollution have created a unique combination of abiotic and biotic stresses to zooplankton. However, little information is available on the effects of antipsychotic drugs commonly used to treat psychosis, such as chlorpromazine (CPZ), on non-target aquatic organisms in light of global warming. This study investigated how dopamine concentrations (DAC), acute toxicity and chronic toxicity of  Psychoactive substances (PSs) can alter mental functions such as thinking, emotion, and volitional action after being absorbed into the human body. With the increasing number of those suffering from mental illness and the global illicit drug trade, the demand for and use of PSs has risen rapidly in the last decade, and PS misuse has progressively become a global issue . The unmChlorpromazine (CPZ) is a low-cost phenothiazine psychoactive substance. As a first-generation antipsychotic, CPZ is commonly used to treat psychotic disorders such as delusions, hallucinations, mania, schizophrenia, and thinking disorders in humans . CPZ redOncorhynchus mykiss depends on temperature [Chironomus tentans [® to the estuarine grass shrimp Palaemonetes pugio [In the past decade, scholars have emphasized that climate change poses a direct or indirect threat to mental health ,19. Shorperature . It was  tentans , fungicies pugio . HoweverDaphnia magna is among the most widely used zooplankton species in ecotoxicology, little is known about the toxicity of CPZ to this model system. It was found that the 48 h 50EC of CPZ was 1.805 mg/L in D. magna [Brachionus calyciflorus, B. plicatilis and B. havanaensis, have been widely used for toxicity assessment as model organisms because of their small size, ease of culture, rapid reproduction rate, and sensitivity to toxicity [B. calyciflorus has a parthenogenetic reproduction mode, which provides a basis for us to test individuals with the same genetics [B. calyciflorus has been widely utilized to assess the potential toxicity of pharmaceuticals and their metabolites [Zooplankton occurs widely in freshwater ecosystems and is often used as a bioindicator to evaluate the potential health risks of exposure to trace elements and toxic chemical compounds ,29. AlthD. magna , and itsD. magna . Rotifertoxicity ,33. Addigenetics . Consideabolites ; howeverB. calyciflorus as well as its life history and population growth parameters at high temperatures. The current study aims to (1) test the acute toxicity of CPZ to rotifers at different temperatures; (2) further understand the response of neurotransmitter DA in rotifers to CPZ toxicity at higher temperature; (3) determine the chronic effects of sublethal CPZ concentrations on rotifer development and reproduction at higher temperatures. The current investigation will reveal the toxic effects of CPZ on freshwater invertebrates in a systematic manner, which is critical for a comprehensive assessment of the environmental risk of antipsychotics to aquatic ecosystems.We looked at the effects of CPZ on the neurotransmitter DA of B. calyciflorus were hatched from a single resting egg collected from the sediments of Lake Longwo  in Wuhu city [Tetradesmus obliquus  at a food density of 1.5 × 106 cells/mL. Algae were grown in semi-continuous culture using HB-4 medium [Monoclonal cultures of uhu city , and weruhu city . Rotiferuhu city  at 25 °C4 medium . Rotifer4 medium .Chlorpromazine hydrochloride (CAS: 69-09-0) was purchased from Sangon Biotech  with 98% of purity. The degradation of CPZ in the dark at room temperature was almost negligible: the half-life was estimated as 87.3 weeks. Meanwhile, the half-life of CPZ in darkness at 70 °C has been calculated as 4.55 weeks . A stock6 cells/mL of T. obliquus. The number of dead rotifers was counted after 24 h, and the 50LC was calculated using the Probit method [For acute toxicity assessment, seven concentrations (0 (control), 0.8, 1.2, 1.6, 2.0, 2.4, 2.8 and 3.2 mg/L) were included for each temperature . Ten rotifer juveniles (<4 h old) were chosen at random and placed in a glass jar containing 5 mL of test solution. Four replicates were performed for each treatment. Rotifers were cultured under darkness with 1.5 × 10t method .B. calyciflorus neurotransmitters in light of global warming, we set up groups of 0, 0.125, 0.25 and 0.5 mg/L CPZ treatments based on 24 h 50LC, and measured changes in DA concentration (DAC). First, rotifers pre-cultured at 18, 25 and 32 °C were collected in three 2000 mL glass beakers according to temperature, and then rotifers in each temperature group were equally distributed to twelve 50 mL glass beakers. Each beaker held approximately 8000 rotifers and 50 mL of test solution. These beakers were kept at temperatures of 18, 25 and 32 °C for 12 h, with no food provided during this period. The rotifers were harvested by filtration through a 25 µm pore-sized mesh in a cryovial (washed with EPA medium and made up to 5 mL), rapidly frozen with liquid nitrogen and then stored at −80 °C for determination of DA levels. Each treatment was carried out three times.To explore the effects of CPZ on g for 10 min at room temperature. The supernatant was collected and subjected to assessment concentration using ELISA kits produced by Shanghai Elisa Biotech Co., Ltd. , following the manufacturer’s specifications. In brief, the DA level was determined using an enzyme-linked immunosorbent assay, and the absorbance at 450 nm was measured with an RT-6100 enzyme-labeled instrument .Rotifers were homogenized for 10 min at 4 °C using a Bullet Blender Tissue Homogenizer  at 2.0 m/s. The mixtures were then centrifuged at 3000× 50LC. Each treatment was replicated three times. In total, 36 glass jars (three temperatures × four CPZ concentrations × three replicates) were used. The experiment was conducted in 8 mL glass jars. For each glass jar, ten juveniles (<4 h old) were introduced to a 5 mL test solution containing 1.5 × 106 cells/mL of T. obliquus. Every 24 h, 80% of the test solutions were replaced with freshly prepared solutions containing designated concentrations of algae. The cultured rotifers were checked every 12 h to record the numbers of neonates and dead individuals which were removed until all the experimental animals died.For the life table experiments, four CPZ concentrations (0 (control), 0.125, 0.25 and 0.5 mg/L) and three temperatures  were tested based on the 24 h xl) and age-specific fecundity (xm) were calculated by using the conventional life table technique [e0), generation time (T), net reproduction rate (R0) and intrinsic rate of population increase (mr) were calculated according to Krebs [Based on the data collected, age-specific survival  was counted daily and transferred into a freshly prepared test solution. T. obliquus deposited at the bottom of each test tube was resuspended with a micropipette every 12 h. The experiments were terminated when the population density peaked and continued to decline for 2~3 days.The temperature and CPZ concentration settings are the same as in r) of the rotifers was calculated as follows:t is the culture day in which the rotifer density was the highest, and N0 and tN are the initial and highest rotifer densities, respectively [The population growth rate . A statistically significant difference was set as p < 0.05. All data were statistically analyzed using SPSS 22.0 .All data were expressed as the mean values with standard errors (mean ± SEs). The normality and homogeneity of data were tested using Kolmogorov–Smirnov’s tests and Levene’s tests, respectively. The 24 h B. calyciflorus reacted to each treatment by slowing down its swimming speed, forming clusters of two or three individuals, and weakening its response to external stimuli. The CPZ concentration at which rotifers began to die decreased from 1.2 to 0.4 mg/L as the temperature increased, and there was a significant linear relationship between CPZ concentration and rotifer mortality, respectively  when compared to controls. Temperature was significantly correlated with DAC in rotifers exposed to 0.125 mg/L CPZ treatments and controls, with DAC being highest at 25 °C , but the CPZ × temperature interaction had no effect , but treatments with CPZ at 0.25 and 0~0.5 mg/L did not significantly affect the peak values of xm at 25 and 32 °C  exposed to different concentrations of CPZ. At 18, 25 and 32 °C, CPZ concentration had a significant effect on e0, T, R0 and mr . Treatments with CPZ at 0.25~0.5, 0.25~0.5 and 0.125~0.5 mg/L significantly decreased the e0 at 18, 25 and 32 °C, respectively; treatments with CPZ at 0.5 mg/L significantly shortened T at 18, 25 and 32 °C, respectively; treatments with CPZ at 0.25~0.5 mg/L significantly decreased the mr values at 18 and 32 °C, and treatments with CPZ at 0.125 and 0.5 mg/L significantly decreased the mr values at 25 °C . Except for treatments with CPZ at 0.125 mg/L at 32 °C and 0.25 mg/L at 25 °C, which were not significantly different from controls , CPZ significantly inhibited R0 in all treatments . At 0~0.5 mg/L CPZ concentration, temperature had a significant effect on e0, T, R0 and mr . Compared to controls, and at 18 °C, CPZ treatments of 0.5 and 0~0.5 mg/L significantly increased the e0 and T of rotifers, respectively, and the R0 and mr were significantly decreased in CPZ treatments of 0~0.5 mg/L ; similarly, at 32 °C, CPZ treatments of 0~0.5, 0~0.5 and 0.25~0.5 mg/L significantly decreased the e0, T and R0 of rotifers, respectively, and the R0 and mr were significantly increased in CPZ treatments of 0.25 and 0~0.5 mg/L .Using standard life table methods, we compared the e0, R0, and T of the rotifers , but the CPZ × temperature interaction did not , and mr of the rotifers were significantly affected by CPZ concentration, temperature and their interaction . At three temperatures of B. calyciflorus, significant nominal concentration–response relationships existed between CPZ concentration and e0, T, R0 and mr , whereas the population of 0.25 mg/L CPZ treatments grew slowly and peaked on day 11 (68.67 ± 1.76 ind./mL). Similarly, the r of rotifers decreased significantly as CPZ concentration increased . When the temperature was increased to 25 °C, all treatments reached their highest peaks on days 11, 12 and 10, respectively , but the r of rotifers significantly decreased in comparison to the control . At 32 °C, both treatments with CPZ at 0.125 mg/L and the control reached the peak on day 12 (103.33 ± 5.29 and 116.20 ± 4.39 ind./mL), and treatments with CPZ at 0.25 mg/L reached a peak on day 13 (91.17 ± 2.60 ind./mL), the r of rotifers exposed to 0.25 mg/L CPZ was significantly lower than the controls ; the r was significantly lower at 18 °C only in the 0.25 mg/L CPZ treatments, with no significant differences between the other treatments compared to the controls.The population growth curves of B. calyciflorus as the test organism to study the survival, population growth and dopamine concentration of rotifers under CPZ and temperature stress, in an attempt to provide a basis for environmental risk assessment of CPZ.The first clinical application of chlorpromazine in France in 1952 was considered to be the beginning of psychopharmacology . Since i50LC obtained for CPZ (0.833–1.795 mg/L) with B. calyciflorus is higher than those obtained for aquatic organisms such as the protozoan Spirostomum ambiguum (24 h 50EC of 0.35 mg/L), the freshwater goldfish Carassius auratus (48 h 50EC of 0.43 mg/L) and the crustaceans Thamnocephalus platyurus (24 h 50LC of 0.62 mg/L), similar to the D. magna (48 h 50EC of 1.805 mg/L) [B. calyciflorus to CPZ is comparable to that of D. magna, but more sensitive than that of fish and protozoa. Some researchers investigated the approximate lethal doses of 58 compounds at different temperatures in rats and discovered that the majority of the compounds were 2–17 times more toxic at 36 °C than at 26 or 8 °C [B. calyciflorus at 32 °C than at 25 or 18 °C.Acute toxicity of CPZ to aquatic organisms has been documented. The acute 24 h 05 mg/L) ,30,48. O05 mg/L) ,50,51. I or 8 °C . InteresThe DA system was prevalent in both vertebrates and invertebrates and performs more of a neurotransmitter function. In vertebrates, DA was involved in many functions, including locomotion ,54,55, cT at 0.125 mg/L CPZ treatments compared to the control in the current study. Previous research had shown that low toxicant exposure improved performance, a phenomenon known as hormesis [e0, R0 and mr were significantly reduced in treatments with 0.25 mg/L CPZ at 18 and 32 °C. Consistently, the e0, R0, T and mr were significantly lower in treatments with 0.5 mg/L CPZ than in the control at all temperatures. Higher concentrations of CPZ had more toxic effects on e0, R0, T and mr were obvious in all three temperatures, which was consistent with previous studies [T shortened significantly and the mr increased significantly in all treatments. In our experiments, we discovered that rotifers became significantly smaller as the temperature rose, so we hypothesized that rotifers were allocating more energy to reproduction rather than growth and development, which was also consistent with previous research [mr of rotifers, implying that temperature may affect CPZ toxicity. More specifically, when compared to the control, e0 in 0.125 mg/L CPZ treatments with did not change at 18 °C but significantly decreased at 32 °C. Similarly, R0 in 0.125 mg/L CPZ treatments did not change at 25 °C but significantly decreased at 32 °C when compared to the control. These findings suggested that high temperatures may increase CPZ toxicity to rotifers. Similarly, the toxicity of nTiO2 and imidacloprid to B. calyciflorus with increasing temperature [Palaemonetes spp., Cyprinodon variegatus and Isonychia bicolor [Oncorhynchus kisutch [Life table experiments on rotifers are common in environmental risk assessment, ecological and aquaculture research . At all hormesis . The e0, studies ,64. Withresearch . Two-wayperature ,67, as w bicolor ,69, and  kisutch . In liner have been widely used as reliable indicators to demonstrate reliable various environmental stressors such as chlordecone, fipronil, chloramphenicol and oxytetracycline in the genus Brachionus  [r are critical at the trophic level [r at all temperatures. On the sixth day, population growth was slowed and extinction was particularly observed in B. calyciflorus exposed to 0.5 mg/L CPZ. The effects of high CPZ concentrations on rotifer population dynamics could be explained by the following factors. To begin, the swimming speed of the rotifer determines its water filtration rate and feeding rate [So far, adverse effects on population demographic factors such as iflorus) ,72,73. Wiflorus) . For zooic level . In thisic level . CPZ hading rate , and roting rate , but theB. calyciflorus, and the toxicity increased with increasing temperature within a certain range. Dopamine concentrations in rotifers decreased significantly as CPZ concentrations increased at different temperatures. The life expectancy, net reproduction rate, generation time, intrinsic growth rate and population growth rate of B. calyciflorus were all significantly affected by different temperatures and CPZ concentrations. Overall, CPZ pollution may not be acutely toxic to rotifers, but it may still have an impact on rotifers in light of global warming, which may then influence community structure in natural waters.According to the findings of this study, CPZ was highly toxic to


Pantoea agglomerans PCI05, against food-borne pathogens. Amylase exhibited stability in a wide pH range and retained 50% of its activity at temperatures as high as 100°C. Thermal analysis of the enzyme produced showed thermal stability, up to 130°C. From these findings, it can be envisaged that the alpha-amylase produced from P. agglomerans can be used for starch liquefaction; it was also evaluated for antibiofilm activity. Amylase from this marine bacterium was found to efficiently disrupt the preformed biofilms of food-borne pathogens such as Bacillus cereus, Serratia marcescens, Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella enterica enterica serotype Typhi based on the value of biofilm inhibitory concentrations.Bacterial biofilms are a big menace to industries and the environment and also in the health sector, accumulation of which is a major challenge. Despite intensive efforts to curb this issue, a definitive solution is yet to be achieved. Enzyme-templated disruption of the extracellular matrix of biofilm and its control and elimination are emerging as an efficient and greener strategy. The study describes the antibiofilm potential of alpha-amylase from the marine microorganism  This mi earlier –4. Theseive site .Alpha-amylase is a hydrolase found in a wide range of microorganisms and animals . The polBacterial biofilms pose a serious risk to both industry and the environment, as well as having clinical implications , 14, 15.However, complete elimination of biofilms by these methods is difficult to attain because of the presence of EPS. Utilizing enzymes for the removal of biofilms through the destruction of the physical integrity of the matrix of the latter, that is, EPS, would be an efficient strategy . A couplPantoea agglomerans is a Gram-negative aerobic bacillus that can be found in a variety of biological niches such as aquatic habitats, soils, and sediments. This bacterium has several uses, including biological control agents [P. agglomerans has previously been shown to produce alpha-amylase from pea leaves [P. agglomerans. We believe that the amylase generated by marine P. agglomerans has extensive antibiofilm action. The objective of this study was to isolate and characterize the antibiofilm action of alpha-amylase produced by this marine bacterium against food-borne pathogens.Enzymes are environmentally benign since they degrade quickly. l agents  and prodl agents . This bal agents . P. aggla leaves . HoweverDuring September 2019, surface seawater samples were taken aseptically from the Arabian Ocean's intertidal zone, Thirumullavaram (08°54′ N and 76°38′ E) of Kerala's Kollam district on India's southwest coast . The average surface temperature was 29°C, the pH was 7.7, the mean dissolved oxygen was 8.93 mg/L, and the salinity was 32 ppt at the collection site. The samples were immediately transported to the laboratory in a cold box, filtered through Whatman No. 1 filter paper, and one milliliter (without dilution) was inoculated onto Zobell marine agar plates  and incubated for two days at 30°C . Pure cuIsolated colonies were streaked onto soluble starch agar medium prepared by dissolving 20.0 g starch in one liter of distilled water with a pH of 7.0 and adding 10.0 g casein enzyme hydrolysate, 20.0 g sodium chloride, and 15.0 g agar. To achieve sufficient growth, inoculated plates were kept at 37°C for two days. By flooding the plates with Gram's iodine solution and evaluating colonies with clear zones as amylase producers, the isolates' starch hydrolyzing capacity was observed .http://www.ncbi.nlm.nih.gov/) [Standard procedures were used to determine the morphological, physiological, and biochemical properties of the amylase producer PCI05 . The folih.gov/) , 34. Theih.gov/) .2PO4 1.0 g, NaNO3 3.0 g, MgSO4.7H2O 0.5 g, ZnSO4.2H2O 0.1 g, CuSO4.5H2O 0.1 g, FeSO4.7H2O 0.1 g, FeSO4.7H2O 0.1 g, FeSO4.7H2O 0.1 g, FeSO4.7H2O 0.1 g, FeSO4.7H2O 0.1 g, FeSO. The production medium was then inoculated with 1 ml of a two-day-old bacterial suspension cultured in Zobell marine isolation broth. The flasks were then inoculated and incubated at 37°C for two days with intense aeration at 250 rpm. After incubation, the fermented production media were centrifuged at 11,000 × g for 20 minutes, and the resulting cell-free supernatant (CFS) was filtered through a 0.22 m filter.Submerged fermentation was carried out in a 250 ml Erlenmeyer flask with 100 mL of basal medium (production medium) containing KCl 0.5 g, KHAll experiments in triplicate were performed under varying conditions of temperature; pH; concentrations of carbon (fructose), nitrogen (ammonium nitrate) sources, and sodium chloride; and incubation period . The datIt was determined by incubating equal volumes of precisely diluted enzyme and substrate (1% w/v soluble starch in pH 7.0 phosphate buffer) for 30 minutes at 37°C. The reaction was stopped by adding 2 ml of 3,5-dinitrosalicylic acid, and the amount of glucose released was calculated using a UV/Vis double beam scanning spectrophotometer (SPECTRONIC) at 540 nm .At 4°C, ammonium sulfate was precipitated by adding different amounts of solid sulfate (20 to 80 %) to the CFS and centrifuging the concentrate at 11,000 × g for 15 min. The precipitate was then dissolved in a 10 mM Tris-HCl buffer with a pH of 7.4 and dialyzed against the same buffer overnight at 4°C before being kept at −20°C for further studies.P. agglomerans, PCI05, was purified in three steps: 0–80% ammonium sulfate precipitation, dialysis, and gel filtration chromatography utilizing a Sephadex G-75 column (3 × 45 cm) . The dialyzed material was loaded onto a DEAE Sephadex G-75 ion exchange column, which was subsequently equilibrated with 10 mM Tris-HCl buffer, pH 7.4, and dialyzed again. The sample was slowly placed on top of the column at equilibrium, to allow effective diffusion coupled with binding, and then washed stepwise with the same buffer at a constant flow rate of 0.5 ml/min with increasing salt concentration . As previously stated in this paper, eluted fractions were tested for amylolytic activity. Active fractions were blended and freeze-dried, producing a 15% yield. SDS-PAGE with 4% stacking gel , (NH4) 2S2O8 1% (w/v)) and 12% resolving gel  (NH4) 2S2O8 1% (w/v)), stained with silver nitrate, and the molecular weight of pure amylase was then estimated by comparing it to the relative mobility of standard molecular weight markers  .   and three denaturing agents . Any of the metal ions or chemical molecules listed above are included in the buffer.The amylase activity was examined with various pH values, temperatures, metal ions, and incubation periods. The stability of the isolated enzyme was evaluated at various pH values  and temperatures . It was examined after an hour of preincubation in Tris-HCl buffers ranging from 1.0 to 10.0 with the same ionic strength and pH. The residual activity was determined immediately after the aliquots were processed as indicated previously. The thermal stability was investigated by preincubating it at various temperatures for an hour, and the residual activity was assessed as described elsewhere . As a coμl aluminum pan, approximately 5 mg of the sample was loaded. Weight loss was measured as a function of temperature throughout a temperature range of 40°–200°C at a heating rate of 10ºC/min; the flow rate of the gas was 100 ml/min.A combined DSC-TGA employing Shimadzu TG 60 and DSC 60, Japan, was used to investigate the thermal stability and weight loss of alpha-amylase. In a standard 70 −1) on a Thermo Nicolet 6700 FT-IR spectrophotometer. A thin pellet was made by mixing the sample with dried KBr powder and grinding it to a fine powder. All scans were subtracted from pure water and CO2 standards, and each spectrum was an average of 32 scans.Using a reversed-phase C18 Nova-Pak column , RP-HPLC was performed to assess the purity and homogeneity of the protein recovered from the DEAE Sephadex column. In this technique, two buffers were used: buffer A is 0.1% (v/v) trifluoroacetic acid (TFA) in water and buffer B is 99.9% acetonitrile with 0.1% (v/v) TFA. The former was used to equilibrate the column, which was subsequently operated at a flow rate of 1 ml/min at 220 and 280 nm with the blank (mobile phase). After obtaining a steady line, the sample was injected and eluted with a linear buffer B gradient (0–100%). The KBr pellet approach was used to record the infrared spectrum (4000–500 cmListeria monocytogenes (MTCC 1143), Salmonella enterica enterica serotype Typhi (MTCC 8767), Vibrio parahaemolyticus (MTCC 451), Bacillus cereus (MTCC 1272), and Serratia marcescens (MTCC 97)) were developed in nutrient broth (HiMedia), and their microtiter plate adherence was determined according to the methodology described elsewhere [µg·mL−1 CuSO4. All three wells were filled with different concentrations of column-purified amylase, such as 50, 100, 150, 200, and 250 μl·mL−1. The trials were carried out six times to statistically validate the results, with the control and treated wells arranged in a Latin square layout. To obtain dynamic culture conditions, plates were placed on a platform shaker. Planktonic cells and spent media were removed, and adherent cells were gently washed twice in deionized water before being stained. Biofilms were stained for 10 minutes with 200 mL of 0.4 percent crystal violet solution (w/v) and then rinsed twice with deionized water. To assess the extent of biofilm destruction, the optical density of the plates was measured at 595 nm in an ELISA reader (Labnics), and the plates were observed via a phase-contrast microscope with a 40x magnification (Optica).Bioﬁlms of food-borne microbial-type culture bacterial pathogens  of amylase for 1 hour at 30°C to determine the amount of rupture. The exposed coverslip was dyed with a 0.4% crystal violet solution (w/v) and then microscopically examined as previously described.The results were confirmed by an S. Typhi, B. cereus, and L. monocytogenes biofilms were allowed to grow on glass surfaces for 24 hours. Based on preliminary microscopic examinations, food-borne bacterial pathogens were chosen, and biofilms were treated with amylase at the pathogen's minimum inhibitory concentration (MIC) and then incubated for another 24 hours. Glass slides were then removed, rinsed with phosphate buffer solution (PBS), air-dried, and treated with 4% glutaraldehyde  for 2 hours before being dehydrated with various grades of alcohol (33–99.9%). After air-drying, the samples were coated with platinum vapor and examined using a scanning laser electron microscope ; untreated biofilms were used as controls.The Twelve (12) strains were isolated and checked for extracellular alpha-amylase production using a plate assay supplemented with 1% starch based on colony appearance and stability during subculturing. Six isolates showed clear zones around the colonies, including PCI05, which demonstrated significant hydrolytic activity and was chosen for additional optimization methods aimed at increasing extracellular alpha-amylase production and biofilm rupture.P. agglomerans based on its physiological, biochemical, and taxonomic characteristics. The strain has been cultivated in a variety of environments, including temperatures ranging from 27 to 50°C and a range of sodium chloride solution concentrations. However, optimal growth was observed at 30°C and sodium content of 7% (w/v).The strain PCI05 was identified as P. agglomerans produced the most alpha-amylase, and the process was found to be sensitive to the type of nitrogen source employed and its quantity in the medium. In addition to ammonium nitrate, the presence of both organic and inorganic nitrogen sources influenced the production of alpha-amylase by P. agglomerans. In a medium treated with ammonium chloride, the greatest inhibition was detected. Increases in salt content increased alpha-amylase production, with the optimum occurring while 7% sodium chloride was employed; however, subsequent increases in salt concentration resulted in a drop in enzyme production.Carbon and nitrogen sources, pH, temperature, salt concentration, and incubation period all had an impact (one-factor-at-a-time approach). These variables were found to have a substantial influence on alpha-amylase production based on the results of optimization experiments. In a basal medium enriched with fructose, the strain −1 is attributed to hydrogen-bonded O-H-stretching vibrations, while the peak between 3100 and 2900 cm−1 is attributed to C-H-stretching vibrations. The C-O-C-stretching vibrations appeared as sharp peaks at 1263 and 949 cm−1; sharp peaks at about 1138 and 1068 cm−1 showed the presence of C-O-stretching vibrations of tertiary and primary alcohols, respectively. The bending motion (rocking) associated with -CH2- groups appear at about 863 and 822 cm−1.The column was injected with a purified alpha-amylase sample (pooled after Sephadex G-75 fractionation), and the profile showed a single peak  water molecules. At 60 to 120°C, the Tm of the enzyme appeared to be stable with a loss of only 2.5% of the weight. A sharp endothermic peak was observed at 131.5°C with a weight loss of 6.3%. However, when the temperature further increased, the weight of the sample started to decline sharply due to exothermal degradation , indicatP. agglomerans was evaluated for its antibiofilm activity corresponding to various volume levels such as 50, 100, 150, 200, and 250 μl. With crystal violet staining, the BIC was assessed microscopically as well as spectrophotometrically. The percentage of BIC against different bacteria such as B. cereus (150 µl·mL−1), V. parahaemolyticus (100 µl·mL−1), S. marcescens (150 µl·mL−1), L. monocytogenes (150 µl·mL−1), and S. Typhi (150 µl·mL−1), respectively, were 86.83, 77.83, 73.34, 85.47, and 84.09%  was significantly reduced when alpha-amylase was added to their BIC. After treatment with alpha-amylase, SEM images  were used to investigate the effect of amylase on the degree of biofilm development by food-borne pathogens. Biofilm generation by M images  revealedα-amylase-producing strain of P. agglomerans was isolated and characterized from surface seawater samples collected from India's southwest coast. When fructose was used as the carbon source in a base medium and the temperature range was 10 to 30°C, the maximum amount of enzyme was produced. The synthesis of enzyme reduced as the incubation temperature increased, peaking at 50°C. The growth of bacteria was considerably reduced at higher temperatures, resulting in a decrease in enzyme formation [P. agglomerans also produced the most alpha-amylase at a pH of 7.0. The enzyme's synthesis and secretion are thought to be affected by the medium's initial pH. The extracellular pH affects several enzymatic activities as well as the transport of many components through the cell membrane; therefore, microbial strains' production of alpha-amylase is greatly influenced by it [In this study, an ormation . In addied by it . Enzyme Bacillus sp. MD 124 corresponded to an optimum pH value of 6.0. The majority of organisms studied previously produced the most extracellular alpha-amylase at an optimum pH of 5.0–7.0 or 6.0–7.0 [-l sodium chloride, the activity of a salt-tolerant extracellular alpha-amylase from Bacillus dipsosauri was maintained. Although Halomonas meridiana was active in a 15% sodium chloride solution in a previous investigation, it became inactive at temperatures above 37°C [Penicillium camemberti, PL21, were found to be 1.67 and 1% according to two independent studies [P. agglomerans can be classified as a thermostable enzyme, and it was found to be stable even at 100°C, retaining 50% of its activity. Similarly, thermostable alpha-amylase has been obtained and purified from Bacillus sp. MD 124 earlier [Thermostable salt-tolerant alpha-amylase production from  6.0–7.0 –44. As tove 37°C . Upon an studies , 47. BasCryptococcus flavus, S. alluvius ATCC 26074, L. kononenkoae, and C. antartica CBS 667 [T. gondii [Lactobacillus manihotivorans [Thermomyces lanuginosus [The best activity of alpha-amylases is achieved at 40°C, according to a literature review; however, the optimal temperature varies widely, for example, 50°C for  CBS 667 . T. gond. gondii  needs a uginosus , as welluginosus , 53, req2+ and Ca2+ had a significant impact on alpha-amylase activity in the current investigation. Ni2+, Cd2+, Zn2+, and Hg2+ inhibited alpha-amylase from Bacillus sp. strain KSM-1378 [Bacillus firmus [2+, Ag+, Cu2+, and Fe2+ inhibited alpha-amylase from B. subtilis, B. amyloliquefaciens I, and B. amyloliquefaciens II [The enzyme was functional in a wide pH range pH 5–10) and was active in both acidic and alkaline environments. In general, the pH value corresponding to the efficiency of alpha-optimal amylase varies greatly, with most bacteria and fungi's activities peaking in the acid to neutral range, 6-7 –10 and w. CationsKSM-1378  and Bacis firmus , whereasciens II .L. monocytogenes, E. coli, S. Typhi, B. cereus, S. marcescens). Earlier research on a range of enzymes such as proteases, papain, alpha-amylase, and cellulase suggested that hydrolases' antibiofilm activity could be attributed to the hydrolysis of a substrate implicated in bacterial adherence, such as EPS [The antibiofilm activity of amylase was tested using common food-borne bacterial pathogens  was associated with a precipitation step comprising 60 to 80% ammonium sulfate. The enzyme was discovered to be stable throughout a wide pH range. The thermal analysis demonstrated that the synthesized enzyme has higher thermal stability. The current findings suggest that the enzyme could be useful in the starch liquefaction process. The antibiofilm activity of P. agglomerans crude alpha-amylase was further investigated, and it was discovered to efficiently disrupt preformed biofilms of food-borne pathogens.Alpha-amylase was isolated from