Recent 31P-MRS techniques using 7T systems, e.g. long mixing time stimulated echo acquisition mode (STEAM), allow deeper insight into cardiac metabolism through assessment of inorganic phosphate (Pi) content and myocardial pH, which play pivotal roles in energy production in the heart. Therefore, we aimed to further explore the cardiac metabolic phenotype in T2DM using STEAM at 7T. Seventeen patients with T2DM and twenty-three healthy controls were recruited and their cardiac PCr/ATP, Pi/PCr and pH were assessed at 7T. Diastolic function of all patients with T2DM was assessed using echocardiography to investigate the relationship between diastolic dysfunction and cardiac metabolism. Mirroring the decreased PCr/ATP , the cardiac Pi/PCr was increased  in T2DM patients in comparison to healthy controls. Myocardial pH was not significantly different between the groups . There was a negative correlation between PCr/ATP and diastolic function  in T2DM. No correlation was observed between diastolic function and Pi/PCr and . In addition, we did not observe any correlation between cardiac PCr/ATP and Pi/PCr (p = 0.19). Using STEAM 31P-MRS at 7T we have for the first time explored Pi/PCr in the diabetic human heart and found it increased when compared to healthy controls. The lack of correlation between measured PCr/ATP and Pi/PCr suggests that independent mechanisms might contribute to these perturbations.Phosphorus magnetic resonance spectroscopy ( Type 2 diabetes mellitus (T2DM) is a global health concern and the cardiac consequences of T2DM are numerous. Their development is typically through an asymptomatic phase of diastolic dysfunction , which cIn T2DM there is an increasingly recognised pathological metabolic phenotype . As the 31P-MRS) allows non-invasive assessment of high-energy phosphate metabolites, e.g., ATP and phosphocreatine (PCr), and hence allows tissue metabolism assessment in vivo , whereas resting heart rate was higher in patients with diabetes . All T2DM patients had a Hb1Ac over 6% (mean 7.2 ± 1.2%) while their LVEF was in the normal range (mean 59 ± 4%). However, their E/e’ (average) was already mildly elevated (mean 6.9 ± 2.0) indicating low grade diastolic dysfunction. Further baseline data is given in Seventeen patients with type two diabetes , and twenty-three healthy controls  were recruited. Patients with diabetes had a significantly higher BMI . In the opposite direction and demonstrated for the first time here, cardiac Pi/PCr was significantly increased in T2DM patients . The median (interquartile range) Pi CRLBs were 33.1% (22.9 to 50.3), and were not different between the two groups, i.e. 32.1% (23.5 to 50.2) vs 35.4% (22.4 to 58.3) for healthy controls and patients with diabetes, respectively. in vivo31P-MRS at 7T. Our results show that, as the PCr/ATP drops in T2DM patients, their cardiac Pi/PCr increases. At the same time, the myocardial pH does not differ between T2DM and healthy controls. Interestingly, we have found no correlation between PCr/ATP and Pi/PCr suggesting some level of mechanistic independence between them.We investigated cardiac inorganic phosphate reserves and myocardial pH in patients with type 2 diabetes mellitus, using Our PCr/ATP result is in good agreement with literature, where the drop in PCr/ATP in diabetes has been extensively investigated ,27,28. CM STEAM at 7T technique allowed us to measure for the first time cardiac Pi/PCr in T2DM patients in vivo. We found significantly higher Pi/PCr values in the heart of T2DM patients in comparison to healthy controls, i.e. 0.13 ± 0.07 vs. 0.10 ± 0.03 (p = 0.02). This increase in Pi/PCr mirrors the drop in PCr/ATP discussed above, potentially limiting the available energy from ATP hydrolysis. If this progresses, diastolic dysfunction will progress and ultimately the systolic function will likely become impaired as well. Our reported values of Pi/PCr in healthy volunteers are in good agreement with literature reports, i.e. 0.10 ± 0.07 [The recently described long T0 ± 0.07  or 0.10 0 ± 0.07 . Similar0 ± 0.07 . The Pi/0 ± 0.07  or 0.24 0 ± 0.07 . This is0 ± 0.07 . This fuin vivo measured myocardial pH in T2DM patients. Our results show that the myocardial pH of T2DM patients is not statistically different than that of healthy controls, i.e. 7.14 ± 0.12 vs. 7.10 ± 0.12 (p = 0.31). This is in agreement with previous literature reports assessing myocardial pH in patients with cardiomyopathies, where similar values of 7.06 ± 0.03 [Alongside the Pi/PCr, we also report the first 6 ± 0.03  or 7.12 6 ± 0.03  were sho6 ± 0.03  or 7.14 6 ± 0.03  found in2 = 0.16, p = 0.21) in T2DM in this study. On the other hand, E/e’ (mean) did negatively correlate with the cardiac PCr/ATP . This is in agreement with a recent study showing negative correlation between cardiac PCr/ATP and E/e’ over several stages of heart failure with preserved ejection fraction including T2DM [We did not find a correlation between diastolic dysfunction and the cardiac Pi/PCr , PCr content is more sensitive to change. Hence, it is unclear whether the observed Pi/PCr increase represents only a decrease in PCr or also an increase in cardiac Pi. In addition, our patient group was relatively small. Hence, further investigation of cardiac Pi metabolism in a larger cohort of patients with T2DM using recent absolute quantification techniques  will be 31P-MRS at 7T we have observed for the first time increased Pi/PCr in the diabetic human heart in comparison to healthy subjects. While spectral SNR proved to be a limiting factor in patients with high BMI, good quality data were acquired in 12/17 patients. PCr/ATP and Pi/PCr were not significantly correlated, which hints that multiple mechanisms might contribute to these perturbations.In Conclusion, using STEAM


Lemna species are cosmopolitan floating plants that have great application potential in the food/feed, pharmaceutical, phytoremediation, biofuel, and bioplastic industries. In this study, the effects of exogenous melatonin  on the growth and production of various bioactive metabolites and intact lipid species were investigated in Lemna aequinoctialis culture.Lemna aequinoctialis culture. Melatonin treatment also increased cellular production of metabolites including β-alanine, ascorbic acid, aspartic acid, citric acid, chlorophyll, glutamic acid, phytosterols, serotonin, and sucrose, and intact lipid species; digalactosyldiacylglycerols, monogalactosyldiacylglycerols, phosphatidylinositols, and sulfoquinovosyldiacylglycerols. Among those metabolites, the productivity of campesterol (1.79 mg/L) and stigmasterol (10.94 mg/L) were the highest at day 28, when 10 µM melatonin was treated at day 7.Melatonin treatment significantly enhanced the growth  of the L. aequinoctialis cultivation as a resource for food, feed, and pharmaceutical industries.These results suggest that melatonin treatment could be employed for enhanced production of biomass or various bioactive metabolites and intact lipid species in large-scale The online version contains supplementary material available at 10.1186/s12870-022-03941-x. Lemna aequinoctialis is a member of the aquatic monocotyledonous Lemnaceae family (duckweeds) and is one of the simplest and smallest plants trifluoroacetamide; Sigma-Aldrich, St, Louis, MO, USA) containing 1% trimethylchlorosilane, and 10 µL of 2,000 µg/mL myristic-d27 acid  in pyridine  as an internal standard were added to the dried samples and the mixtures were incubated in a 65℃ water bath for 60 min. One microliter of each sample was then injected into the GC-MS  with a DB-5 MS column , and the auxiliary, MS source, and quadrupole temperatures were set to 280, 230, and 150℃, respectively. The initial oven temperature was set to 60℃ and elevated up to 310℃ at 5℃/min.Freeze-dried samples were ground with a mortar and pestle, and 20 mg of each d method . Brieflyd method . BrieflyL. aequinoctialis sample was separately weighed, and extraction and nanoESI-MS analysis of the intact lipid species of L. aequinoctialis were performed as previously reported trifluoroacetamide; Sigma-Aldrich, St. Louis, MO, USA) containing 1% trimethylchlorosilane, and 10 µL of 2,000 µg/mL myristic-d27 acid  in pyridine  as an internal standard were added to the dried samples and then incubated in a 65℃ water bath for 60 min. One microliter of each sample was injected into the GC-MS  with a DB-5 MS column , and the auxiliary, MS source, and quadrupole temperatures were set to 280, 230, and 150℃, respectively. The initial oven temperature was set to 60℃ and elevated up to 310℃ at 5℃/min. The standard calibration curves, LOD, and LOQ were also determined according to the previously reported method [Absolute quantification of phytosterols was accomplished using the calibration curves of each standard compound. Standard curves were generated with 0.78125, 1.5625, 3.125, 6.25, 12.5, and 25 mg/L of campesterol; 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, and 50 mg/L of β-sitosterol; and 3.125, 6.25, 12.5, 25, 50, and 100 mg/L of stigmasterol. GC-MS analysis for absolute quantification was performed according to the previously described method . Brieflyd method .SPSS Statistics 26 software  was used for the Kruskal-Wallis test, Mann-Whitney test, and Bonferroni’s correction. Principal component analysis (PCA) was performed using SIMCA software , and mean-centered and Pareto scaling were adapted for preprocessing.Additional file 1: Figure S1. Growth of L. aequinoctialis culture cultivated for 35 days. Total number offronds (A) and dry weight (B).Dataare mean values, and the vertical bars represent the standard deviation fromthree biological replications. TableS1.Identification of various L.aequinoctialis culture metabolites by gas chromatography-mass spectrometry(GC-MS) analysis.The base peak ineach compound among ion fragments is shown in bold characters. RT, retentiontime; TMS, trimethylsilylation; MEOX, methoxylamine hydrochloride. TableS2 Relativelevels (relative intensity/g) of various L.aequinoctialis culture metabolites cultivated under various melatoninconcentrations at day 14.Data aremean ± standard deviationvalues of nine measurements from three biological replicates and threetechnical replicates of the control group and the 0.1, 1, and 10 μM melatonin treatment groups on day 14. The valueswithin a column with different letters  mean statisticallysignificant differences evaluated by using the Kruskal-Wallis test followed by theMann-Whitney test as a post hoc analysis with Bonferroni's correction (p < 0.0083). TableS3.Relative levels (relative intensity/g) of various L. aequinoctialis culture metabolites cultivated under variousmelatonin concentrations at day 28.Dataare mean ± standard deviation values of nine measurements from three biologicalreplicates and three technical replicates of the control group and the 0.1, 1,and 10 μM melatonin treatment groups on day 28. The values within a column withdifferent letters  mean statistically significant differencesevaluated by using the Kruskal-Wallis test followed by the Mann-Whitney test asa post hoc analysis with Bonferroni's correction (p < 0.0083). TableS4.Identification of various intact lipid species in L. aequinoctialis culture by nano-electrospray ionization-mass spectrometry(nanoESI-MS) analysis. TableS5 Relativelevels (relative intensity/g) of various intact lipid species of L. aequinoctialis culture cultivatedunder various melatonin concentrations at day 14. Data are mean ± standarddeviation values of nine measurements from three biological replicates and threetechnical replicates of the control group and the 0.1, 1, and 10 μM melatonintreatment groups on day 14. The values within a column with different letters mean statistically significant differences evaluated by using theKruskal-Wallis test followed by the Mann-Whitney test as a post hoc analysiswith Bonferroni's correction (p < 0.0083). TableS6.Relative levels (relative intensity/g) of various intact lipid species of L. aequinoctialis culturecultivated under various melatoninconcentrations at day 28.Data aremean ± standard deviationvalues of 9 measurements from 3 biological replicates and 3 technicalreplicates of control group and 0.1 μM, 1 μM, and 10 μMmelatonin treatment groups on day 28. The values within a column withthe different letters  mean statistically significant differencesevaluated byusing the Kruskal-Wallis test followed by Mann-Whitney test as post hocanalysis with Bonferroni's correction (p <0.0083).


In comparison to I.P. anesthesia with room air (21% O2), supplying 100% O2 improved 7-day survival by cardiovascular stabilization, reducing lactic acidosis and preventing vascular leakage. However, this had no effect on the intestinal epithelial damage, permeability, and inflammatory response observed after intestinal IRI. In contrast to I.P. + 100% O2, isoflurane anesthesia reduced intestinal IRI by preventing ongoing low-flow reperfusion hypotension, limiting intestinal epithelial damage and permeability, and by having anti-inflammatory effects. When translating the aforementioned results of this study to clinical situations, such as intestinal ischemia or transplantation, the potential protective effects of hyperoxygenation and volatile anesthetics require further research.Animal research in intestinal ischemia-reperfusion injury (IRI) is mainly performed in rodent models. Previously, intraperitoneal (I.P.) injections with ketamine–xylazine mixtures were used. Nowadays, volatile anesthetics (isoflurane) are more common. However, the impact of the anesthetic method on intestinal IRI has not been investigated. We aim to analyze the different anesthetic methods and their influence on the extent of intestinal IRI in a rat model. Male Sprague–Dawley rats were used to investigate the effect of I.P. anesthesia on 60 min of intestinal ischemia and 60 min of reperfusion in comparison to hyperoxygenation (100% O Intestinal ischemia-reperfusion injury (IRI) is a frequent clinical entity, typically observed in patients suffering from intestinal ischemia but also as an inevitable part of intestinal transplantation (ITx) ,2.Ischemia causes the depletion of adenosine triphosphate (ATP) and the accumulation of radical oxygen species (ROS) precursors . When blResearch to unravel the pathophysiologic mechanisms and potential interventions in intestinal IRI is mainly performed in rodent models ,12. PrevHowever, to our knowledge, the impact of these anesthetic methods in se has not been investigated in intestinal IRI. Therefore, we aimed to analyze the different potential modulators, which might influence the outcome of intestinal IRI in a rodent model. As volatile anesthetics are provided with oxygen supplementation, we split the experimental setup to investigate, on one hand, the effect of oxygen supplementation, followed, on the other hand, by the additional potential protective effect exerted by isoflurane.2 increased the oxygen saturation and had a stabilizing effect on the heart rate during the experiment (2) measurements at sacrifice showed hypoxia in non-oxygenated animals, while 100% O2 administration led to hyperoxia: sham ischemia with intraperitoneal anesthesia (I.P.) (sham I.P. + 21% O2) 52.38 ± 15.95 mmHg and I.P. + 21% O2 73.48 ± 20.84 mmHg vs. I.P.+100% O2: 268 ± 22.31 mmHg (both p < 0.001) (2 at the moment of sacrifice after 60 min of reperfusion: 7.31 ± 0.02 vs. 7.16 ± 0.10 (p = 0.02), whilst this was not different from I.P. + 100%: 7.19 ± 0.08  (p < 0.001)  B. When l< 0.001) D. Signifctively) E. By ana< 0.001) F.2 group was 5.83 ± 0.41, which was not altered following oxygen supplementation (I.P. + 100% O2 6.00 ± 0.00) (p > 0.99). This was confirmed by the villus height, which measured 95.2 ± 19.3 µm with I.P. + 21% O2 versus 105.1 ± 9.2 µm for I.P. + 100% O2 (p > 0.99). When looking at the intestinal epithelial permeability to ions, by measuring the transepithelial electrical resistance (TEER) in an Ussing chamber setup, no difference was noted: 7.5 ± 2.8 Ω *cm2 in I.P. + 21% O2 versus 8.6 ± 1.8 Ω *cm2 in the I.P. + 100% O2 group . Plasmatic heparan sulfate levels showed a significant increase after IRI in I.P. + 21% O2 in contrast to sham: 233.5 ± 109.1 ng/mL vs. 46.63 ± 54.35 ng/mL (p = 0.05) (p = 0.02), (ii) increased reperfusion edema (wet/dry ratio): 6.15 ± 1.18 vs. 4.41 ± 0.397 (p = 0.001), and (iii) increased plasmatic endotoxin levels: 83.13 ± 32.89 vs. 20.14 ± 8.94 mU/mL (p < 0.001)  A,B. This< 0.001) C,E.p = 0.001). Oxygen supplementation had no impact on IL-6 levels: 1022 ± 568.3 pg/mL  (2) vs. 6.837 ± 3.572  and vs. 8.497 ± 3.269  (2) vs. 14.58 ± 14.03  and vs. 23.77 ± 16.38  (2) vs. 4.211 ± 1.141  and vs. 5.288 ± 1.330  -6 significantly increased in contrast to sham treatment: 1540.0 ± 856.3 vs. 334.0 ± 165.6 pg/mL (ctively) A. This i< 0.001) B. Simila = 0.01) C, and TN< 0.001) D.2) or volatile (isoflurane), no differences were seen in oxygen saturation or heart rate if 100% of oxygen was applied during the experiment (2 measurements showed more pronounced hyperoxia in the isoflurane ischemic rats (isoflurane + 100% O2): sham ischemia with isoflurane (sham isoflurane + 100% O2) 249.0 ± 70.57 mmHg and I.P. + 100% O2 268.0 ± 22.31 mmHg vs. isoflurane + 100% O2: 354.7 ± 63.84 mmHg  (2: 7.322 ± 0.03 vs. 7.193 ± 0.08 (p = 0.04), whilst this was not different from isoflurane + 100% O2: 7.238 ± 0.04   B. Howevectively) D. A signctively) E. Lactatctively) F.2) vs. 6 ± 0 (I.P. + 100% O2) (p = 0.47) (p = 0.77)  A,D–F. Th = 0.77) B. The me2) vs. 20.14 ± 8.935 mU/mL (p = 0.67)  E.2) vs. 1022.0 ± 568.3 pg/mL (I.P. + 100% O2) (p = 0.02) (2) vs. 8.497 ± 3.269  (Plasmatic levels of IL-6 were significantly reduced with isoflurane anesthesia: 136.7 ± 52.52 pg/mL (gas + 100% O = 0.02) A. This w= 0.003) B. This e= 0.003) C–D.p < 0.0001)  in contrast to 70% survival with oxygen supplementation and even 90% when isoflurane was used instead ( 0.0001) .In this experimental rodent study on intestinal IRI, we studied the effect of oxygen supplementation and isoflurane anesthesia (volatile) compared to I.P. ketamine–xylazine anesthesia.2) in intestinal IRI experiments have been shown before. The main effects were attributed to decreased enterocyte apoptosis and decreased neutrophil recruitment by the downregulation of E-selectin production [Oxygen supplementation clearly ameliorated the detrimental effects of intestinal IRI with ketamine–xylazine anesthesia, with significantly improved 7-day survival. The protective effects of hyperoxia , weighing 275–350 g, 6 weeks old, were housed in the KU Leuven animal facility under specific pathogen-free conditions, with 2–3 rats per cage. The rats were acclimatized for 5–7 days before any intervention. The animals were kept in 14/10 h light/dark cycles, controlled temperature, and they received rat chow and water ad libitum. The animals were not fasted before surgery. Institutional animal research ethical committee approval—following the EU directive for animal experiments—was granted under the number P122/2019. The reported animal study is in compliance with the ARRIVE guidelines 2.0 .Intraperitoneal (I.P.) injection: mixture of 57.14 mg/kg body weight (BW) of ketamine  and 5.71 mg/kg BW of xylazine  .2 by usage of an isoflurane vaporizer .Gas: isoflurane , induction with 5% and maintenance with 1.5–1.75% at 1 L/min, and 100% OIRI was performed with 60 min of ischemia and 60 min of reperfusion. Intestinal IRI was induced, after median laparotomy of 4 cm on the linea alba, by isolated atraumatic clamping of the superior mesenteric artery. Ischemia was checked by pulselessness in the mesentery and discoloration/dysmotility of the bowel. The laparotomy wound was temporarily closed during the experiment. At reperfusion, 1 mL of warmed saline (37 °C) was administered intraperitoneally to compensate for fluid loss by evaporation. Reperfusion was checked by recovery of arterial pulsations in the mesentery, recoloration, and regain of motility. At the end of the experiment, all animals were sacrificed by exsanguination, followed by blood and intestinal tissue collection. The animals were under anesthesia until exsanguination. All experiments were performed by the same researcher (M.C.).n = 6/group) (Rats were randomly divided into five groups (6/group) :For survival analysis, 10 additional animals were included in each IRI group and observed daily for 7 days. The laparotomy wound was closed subcutaneously in 2 layers with Prolène 4-0 , and 0.5 mL of ropivacaine  was administered in the wound edges for local analgesia.In accordance with animal welfare, rats were monitored at least 3 times daily, and buprenorphine subcutaneous  was used for analgesia, once preoperatively and twice daily postoperatively, during the first 3 days.At the end of the experiment, rats were anesthetized with pentobarbital before sacrifice .Vital parameters were measured, every 15 min, from 5 min before onset of ischemia until awakening/sacrifice/death. Rectal temperature was measured by usage of a clinical thermometer . Heart rate and saturation were measured on hind paws by usage of OxiPen . Tail cuff blood pressure measurements were performed by usage of the Coda non-invasive blood pressure system . Ten blood pressure measurements were taken at each time point, and the median was used for analysis.Heparinized blood samples were collected after puncture of the aorta for blood gas analysis (0.4 mL) in 2 EDTA tubes. The tubes were spun at 3500 rpm for 10 min at 4 °C. Plasma was snap frozen in liquid nitrogen and stored at −80 °C. One ileal tissue sample of 5 cm was taken just proximally of the ileocaecal valve, kept in glucose buffer on ice, and mounted in the Ussing chambers. Ileal samples were collected proximally of the previous and preserved in 4% buffered formalin for histological evaluation and snap frozen, after feces removal, for molecular analysis.2) (mmHg), arterial carbon dioxide partial pressure (pCO2) (mmHg), oxygen saturation (%), and plasmatic L-lactate release (mmol/L) were analyzed by blood gas analyzer .Hemoglobin levels (g/dL), hematocrit (%), pH, arterial oxygen partial pressure  on four fields per section by usage of the Park–Chiu score ,41.Villus length—defined as the distance between the mouth of the crypts and the tip of the villi—was measured in 4 different fields per tissue section, and the average was calculated to avoid the potential impact of patchy necrosis.2 by a blinded, experienced researcher (A.A.). Each half chamber was filled with 3 mL Krebs solution with 10 mM mannitol at the mucosal side and 10 mM glucose at the serosal side. Both buffers were maintained at 37 °C and continuously oxygenated with 95%/5% O2/CO2 and stirred by gas flow in the chambers. In this setup, data sampling and pulse inductions are computer-controlled using Clamp software . Transepithelial electrical resistance (TEER) was measured by averaging 90 min of measurement after initial 30 min of stabilization. All tissue was mounted within 10 min after the exsanguination of the animal.Full-thickness ileal tissue  was mounted, in triplicate, in a standard vertical Ussing chamber  with an opening of 9.60 mmIn our particular setting of intestinal IRI, leading to a diminished/denudated mucosal surface area , TEER wTM, Lonza, Belgium), according to the manufacturer’s instructions. Absorbance was measured spectrophotometrically by FLUOstar Omega  at 410 nm. Corrections were made by the subtraction of the absorbance of the sample without the addition of LAL.Quantification of plasma endotoxin levels was obtained by the colorimetric limulus amebocyte lysate test (LAL QCL-1000Tissue water content (edema) was assessed by the ratio between the weight before and after drying. Snap-frozen, whole-thickness ileal tissue samples were weighed just before and immediately after drying them for 3 h at 80 °C in a drying oven with forced convection . The results were expressed as a wet/dry ratio and a percentage of water in the tissues ((1-(Dry weight/Wet weight))*100).Plasma Syndecan-1  concentration was measured by ELISA according to the manufacturer’s instructions.Plasma heparan sulfate  concentration was measured by ELISA according to the manufacturer’s instructions.Plasma IL-6 concentration was measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions .The relative expression of pro-inflammatory cytokines (interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and anti-inflammatory cytokine (IL-10)) were determined by qRT-PCR. Tissue was homogenized in TRIzol reagent , and total ribonucleic acid  was extracted using the RNeasy isolation kit , according to the manufacturer’s instructions. cDNA was synthesized from 200 ng total RNA using M-MLV transcriptase . Next, real-time PCR reaction was performed on a LightCycler 96W  with Taqman Fast Universal PCR Master Mix and Taqman gene expression assays  (IL-1β (Rn00580432_m1), TNF-α (Rn00562055), and IL-10 (Rn00563409)). A three-step amplification program was used: 95 °C for 10 min, followed by 45 cycles of amplification . Target messenger RNA (mRNA) expression for cytokines was quantified relative to the housekeeping gene GAPDH  and to the sham using the “-ΔΔCt method”.p-value < 0.05 was considered statistically significant .All data were expressed as mean ± standard deviation and represented in scattered plots. The line in the middle is plotted at the mean. The whiskers indicate the standard deviation. Data were checked for outliers by the ROUT method with Q = 1% and subjected to (log)normality testing (Shapiro–Wilk test). Comparisons between multiple groups were performed with one-way ANOVA and post-hoc Tukey’s test in the case of normal distribution or the Kruskal–Wallis and post-hoc Dunn tests for non-normal distribution. Survival analysis was performed by the Kaplan–Meier test (log-rank test). A In experimental rodent models, different types of anesthesia are commonly used. In this study on intestinal IRI, it was shown that oxygen supplementation had a protective effect on the vascular permeability of the intestine. Isoflurane anesthesia attenuated the detrimental effects of intestinal IRI even more by reducing intestinal epithelial permeability and the inflammatory cascade compared to ketamine–xylazine anesthesia. The type of anesthesia used in these experimental models can influence the outcome parameters analyzed and should be taken into account. The potential clinical implications should be investigated in ischemic and transplant patients.


The control (CK) did not receive N fertilizer. Maize yield, N uptake and use, changes in soil inorganic N content, and N balance were investigated. Compared with the single basal application (N1), split-N applications  increased maize yield from 13.8% to 24.5% by increasing kernel number per ear and 1000-kernel weight and also improved N accumulation from VT to physiological maturity (PM) stages and its contribution to grain N uptake. In addition, compared with N1, split-N applications also decreased N losses by increasing inorganic N contents in the 0–40 cm soil layer and by decreasing N leaching in the 60–200 cm soil layer. Regression analysis demonstrated that N accumulation after the VT stage was positively related with maize yield. Among treatments, N4 had the highest yield, N recovery efficiency, agronomic efficiency, and partial factor productivity, with respective increases of 24.5%, 14.7 percentage point, 11.4 kg kg−1, and 11.4 kg kg−1 compared with those in N1. As a result, N losses were also reduced by 33.7% in N4 compared with those in N1. In conclusion, the split-N management strategy with four N applications under film-mulched drip irrigation has great potential to improve maize yield, increase N use efficiency, and reduce N loss in the semiarid region of Northeast China.Film-mulched drip irrigation has become an important strategy in maize cultivation in the semiarid region of Northeast China. Most farmers concentrate nitrogen (N) fertilizer use early in maize growth, which leads to low N use efficiency and large N losses. Therefore, a three-year (2018 to 2020) field experiment was conducted to determine the optimal N management strategy for maize under film mulch with drip irrigation in the semiarid region of Northeast China. The experiment included five treatments with the total amount of N fertilizer (210 kg N ha HoweverEffective N management with the “4R” strategy  has been advocated to ensure synchronization between soil N supply and crop N requirements and thereby maximize crop yield and N use efficiency and minimize negative environmental effects . Split-N7 ha of arable land, and it is one of the important maize growing regions in China  determine the effects of split-N applications on maize yield and N use efficiency, (2) analyze changes in soil inorganic N and determine apparent N balance and N losses, and (3) recommend the optimal N fertilizer application strategy for film-mulched drip-irrigated maize in semiarid areas of northeastern China.22.1−1; total N 1.2 g kg−1; Olsen-phosphorus (P) 14.98 mg kg−1; ammonium acetate-potassium (K); and pH 8.06. Before planting, the soil nitrate-N (NO3−–N) and ammonium-N (NH4+–N) concentrations were 16.91 and 3.13 mg kg−1 at 0–20 cm; 15.28 and 3.18 mg kg−1 at 20–40 cm; 11.28 and 2.92 mg kg−1 at 40–60 cm; 9.32 and 3.11 mg kg−1 at 60–80 cm; 8.39 and 2.66 mg kg−1 at 80–100 cm; 8.93 and 2.96 mg kg−1 at 100–120 cm; 7.28 and 3.24 mg kg−1 at 120–140 cm; 7.61 and 3.13 mg kg−1 at 140–160 cm; 6.35 and 3.17 mg kg−1 at 160–180 cm; and 6.27 and 2.73 mg kg−1 at 180–200 cm, respectively.The field experiment was conducted from 2018 to 2020 at the experimental station of Jilin Academy of Agricultural Sciences, Qian’an County, Jilin Province, Northeast China . The station is in the northwestern part of Jilin Province, which has a cool temperate climate with a single cropping season. Average annual precipitation is 400 mm, and average annual evaporation is greater than 1,500 mm Figure 12.2−1, which was determined in a previous study in the region (−1) applied in different proportions at sowing, sixth-leaf (V6), twelfth-leaf (V12), tasseling (VT), and blister (R2) stages of maize growth: N1, 100-0-0-0-0; N2, 50-50-0-0-0; N3, 30-50-20-0-0; N4, 20-30-30-20-0; and N5, 10-20-30-20-20 (2O5 ha−1) and K (100 K2O ha−1) fertilizers were applied in each treatment as basal fertilizer before sowing. Fertilizers were urea (N 46%), calcium superphosphate (P2O5 46%), and potassium chloride (K2O 60%).The N fertilizer rate in drip-irrigated maize was 210 kg N hae region . The exp30-20-20 Table 1.−1. The cultivation mode was large ridge with double rows under film mulching. Row spacing on the ridge was 40 cm, and row spacing between ridges was 90 cm. Treatment plots were 60 m2 . Maize was planted on May 5, May 8, and May 6 and was harvested on September 30, October 2, and October 5 in 2018, 2019, and 2020, respectively. Before mulching, herbicides were sprayed on the soil surface to control weeds. Mulching was synchronized with laying the drip irrigation belt. The plastic film was a polyethylene agricultural transparent film with a width of 120 cm and a thickness of 0.08 mm. Drip irrigation belts used inner inlaid pieces, and spacing of drippers was 30 cm. Drip irrigation belts were placed in the middle of ridges, and each drip irrigation belt watered two rows of maize. Based on a previous study , 225 m3 ha−1 for V6, 375 m3 ha−1 for V12, 450 m3 ha−1 for VT, and 375 m3 ha−1 for R2 stages. A separate water meter was used to control the irrigation amount in each treatment. Each plot was equipped with a separate 18-L differential pressure fertilizer tank. Duration of fertilizer application was 120 min, with irrigation with water without fertilizer 30 min before and after fertilization. Other field measures were optimized and managed according to local agricultural technicians.Maize variety Huanong101 was planted at a density of 70,000 plants haus study , the dri2.3nd and 7th rows. All samples were fixed at 105°C for 30 min and then dried at 70°C to constant weight. Plant biomass was weighed for each treatment. Plant materials were crushed, and N contents of straw and grain were determined following digestion with H2SO4–H2O2 according to a Kjeldahl method.Maize samples were collected from three-leaf (V3), V6, V12, VT, R2, and physiological maturity (PM) stages. In each plot, five plants were collected and divided into straw and grain parts in the 22 (between the 3rd and 6th rows) in each plot and ultimately converted to 14% standard water content. In addition, 30 representative plants were selected to measure kernel number per ear and 1000-kernel weight.At harvest, grain yield was measured in 30 m3−-N and NH4+-N contents, soils were sieved through a 5-mm screen and then extracted using 1 mol L−1 KCl solution (soil:liquid ratio 1:5) with shaking. Extracts were analyzed for NO3−-N and NH4+-N on a flow injection analyzer . The soil bulk density was determined using the cutting ring method. For each plot, five points were evaluated in the soil profile within 100 cm and cutting-ring soil samples were collected at 20 cm intervals throughout the soil layer. The soil was dried at 105°C for 24 h.Soil samples at 20 cm intervals from 0 to 200 cm were collected before planting in 2018 and after harvesting in 2020. Soil was randomly sampled at eight points, and samples were mixed into one composite sample per plot. To determine water content, soils were oven-dried at 105°C for 24 h. To determine NO2.4Contribution of accumulated N after the VT stage to grain N accumulation under different split-N treatments was assessed as follows :Accumulated recovery efficiency (REN), agronomic efficiency (AEN), and partial factor productivity (PFPN) under different N treatments were calculated as follow:i is the season ; FU and CKU are N uptake in aboveground dry matter with and without N fertilizer application (kg ha−1), respectively; FY and CKY are maize yield with and without N fertilizer application (kg ha−1), respectively; and NF is the N fertilizer application amount (kg ha−1).where 3−-N and NH4+-N in the top 100 cm of soil were used in N budget calculations. Apparent N mineralization and apparent N loss were determined according to the following formula:To evaluate environmental effects of different N application modes, the apparent N balance between N input and output was estimated in the soil–plant systems. Contents of NO−3), and Ninorganic is the soil inorganic N content, which is the sum of NO3−-N and NH4+-N contents (mg kg−1). Soil bulk density was 1.24 g cm−3 at 0–20 cm, 1.31 g cm−3 at 20–40 cm, 1.41 g cm−3 at 40–60 cm, 1.40 g cm−3 at 60–80 cm, and 1.38 g cm−3 at 80–100 cm.where T is the thickness of the soil layer (cm); BD is the soil bulk density  is estimated based on the CK; uptakeN is the N uptake in aboveground dry matter (kg ha−1); residualN and initialN are soil inorganic N accumulation after harvest and before sowing, respectively, in the 0 to 100 cm soil layer.where apparent N mineralization  among N application treatments were determined using Tukey’s multiple range tests in SPSS 19.0 software .Significant differences (33.1P< 0.001) compared with that with single basal fertilization (N1), with average increases of 13.8 to 24.5% over the three years. In 2018, increases ranged from 13.3 to 25.1%; in 2019, they ranged from 11.9 to 21.9%; and in 2020, they ranged from 16.2 to 26.6%. Differences in yield were due to significant increases in kernel number per ear (P< 0.001) and 1000-kernel weight (P< 0.001) in split-N treatments, with average increases ranging from 6.4% to 13.7% and 4.6% to 9.5%, respectively, over the three years.Split-N fertilizer management significantly affected maize yield, kernel number per ear, and 1000-kernel weight Table 2.Among split-N treatments, maize yield, kernel number per ear, and 1000-kernel weight first increased and then decreased with the increase in N application frequency. The highest yield was in N4 treatment, and compared with that in N2, N3, and N5 treatments, average maize yield increased by 11.8%, 3.9%, and 5.1%; kernel number per ear increased by 6.8%, 1.8%, and 2.5%; and 1000-kernel weight increased by 4.6%, 1.8%, and 2.1%, respectively, over the three years.3.2Nitrogen fertilizer applications increased N accumulation from V6 to PM stages compared with that in CK, although there were no differences in the V3 stage Figure 2Nitrogen fertilizer significantly increased the proportion of total N accumulation in VT to PM stages compared with that in CK Figure 3Split-N treatments significantly increased the contribution of accumulated N to grain N accumulation after the VT stage, with average increases of 9.2% to 36.6% compared with the N1 treatment over the three years Figure 43.3−1, and PFPN by 6.5–11.4 kg kg−1 over the three years. Among split-N treatments, the highest N use efficiency was in N4, and compared with N2, N3, and N5, REN increased significantly by 6.7, 2.2, and 3.6 percentage points; AEN by 4.9, 2.2, and 2.9 kg kg−1; and PFPN by 4.9, 2.2, and 2.9 kg kg−1, respectively.Accumulated recovery efficiency (REN), AEN, and PFPN were significantly affected by split-N applications Figure 53.4R2 = 0.799 (yield), 0.713 (REN), 0.653 (AEN), 0.859 (PFPN)] were higher than those of equations for V3 to VT stages , which indicated that N accumulation was more closely related to yield and N use efficiency in VT to PM stages.Regression analyses showed that yield, REN, AEN, and PFPN were significantly positively related to N accumulation in V3 to VT and VT to PM stages Figure 63.53−-N content by 22.9% to 57.1% at 0–20 cm and by 19.8% to 52.7% at 20–40 cm compared with N1  significantly increased soil NO4+-N content in split-N treatments  was significantly higher than that in the N1 treatment at 0–40 cm (P = 0.002–0.037), but no differences were detected at 40–200 cm (P = 0.054–0.723). Compared with contents at the beginning of the experiment, NH4+-N content was not significantly different in different soil layers in any treatment (P = 0.064–0.726), except in N1, which had low NH4+-N content in the 0–40 cm soil layer.Soil NHThe apparent N balance in the soil–plant systems is shown in 44.1Exogenous N supply significantly affects crop yield and its components , and reg4.2Crop growth, root absorption capacity, and changes in soil N availability affect crop accumulation of N . Under oIn maize production, grain yield is closely related to N accumulation in the late growth stage . Thus, t4.33−-N and NH4+-N) is the main form of N uptake by plants and is also easily leached from soil in one of the main pathways of N loss in rain fed maize cropping systems (Soil inorganic N (NO systems . Reducin systems . Soil N  systems . Reducin systems . Therefo systems . In the  systems . By cont systems .5In the semiarid region of Northeast China, a drip irrigation system combined with split-N fertilizer application increased maize yield by increasing kernel number per ear and 1000-kernel weight. It also improved N uptake in the later stage of growth and N use efficiency by matching soil N supply with maize N requirements. Split-N application reduced the risk of soil N leaching by increasing inorganic N content in the tillage layer and decreasing inorganic N content in deep soil. Among split-N treatments, the drip irrigation N application strategy with 20% basal fertilizer + 30% jointing fertilizer + 30% belling fertilizer + 20% tasseling fertilizer obtained the highest maize yield and N use efficiency and also reduced potential N losses. Thus, the N4 treatment has broad application prospects.The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding authors.YH conceived the study and wrote the manuscript. LK and LZ measured experimental samples. YZ analyzed data. XX and ZL designed the study and drafted the manuscript. All authors reviewed the manuscript. All authors contributed to the article and approved the submitted version.


In culture, Namystinia karyoxenos accumulates Ba2+ and Sr2+ 42,000 and 10,000 times higher than the surrounding medium, forming barite and celestite representing 90% of the dry weight, the greatest concentration in biomass known to date. As heterotrophs, diplonemids are not restricted to the photic zone, and they are widespread in the oceans in astonishing abundance and diversity, as their distribution correlates with environmental particulate barite and celestite, prevailing in the mesopelagic zone. We found diplonemid predators, the filter-feeding zooplankton that produces fecal pellets containing the undigested celestite from diplonemids, facilitating its deposition on the seafloor. To the best of our knowledge, evidence for diplonemid biomineralization presents the strongest explanation for the occurrence of particulate barite and celestite in the marine environment. Both structures of the crystals and their variable chemical compositions found in diplonemids fit the properties of environmentally sampled particulate barite and celestite. Finally, we propose that diplonemids, which emerged during the Neoproterozoic era, qualify as impactful players in Ba2+/Sr2+ cycling in the ocean that has possibly contributed to sedimentary rock formation over long geological periods.Barium and strontium are often used as proxies of marine productivity in palaeoceanographic reconstructions of global climate. However, long-searched biological drivers for such correlations remain unknown. Here, we report that taxa within one of the most abundant groups of marine planktonic protists, diplonemids (Euglenozoa), are potent accumulators of intracellular barite (BaSO Depe2+ , 4. The -imaging . In contediments . Indeed, effects . In humaractures . Moreovend fungi , 10.2+ than Ba2+, possibly due to higher solubility and availability, i.e., the concentration of Sr2+ is around 88 μM compared to 40 to 150 nM Ba2+  and strontianite (SrCO3), while Ba2+ forms barite (BaSO4) or witherite (BaCO3) SO4  , 14. Morthareans . Intracethareans , 17, 18.eans , a group of biflagellated heterotrophic protists –31, are –rganisms , 19. In rganisms –34. Althganisms –. We analLacrimia and Namystinia, represented by Lacrimia sp. YPF1808, Lacrima lanifica, and Namystinia karyoxenos, were shown to possess celestite crystals , or calcite (CaCO3)  see  can be e10.1128/mbio.03279-22.1FIG S14 and BaSO4, gypsum (CaSO4), and calcite (CaCO3). Each complex of SO42– with Sr2+, Ba2+, or Ca2+ can be unambiguously identified by the position of the most intense Raman band at around 1,000 cm−1, belonging to the symmetric ν1 vibrational mode of oxygen atoms within the SO42– tetrahedron. Download FIG S1, PDF file, 0.2 MB.Typical Raman spectrum of biogenic celestite crystals compared with the spectra of chemically prepared barite, the equimolar precipitate of SrSOCopyright © 2023 Pilátová et al.2023Pilátová et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03279-22.2FIG S24 precipitates. Download FIG S2, PDF file, 0.1 MB.Typical Raman spectra (A to E) of biogenic celestite crystals found in various diplonemid cells. In terms of Raman frequencies, the spectra were virtually identical; however, minor spectral variability in the relative intensities of Raman bands was observed. Similar variability was also observed for mineral celestite and chemically prepared SrSOCopyright © 2023 Pilátová et al.2023Pilátová et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the N. karyoxenos was examined by light microscopy with differential interference contrast (DIC), the crystalline structures appeared as small birefringent particles moving fast by Brownian motion , and serial block-face scanning electron microscopy SBF-SEM) showed that the crystalline inclusions in two clades of diplonemids differed in their localization and shapes. In semithin resin-embedded sections of  lacunae , and D,  lacunae  and in s lacunae  and B. OF-SEM shot matrix  to L adjt matrix . Three-dt matrix . Less frt matrix  or in cot matrix , while tt matrix  and M or, and sert matrix  and N. A YPF1808 , and I.  YPF1808 , with sm YPF1808 .10.1128/mbio.03279-22.9MOVIE S2Lacrimia sp. YPF1808 taken by SBF-SEM with celestite crystals marked by arrows. PV, posterior vacuole; N, nucleus; FP, flagellar pocket. 3D reconstruction of a single cell via SBF-SEM shows the cytoplasm in yellow, a large posterior vacuole in orange, and celestine crystals in cyan. Download Movie S2, MOV file, 12.9 MB.Visualization of serial sections through a celestite-containing cell of Copyright © 2023 Pilátová et al.2023Pilátová et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Lacrimia sp. YPF1808  SEM micrograph; (B) merged image of the most common elements; (C) merged image of all elements present; (D) map of carbon K line; (E) map of nitrogen K line; (F) map of oxygen K line; (G) map of magnesium K line; (H) map of silicon K line; (I) map of strontium L line; (J) map of phosphorus K line; (K) map of sulfur K line; (L) map of chlorine K lines; (M) map of calcium K line; (N) map of barium L line. (O) Pie chart of the most common elements detected in the area with particular pixel counts as depicted in panel B: oxygen and carbon K lines (blue), oxygen K line (red), oxygen K line, strontium L line, sulfur K line (yellow). Scale bar, 10 μm. Download FIG S3, PDF file, 0.1 MB.Cryo-SEM-EDX analysis of Copyright © 2023 Pilátová et al.2023Pilátová et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03279-22.4FIG S4N. karyoxenos freeze-fractured samples. (A) SEM micrograph; (B) merged image of the most common elements; (C) merged image of all elements present; (D) map of carbon K line; (E) map of nitrogen K line; (F) map of magnesium K line; (G) map of silicon K line; (H) map of strontium L line; (I) map of phosphorus K line; (J) map of sulfur K line; (K) map of chlorine K lines; (L) map of calcium K line. (M) Pie chart of the most common elements detected in the area with particular pixel counts as depicted in panel B: oxygen and carbon K lines (blue), oxygen K line (dark red), oxygen and carbon K lines (yellow). Scale bar, 10 μm. Download FIG S4, PDF file, 0.1 MB.Cryo-SEM-EDX analysis of Copyright © 2023 Pilátová et al.2023Pilátová et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Lacrimia sp. YPF1808 (1 μm-thick sections from resin blocks used for SBF-SEM) and N. karyoxenos (250 nm-thick resin sections examined by TEM) were confirmed by SEM-EDX and TEM-EDX, respectively  with space group Pnma and lattice parameters a = 8.3 Å, b = 5.3 Å, and c = 6.8 Å in L. lanifica. Larger lattice parameters  were observed in N. karyoxenos, which may be explained by the replacement of Sr2+ with larger Ba2+ in the structure of celestite.The identities of celestite crystals in ectively ; Fig. S510.1128/mbio.03279-22.5FIG S5Lacrimia sp. YPF1808 showing the elemental composition of samples used for 3D reconstruction by SBF-SEM. (A) Electron micrograph; (B) merged image of the most common elements; (C) merged image of all elements present; (D) map of carbon K line; (E) map of oxygen K line; (F) map of strontium L line; (G) map of calcium L line; (H) map of barium M line; (I) map of osmium M line; (J) map of nitrogen K line; (K) map of sodium K line; (L) map of sulfur K lines. (M) Pie chart of the most common elements detected in the area with particular pixel counts as depicted in panel B: *, oxygen K line/calcium L line; **, oxygen K line/calcium L line/nitrogen K line; ***, oxygen K line/calcium L line/nitrogen K line/strontium L line/osmium M line. Scale bar, 2 μm. Download FIG S5, PDF file, 0.1 MB.SEM-EDX analysis of Copyright © 2023 Pilátová et al.2023Pilátová et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Lacrimia sp. YPF1808 (3 (−3), and common cellular densities of 0.985 to 1.156 g·cm−3 reported elsewhere  was consistent with the minute 88Sr content measured by ICP-MS. The high values of 88Sr in N. karyoxenos, Lacrimia sp. YPF1808, and L. lanifica corresponded to the abundance of intracellular crystals detected by Raman microscopy, TEM, and SBF-SEM. Since direct measurement of the dry mass was impossible due to the inevitable presence of salts from the medium, the elemental composition analysis by ICP-MS was calculated in atoms per cell or femtomoles per cell. To calculate Sr and Ba content per dry mass, the latter was subsequently estimated by quantitative phase imaging using holographic microscopy  in N. karyoxenos, corresponding to 340 ± 38 mg·g−1. Lacrimia sp. YPF1808 and L. lanifica were also potent 88Sr accumulators, with 370 ± 58 fmol·cell−1 (130 ± 25 mg·g−1) and 54 ± 8 fmol·cell−1 (64 ± 13 mg·g−1), respectively. Depending on the species, the intracellular concentration of 88Sr was 1,200 to almost 10,000 times higher than in the surrounding medium . Download Table S1, PDF file, 0.04 MB.ICP-MS analysis of seawater growth medium and artificial medium without the addition of sulfates, BaCopyright © 2023 Pilátová et al.2023Pilátová et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 88Sr in diplonemids, the naturally co-occurring Ba was present in much lower amounts  in N. karyoxenos, with lower values in Lacrimia sp. YPF1808  and L. lanifica . Altogether, SO4 accumulation reached 91%, 33%, and 14% of dry weight in N. karyoxenos, Lacrimia sp. YPF1808, and L. lanifica, respectively. All strains showed significantly different levels of accumulated Sr or Ba contents , celestite , and mixed forms of SO4   , revealiL. lafinica and Lacrimia sp. YPF1808 contained mostly mixed crystals of SO4. In N. karyoxenos, pure celestite prevailed in the central part of crystals (999 cm−1) while barite dominated at the periphery (988 cm−1), and they gradually overlapped each other  reaches a maximum of 220 mg·g−1 per dry weight , even though the concentration of Ca2+ in the cultivation medium or in the environment is several orders of magnitude higher than that of Sr2+ and Ba2+.Interestingly, when both trace elements are provided in equimolar concentrations, diplonemids form pure celestite and barite and/or mixed forms of SOcleation . The Sr2tiporter , 46. Thea brucei . Althougmparable , 46, somver Ca2+ . When su−3 and 4.5 g·cm−3, respectively, have been repeatedly reported as statoliths in ciliates or charophytic algae   , 48. Indectively . Accordiectively . Hence, ectively . Hence, ectively , 50.2+/Sr2+, which can also be converted to sulfates either on the bacterial extracellular polymeric substances or in the microenvironment of decaying matter of marine snow aggregates in the process of diagenesis , overlapping with the Ediacaran period phosphates, as reported elsewhere  heat-inactivated horse serum (Sigma-Aldrich) and 25 mg LB broth powder (Amresco). The medium was used as rinsing solution for preparation of ICP-MS samples and cell microcrystal depletion to be measured via quantitative phase imaging (QPI) (see below). For Ba2+ loading experiments, BaCl2 was added in equimolar amounts with respect to naturally occurring (88 μM) Sr2+ , 22°C (Namystinia karyoxenos YPF1621), or 13°C . P. papillatum and R. euleeides were isolated from coastal surface waters (United States) in 1985 and 1986, respectively. The remaining species originated either from coastal seawater around Japan or from Enoshima Aquarium  and were continuously maintained in culture for 1 to 7 years prior to the analyses. The identity of not-yet-formally described species was established based on the 18S rRNA sequences as described previously  and processed with GIMP v. 2.10.14, Irfan View v. 4.54, and Image J v. 1.51 software. Polarized microscopy was performed using crossed polarizers installed to a Raman microscope (as specified below).Centropages typicus, Temora longicornis, and Acartia sp. were then picked under a dissection microscope. All experiments were carried out at cultivation temperature of the prey species of diplonemids . Ten copepods were kept in 20 mL of diplonemid culture (105 cells mL−1) for 5 days, after which their fecal pellets were collected under a dissection microscope and immediately analyzed by Raman microscopy (as specified below).Zooplankton was collected in the Bay of Villefranche sur Mer, France  with a 10-min haul from 10 m to the surface, using a 20-μm-mesh-size plankton net. Captured copepods were transferred into 0.5 liters of freshly filtered natural seawater and starved for 12 h. in situ determination of the chemical composition of intracellular structures, a confocal Raman microscope  was used as previously described  equipped with an Olympus SIS charge-coupled-device camera Veleta  and an EDAX windowless EDX detector Apollo XLTW for elemental analysis. The diffraction data were collected by means of 3D electron diffraction (ED)  . The dation (ED) . Structuion (ED) .Cells pellets were high-pressure frozen as described above and transferred into a Leica ACE 600 preparation chamber  precooled at −135°C, fractured with a scalpel, freeze-etched at −100°C for 1 min, and sputter-coated with 2.5 nm of gold-palladium at −125°C. Specimens were transferred under vacuum using transfer system VCT100  and observed with a Magellan 400L SEM  precooled at −125°C (cryo-SEM). Topographical images and EDX measurements were obtained using an EDT detector and EDAX detector , respectively, either at 5 keV/0.1 NA or 10 keV/0.8 NA. The taken spectra were analyzed with EDAX TEAM software and quantified by the eZAF method.Lacrimia sp. YPF1808 by the high-pressure freezing technique followed the protocol for TEM sample preparation. After freeze-substitution, the samples were subsequently stained with 1% thiocarbohydrazide in 100% acetone for 1.5 h, 2% OsO4 in 100% acetone for 2 h at room temperature, and 1% uranyl acetate in 100% acetone overnight at 4°C. After every staining step, the samples were washed 3 times with 100% acetone for 15 min. Samples were then infiltrated with 25%, 50%, or 75% acetone-resin mixture for 2 h at each step, and finally infiltrated in 100% Hard Resin Plus 812 (EMS) overnight and polymerized at 62°C for 48 h. Resin-embedded blocks were trimmed and imaged using an Apreo SEM equipped with a VolumeScope . Serial images were acquired at 3.5 keV, 50 pA, 40 Pa with a resolution of 6 nm, 100-nm slice thickness, and dwell time per pixel of 4 μs. Image data were processed in Microscopy Image Browser v2.702  or Sr- and Ba-free artificial seawater rinsing solution (see above)  to remove Ba2+ and Sr2+ present in the cultivation medium. Cultivated cells were harvested by centrifugation and rinsed twice with 50 mL and once with 2 mL of the rinsing solution, and the resulting pellets were freeze-dried. A 0.5-mL digestion acid mix (425 μL of 70% HClO4 and 75 μL of 69% HNO3) prepared as described elsewhere  was used to prepare standard curves. Analyses were done using an inductively coupled plasma sector-field mass spectrometer (ICP sfMS) Element XR-2 with jet interface  following a described protocol . We calculated the total dry mass of the cells as the sum of crystal-free cells, measured by holographic microscopy, and SrSO4 and BaSO4 amounts measured via ICP-MS. The dry weight ratios of trace elements measured via ICP-MS were calculated based on the total dry weight of corresponding strains.Samples for holographic microscopy were immobilized prior to measurement as described above for Raman microscopy. Imaging was performed at the Q-Phase microscope . The holographic Q-Phase microscope is equipped with halogen lamp illumination through an interference filter  and microscope objective . The numerical reconstruction of acquired data was performed using Q-Phase software . The technique enables automated cell segmentation and quantitative analysis of cellular mass based on the specific proportions of thickness and refractive indices of measured cells in comparison to the reference . Due to post hoc) on an alpha level of 0.05. Calculations of standard errors of the means based on independent methods  with different levels of variability were done according to mathematical conversion using Taylor expansion.Statistical analysis was conducted using SigmaPlot v. 12.5 and SPSS v. 23.0. Logarithmically normalized data were subjected to statistical tests (one-way analysis of variance [ANOVA] and Tukey’s All data generated or analyzed during this study are included in the published article and its supplemental material files.


To explore the effect of maternal body mass index (BMI) on steroid hormone profiles in women with gestational diabetes mellitus (GDM) and those with normal glucose tolerance (NGT).We enrolled 79 women with NGT and 80 women with GDM who had a gestational age of 24–28 weeks. The participants were grouped according to their BMI. We quantified 11 steroid hormones profiles by liquid chromatography-tandem mass spectrometry and calculated the product-to-precursor ratios in the steroidogenic pathway.2 showed higher concentrations of dehydroepiandrosterone (DHEA) (p<0.001), testosterone (T) (p=0.020), estrone (E1) (p=0.010) and estradiol (E2) (p=0.040) and lower Matsuda index and HOMA-β than women with NGT and BMI<25kg/m2. In women with GDM, concentrations of E1 (p=0.006) and E2 (p=0.009) declined, accompanied by reduced E2/T (p=0.008) and E1/androstenedione (A4) (p=0.010) in the BMI>25 kg/m2 group, when compared to that in the BMI<25 kg/m2 group. The values of E2/T and E1/A4 were used to evaluate the cytochrome P450 aromatase enzyme activity in the steroidogenic pathway. Both aromatase activities negatively correlated with the maternal BMI and positively correlated with the Matsuda index in women with GDM.Women with GDM and BMI<25kg/mNGT women and GDM women with normal weight presented with different steroid hormone profiles. Steroidogenic pathway profiling of sex hormones synthesis showed a significant increase in the production of DHEA, T, E1, and E2 in GDM women with normal weight. Additionally, the alteration of steroid hormone metabolism was related to maternal BMI in women with GDM, and GDM women with overweight showed reduced estrogen production and decreased insulin sensitivity compared with GDM women with normal weight. Gestational diabetes mellitus (GDM) is defined as glucose intolerance and hyperglycemia that occur during pregnancy. It is one of the most common complications during pregnancy, which seriously threatens maternal and fetal health , 2. AccoAbnormal metabolism of steroid hormones may induce physiological disorders that lead to complications in obstetrics and gynecology, such as infertility, miscarriage, polycystic ovary syndrome (PCOS), preeclampsia, and GDM –13. It hHyperglycemia during pregnancy is the result of impaired glucose tolerance caused by pancreatic β-cell dysfunction on a background of chronic insulin resistance . PreviouThe aim of the present study was to explore the difference in steroid profiles in GDM patients and normal pregnant women at 24–28 gestational weeks and to investigate the effect of maternal BMI on steroid hormone profiles and steroid metabolic pathway in women with GDM.in vitro fertilization embryo transfer (IVF-ET) (2); personal history of chronic diseases, type 1 Diabetes Mellitus and T2DM, PCOS, autoimmune or chromosomal diseases and liver, kidney, adrenal or thyroid dysfunction (3); diseases that require hormone therapy. First, we compared the differences between women with GDM and those with NGT. The participants were subdivided according to BMI: BMI>25kg/m2 GDM group (n=24), BMI<25kg/m2 GDM group (n=56), BMI>25kg/m2 NGT group (n=12), and BMI<25kg/m2 NGT group (n=67)  with a gestational age of 24–28 weeks were enrolled between April 7, 2020, and May 22, 2020, at the Women’s Hospital of Zhejiang University School of Medicine, China. The pregnant women were between 23 and 35 years old, with a single fetus and normal fetal development at gestational age of 24–28 weeks. The exclusion criteria were as follows (1): p (n=67) Figure 1We used 75-g OGTT at 24–28 gestational weeks for the diagnostic criteria of GDM, which is recommended by the International Association of Diabetes and Pregnancy Study Group (IADPSG)  were dissolved in 10% methanol and 90% water. Internal standard  was dissolved in methanol. Firstly, 200 μL methanol was added to an HLB SPE plate , and was lowly flowed through the plate under low vacuum. Thereafter, 200 μL water was flowed through the SPE plate to balance the plate, after which the 200 μL calibrators, quality controls (QCs), and serum samples were placed into a 1.5 mL tube, wherein 200 μL of an internal standard mixture was added and mixed for 3 min. Finally, we added 400μL water, mixed it for 1min, and centrifuged it at 4°C for 15000 g for 10 min. The supernatant (700 μL) was then added to the SPE plates. A low vacuum causes the supernatant to flow slowly through the SPE plate. The HLB SPE plate was washed once with 200 μL 15% methanol. After 60μL of methanol was eluted into a 96-well plate and mixed with 60μL of water, the extract was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The details on the methods are shown in LC column: Waters HSS T3 , pre column: HSS T3 .LC method A: The mobile phase consisted of 2 mM ammonium acetate, 0.1% formic acid, and water (solvent A) or methanol (solvent B). The liquid chromatographic gradient was as follows: at 0~4 min, 40%~60% solvent B; 4~6.5 min, 60%~75% solvent B; 6.5~7.5 min, 75%~90% solvent B; 7.5~7.6 min, 90%~45% solvent B; and 7.6~8.0 min, 40% solvent B. Column temperature was 45°C, injection volume was 10 μL and flow rate was 0.45 mL/min.LC method B: The mobile phase consisted of a 0.1% ammonia solution and water (solvent A) or methanol (solvent B). The liquid chromatographic gradient was as follows: at 0~0.6 min, 30% solvent B; 0.6~0.7 min, 30%~65% solvent B; 0.7~1.5 min, 65~85% solvent B; 1.5~2.5 min, 85%~98% solvent B; 2.5~2.6 min, 98%~30% solvent B; and 2.6~3.0 min, 30% solvent B. Column temperature was 45°C, injection volume was 10 μL and flow rate was 0.4 mL/min.Detection was performed using LC-MS/MS (Waters TQS) equipped with an electrospray ionization probe and operated by switching between positive and negative ionization modes. The capillary potential was set at 3.2 kV. The ion-source temperature was 150°C and the desolvation gas was heated to 400°C at a flow rate of 600 L/h. The cone gas flow rate was set to 150 L/h. Multiple reaction monitoring was used for quantification, as listed in p<0.05 was considered statistically significant. Spearman correlation analysis was also performed. * p<0.05, ** p<0.01.SPSS software version 22  was used for the data analysis. The student’s t-test was performed using the clinical characteristics between the two groups. The data are presented as mean ± standard error (SEM). The comparison of steroid hormone profiling and product/precursor ratios was made using the non-parametric test of Mann–Whitney U. The data are presented as median . 0×I0) ½ (Gmean×Imean) ½ and HOMA-β=20× I0/(G0-3.5), where G0 is fasting glucose (mmol/L) and I0 is fasting insulin (μU/mL). Gmean (mmol/L) is the mean of fasting glucose, 1-h glucose OGTT, and 2-h glucose OGTT. Imean (μU/mL) is the mean of fasting insulin, 1-h insulin OGTT, and 2-h insulin OGTT. Matsuda index and HOMA-β data were log transformed to meet normality.Matsuda index=10000/(Gp<0.001), 1-h glucose (p<0.001), 2-h glucose (p<0.001), 2-h insulin (p<0.001), hemoglobin A1c (HbA1c) (p=0.040) and triacylglycerol (TG) (p=0.03) than women with NGT. The indexes related with insulin resistance including Matsuda index (p<0.001) and Homeostasis model assessment (HOMA)-β (p=0.045) were decreased in women with GDM (shown in p=0.001), A4 (p=0.023), and T (p<0.001) (shown in p=0.003) and T  compared with those with low levels  (Women with GDM (n=80) and women with NGT (n=79) had similar maternal age, gestational age, blood pressure, total cholesterol, HDL-cholesterol and LDL-cholesterol. Women with GDM had a significantly higher level of fasting glucose (shown in Table 1.2) had higher maternal age (p=0.040) and higher serum levels of fasting glucose (p<0.001), 1-h glucose (p<0.001), 2-h glucose (p<0.001), 2-h insulin (p<0.001), and TG (p=0.007) when compared to the NGT group (BMI<25 kg/m2). Matsuda index and HOMA-β were decreased in the GDM group (p<0.001), T (p=0.020), E1 (p=0.010), and E2 (p=0.040) were increased in the GDM group (BMI<25 kg/m2) compared to those in the NGT group (BMI<25 kg/m2). Next, we used the ratio of product-to-precursor in the steroid hormone metabolic pathway to demonstrate the activity of the enzymes involved in the reaction. The results indicated that DHEA/17OHP5 (p=0.010) and T/A4 (p=0.003) were increased and DHT/T was decreased in the GDM group (p<0.001) (We observed that the GDM group (BMI<25 kg/mDM group Table 4.2 had significantly higher levels of diastolic blood pressure (p=0.023), fasting glucose (p=0.001), fasting insulin (p<0.001), 1-h insulin (p=0.001), 2-h insulin (p=0.003) and HbA1c (p<0.001) than those with BMI<25 kg/m2  and E2(p=0.009) levels were significantly decreased in GDM women with BMI>25 kg/m2. Regarding enzymatic activity, the BMI>25 kg/m2 group showed an increased ratio of E3/E2 (p=0.003) and a decreased ratio of E1/A4 (p=0.010) and E2/T (p=0.008) compared to the BMI<25 kg/m2 group  and E2/T . The Matsuda index was positively correlated with E1/A4  and E2/T . In addition, BMI was positively correlated with E3/E2 , and Matsuda index was negatively correlated with E3/E2   Figure 2In this study, elevated serum glucose, insulin, HbA1c, and TG levels were expected in GDM patients, which is consistent with previous studies . HoweverDisorders of steroid hormone metabolism have been associated with insulin metabolism. A cohort studies has revealed increased serum T and DHEA levels in pregnant women with PCOS, who were always diagnosed with insulin resistance and higher BMI. Furthermore, a higher level of androgen has also been observed in GDM patients with insulin resistance , 31. UzeIn pregnant women who were normal weight, women with GDM showed higher DHEA, T, E1, E2, and 17β-HSD activity than women with NGT. According to recent literature, T, E3, P5, and DHEA might be the differential metabolites for GDM. The genetic variants rs10046 of CYP19A1 and rs2257157 of 17βHSD isoform 3 could predispose to GDM in Chinese women . AdditioDuring pregnancy, the biochemical synthesis of steroid hormone including estrogens, 16α-hydroxylation, and aromatization requires interacting processing in the placenta, the fetal and maternal adrenal glands, and the fetal liver. This interdependent physiological entity is known as the feto-placental unit, which is involved in steroid hormone synthesis and metabolism . Within via binding to nuclear receptors as well as via modulation of ion channels controlling the secretion of pancreatic hormones  for the publication of any potentially identifiable images or data included in this article.YS, BZ designed the study and drafted the manuscript. XM, BY collected the data. KW, YL performed statistical data analyses. DZo, JX performed steroid hormones measurement. XS, DZh and ZM contributed to revising the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Key Research and Development Program of China (2018YFC1005003); National Natural Science Foundation of China ; Key Research and Development Program of Zhejiang Province (2021C03098); Science and Technology Program of Zhejiang Province (2022C01061) and Zhejiang University Education Foundation Global Partnership Fund.We thank all the physicians, technicians, especially the patients involved in their dedication to the study.Authors DZ and JX were employed by Hangzhou BIOZON Medical Laboratory Co.The remaining 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. 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The structures of the new metabolites were elucidated by extensive spectroscopic analysis and the absolute configurations of 1–3 were determined on the basis of comparison of the experimental circular dichroism (CD) and calculated electronic circular dichroism (ECD) spectra. Compounds 1–3 are the first 5,5,6,6,5-pentacyclic spongian diterpenes bearing an β-hydroxy group at C-1. These metabolites were assayed for their cytotoxic, antibacterial, and anti-inflammatory activities. All three compounds were found to exert inhibitory activity against superoxide anion generation in fMLF/CB-stimulated human neutrophils. Furthermore, 1 showed a higher activity against the growth of Staphylococcus aureus in comparison to 2.Three new 5,5,6,6,5-pentacyclic spongian diterpenes, spongenolactones A–C ( Spongia have been proven to be rich sources of structurally diversified secondary metabolites + .White powder, [α]c = 0.20, CH3OH); IR (neat) νmax 3421, 2919, 2849, 1747, 1684, and 1655 cm−1; 1H NMR and 13C data, see m/z 401.1573 [M + Na]+ .White powder, [α]c = 0.35, CH3OH); IR (neat) νmax 3445, 2919, 2849, 1747, 1683, and 1646 cm−1; 1H NMR and 13C data, see m/z 379.1750 [M + H]+ .White powder, [α]The DFT approach at the B3LYP/6-31G  level of theory was applied to simulate the preliminary geometry optimization of conformers . Then, tThe cytotoxicity assay was performed using the methods described previously ,19. Huh7S. aureus were cultured in LB (Lysogeny broth) medium in the shaker incubator at 37 °C for 24 h. The bacterial culture was diluted to an absorbance of 0.04 at 600 nm using a sterile LB medium. Tested compounds (cpd) were then added to bacteria aliquots (100 μL/well of 96-well) with the concentrations at 50 μM, 100 μM, and 200 μM, respectively. Background control (1% DMSO in LB solution), positive control (1% DMSO in the diluted bacteria solution), and known drug control  were run on the same plate. The absorbance at 600 nm (A) was measured right after the testing compounds were added for the basal absorbance and after 16 h incubation at 37 °C. The percentage bacterial growth was calculated as follows: [ − Abackground control]/[ − Abackground control] × 100. The antibacterial assay was performed using the methods described previously . The bac2+-free HBSS buffer .The dextran sedimentation, Ficoll–Hypaque gradient centrifugation, and hypotonic lysis were used to enrich the neutrophils which were isolated from the blood of healthy adult volunteers, and these methods were described in a previous paper . The neuInhibition of Superoxide Anion Generation5 cells/mL) incubated in HBSS  at 37 °C were treated with the DMSO (as a control) or the tested compounds for 5 min. Neutrophils were activated by 100 nM fMLF for 10 min in the pretreatment of cytochalasin B  for 3 min (fMLF/CB). The generation of superoxide anion was spectrophotometrically measured at 550 nm  [H)-benzopyran-4-one] was used as a positive control.Neutrophils  ,21. LY29Inhibition of Elastase Release5 cells/mL) incubated in HBSS  at 37 °C were treated with DMSO (as a control) or the tested compound for 5 min. Neutrophils were activated with fMLF (100 nM)/CB (0.5 μg/mL) for 10 min. The generation of elastase release was spectrophotometrically measured at 405 nm  [Neutrophils  .1–3), were isolated from a Red Sea sponge, Spongia sp. These metabolites are the first 5,5,6,6,5-pentacyclic spongian diterpenes bearing a β-hydroxy group at C-1. In our previous chemical study of the same organism, we also discovered a compound of the same skeleton, 17-dehydroxysponalactone, which was found to potently reduce the superoxide anion generation and elastase release [1–3 exhibited inhibitory activity against the generation of superoxide anion, and 2 also displayed inhibitory activity against elastase release. Furthermore, 1 showed significant inhibition on the growth of S. aureus.Three new 5,5,6,6,5-pentacyclic spongian diterpenes, spongenolactones A–C ( release . Compoun


Gardenia jasminoides Ellis (labeled as GP2a) and its immunomodulatory activity. GP2a is an acidic polysaccharide with a molecular weight of 44.8 kDa, mostly comprising galacturonic acid. Methylation analysis revealed 4-GalpA (74.8%) to be the major sugar residue in GP2a. Nuclear magnetic resonance analysis indicated that its main chain comprised →4)-α-D-GalpA-6-OMe-(1→4)-α-D-GalpA-(1→ and →4)-α-D-GalpA-6-OMe-(1→2)-α-L-Rhap--α-L-Rhap- while enhancing cell viability in a dose- and time-dependent manner. Compared with untreated cells, nitric oxide production and secretion of cytokines, such as tumor necrosis factor-α, interferon-γ, interleukin (IL)-1β, IL-6, and granulocyte macrophage colony stimulating factor, in GP2a-treated cells significantly increased after 48 h. At 300 µg/mL GP2a concentration, there was no significant difference in the cytokine levels in GP2a- and lipopolysaccharide-treated cells (the positive control). In summary, GP2a is a pectic polysaccharide with homogalacturonan and rhamnogalacturonan-I structural regions in the main chain. Based on its immunomodulatory effects in vitro, GP2a may have potential uses in functional food and medicine.Here, we elucidated the structural characteristics of a polysaccharide isolated from  Gardenia jasminoides Ellis), a popular shrub from the Rubiaceae family, is currently used mostly as raw material for extracting natural pigments and pharmacodynamic components. Its desiccative ripe fruit  is a dual-use resource for medicine and food. According to traditional Chinese medicine (TCM), gardenia is a TCM that is bitter in taste and cold in nature and enters the heart, lung, and triple burner meridians. It is capable of clearing away heat, reducing fire, cooling blood, and eliminating stasis   . Howeveral)/Rha] . Future w/v) at 80 °C for 50 min by ultrasonic-assisted extraction. After extraction, the supernatant was filtered and concentrated by rotary evaporator to less than half of the original volume at 65 °C, followed by adding three volumes of ethanol and maintained at 4 °C for 24 h. The precipitate was collected after centrifugation and dried at 60 °C in an oven; then, it was subjected to protein removal , decolorization (using D941 adsorption resin), dialysis , and freeze-drying to obtain the crude polysaccharides. Thereafter, the crude polysaccharides were separated by DEAE-sepharose FF ion-exchange chromatography with dH2O and a gradient of NaCl. The separated components were further purified and enriched using Chromdex 200 PG gel-filtration chromatography with dH2O. The purified polysaccharide was collected and dialyzed, concentrated, and freeze-dried.Before extraction, gardenia desiccative ripe fruits were crushed and soaked in 95% ethanol overnight for defatting. The defatted gardenia were dried until ethanol was completely removed and extracted once with distilled water at a 1:20 ratio  at a wavelength of 4000–400 cm3 aqueous solution containing 0.02% NaNO3 at a final concentration of 1 mg/mL, then filtered through a 0.45 μm microporous filtering film. The homogeneity and molecular weight of various fractions were measured using SEC-MALLS-RI. The molecular weight of various fractions in the sample solution were measured on a DAWN HELEOS-II laser photometer  equipped with three tandem columns , which were held at 45 ℃ with a model column heater, at a flow rate of 0.4 mL/min. The differential RI detector  was simultaneously connected to obtain the concentration of fractions and the dn/dc value. The dn/dc value of the fractions in the NaNO3 aqueous solution was determined to be 0.141 mL/g. ASTRA 6.1 software (Wyatt Technology) was used to acquire and process data.The GP2a sample was dissolved in 0.1 M NaNO2) and then washed with methanol. We repeated methanol washing 2–3 times. The residue was redissolved in deionized water and filtered through 0.22 μm microporous filtering film for measurement. The treated sample was analyzed and detected by HPAEC on a Dionex™ CarboPac™ PA-20  anion-exchange column connected with a pulsed amperometric detector . A total of 5.0 μL of the treated sample was injected into the column (30 °C) for gradient elution using a mixed solvent system  at a flow rate of 0.5 mL/min. Gradient program was performed as follows: A/B  at 0 min, A/B  at 30.1 min, A/B  at 45 min, A/B (95∶5 v/v) at 45.1 min, A/B  at 60 min. Data were acquired on the ICS5000 (Thermo Scientific), and processed by Chromeleon 7.2 CDS (Thermo Scientific).The GP2a samples (5.1 ± 0.1 mg) were hydrolyzed with trifluoroacetic acid (TFA) (2.0 M) at 121 °C for 2 h in a sealed tube. Next, the sample was dried using nitrogen  were using for quantification by external standard method.4 and 1.0 mL NaBD4 (as the control), respectively. After 3 h, 0.1 mL acetic acid was used to terminate the reaction. The reduced sample and 1.0 mg NaOH were dissolved in anhydrous dimethyl sulfoxide (DMSO) and reacted for 30 min. Then, the solution was alkylated with methyl iodide for 1 h. This chemical reaction was terminated with 1.0 mL ultrapure water and extracted with 2.0 mL CH2Cl2. The methylated sample was obtained from the CH2Cl2 phase and dried with a rotary evaporation, and then hydrolyzed by TFA (2.0 M). Aqueous ammonia (2.0 M) and NaBD4 (1.0 M) were successively added to the hydrolyze sample for reduction. The reaction was terminated using acetic acid and then acetylated with acetic anhydride. Finally, the samples were extracted with CH2Cl2 again and then analyzed by GC-MS  fitted with an electron bombardment ion source and a MassHunter workstation, on a BPX70  column.Polysaccharide was pre-treated by the following derivatization : A totalm/z).The operation conditions are as follows: 10:1 of the split ratio and 140 °C as initial temperature for 2.0 min; then, the temperature was increased to 230 °C in steps of 3 °C/min for 3 min. All detections were performed under a full-scan acquisition mode with the mass scanning range of 30–600  equipped with two second electron detectors (in-lens Duo & Everhart Thornley) was used to observe the surface morphology of the GP2a sample under the STEM mode. All images of the GP2a sample were acquired and analyzed by Zeiss ZEN Connect software.2O (99.96%). High-resolution one-dimensional (1D) NMR  and two-dimensional (2D) NMR  spectra were recorded by a Bruker AVANCE HD III 600MHz NMR spectrometer operating at room temperature. Chemical proton shifts in HDO (δH = δ 4.70 ppm) and methyl carbon shifts in TMSP (δC = −1.80 ppm) were used for 1H and 13C calibration, respectively.The GP2a sample was placed in an NMR tube and dissolved with 0.5 mL D2, with subculturing every 2–3 days [RAW 264.7, a murine macrophage cell line, was obtained from the American Type Culture Collection (ATCC TIB-71). Cells were cultured with Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum and 1% penicillin–streptomycin , and incubated at 37 °C and 5% CO2–3 days .4 cells/mL (100 µL/well) and incubated overnight. Thereafter, a culture medium with GP2a at various concentrations (7.5–300 µg/mL) was added and incubated for 24, 48, or 72 h. Meanwhile, untreated cells were used as blank control and the cells treated with 2 µg/mL LPS (MedChemExpress) were used as positive control. Following intervention, 10 µL CCK-8 solution (MedChemExpress) were added to each well followed by incubation at 37 °C for 45 min. The absorbance was detected using an ELISA reader  by measuring the optical density value at 450 nm.RAW 264.7 cells were seeded in 96-well culture plates at a density of 5 × 104 cells/mL overnight. According to the cell viability assay, cells were treated with GP2a at 120, 240, and 300 µg/mL for 48 h, respectively. The blank control and positive control were the same as above. After incubation, supernatants were collected and mixed immediately with Griess Reagent  at room temperature. The absorbance at 540 nm was read using an ELISA reader. To calculate the NO concentration, a standard curve was plotted by determining the absorbance of a standard NaNO2 solution  at 540 nm. Additionally, the secretion levels of TNF-α, IFN-γ, IL-1β, IL-6, and GM-CSF were all measured using commercial regents . These assays were performed as per manufacturer’s instructions.To detect the production of NO and cytokines, RAW 264.7 cells were seeded in 6-well culture plates at a density of 5 × 10p < 0.01 was considered to indicate a statistically significant difference, whereas a value of p < 0.05 was considered to indicate a trend.All data were analyzed by one-way analysis of variance using IBM SPSS 20.0 software; all values are expressed as means ± standard deviation. A value of