First, thirty compounds were considered as candidate bioactive compounds based on our previous studies including pharmacological evaluation, chemical profiles, and metabolic profiles. Second, the characteristics of estrogen glucuronidation by uridine diphosphate glucuronic acid (UDPGA)-supplemented human liver microsomes (HLM), human intestine microsomes (HIM), and expressed UGT enzymes were determined, and the incubation systems of their key UGT enzymes were optimized. Then, inhibitory effects and mechanisms of XLGB and its main compounds toward the key UGT isozymes were further investigated. As a result, estrogen underwent efficient glucuronidation by HLM and HIM. UGT1A10, 1A1, and 2B7 were mainly responsible for the glucuronidation of estrone, β-estradiol, and estriol, respectively. For E1 and E2, UGT1A10 and 1A1 tended to mediate estrogen-3-O-glucuronidation, while UGT2B7 preferred catalyzing estrogen-16-O-glucuronidation. Furthermore, the incubation system for active UGT isoforms was optimized including Tris-HCl buffer, detergents, MgCl2 concentration, β-glucuronidase inhibitors, UDPGA concentration, protein concentration, and incubation time. Based on optimal incubation conditions, eleven, nine, and nine compounds were identified as the potent inhibitors for UGT1A10, 1A1, and 2B7, respectively (IC50 < 4.97 μM and Ki < 3.35 μM). Among them, six compounds  simultaneously demonstrated potent inhibitory effects against these three active enzymes. Prenylated flavanols from Epimedium brevicornu Maxim., prenylated flavonoids from Psoralea corylifolia L., and salvianolic acids from Salvia miltiorrhiza Bge. were characterized as the most important and effective compounds. The identification of potent natural inhibitors of XLGB against the glucuronidation of estrogen laid an important foundation for the pharmacodynamic material basis.Xian-Ling-Gu-Bao (XLGB) capsule, a well-known traditional Chinese medicine prescription, is widely used for the treatment of osteoporosis. It could significantly increase the levels of estrogen in ovariectomized rats and mice. However, this working mechanism has not been well elucidated. Considering that UDP-glucuronosyltransferase (UGT) enzymes are the important enzymes that inactivate and regulate estrogen activity   Among the sex steroid hormones, estrogens  distinguish themselves for the variety of their target tissues. Indeed, estrogen receptors are present in most cells in mammals, thus enabling these hormones to regulate or interfere with many metabolic pathways, especially in maintaining bone homeostasis and regulating remodeling . The imbEpimedium brevicornu Maxim.) (70%), Dipsaci Radix  (10%), Psoraleae Fructus  (5%), Anemarrahenae Rhizoma (the dried rhizomes of Anemarrhena asphodeloides Bge.) (5%), Salviae Miltiorrhizae Radix et Rhizoma  (5%), and Rehmanniae Radix (the dried roots of Rehmannia glutinosa Libosch.) (5%) (Xian-Ling-Gu-Bao (XLGB) capsule consists of six commonly used herbs: Epimedii Folium (the dried leaves of h.) (5%) . Its abuh.) (5%)  and a sih.) (5%) . Its safh.) (5%) . RemarkaPrenylated flavonoid glycosides , saponins, and coumarins were likely the main active compounds responsible for the therapeutic effect . Their rThe clinical investigation of XLGB for the treatment of menopausal syndrome found that it could significantly improve the clinical symptom and raise the level of serous estradiol while lowering the risk of endometrial hyperplasia . In addiin vivo levels of estrogen , and the conjugated glucuronides were inactive products for estrogens . Therefoestrogen . So far,ucidated . This st1), β-estradiol (E2), estriol (E3), estrone 3-O-glucuronide sodium salt (E1-3-O-G), β-estradiol 3-O-glucuronide sodium salt (E2-3-O-G), estriol 3-O-glucuronide sodium salt (E3-3-O-G), 4-methylumbelliferone (4-MU), 4-methylumbelliferone glucuronide (4-MU-G), zidovudine (AZT), atazanavir, diclofenac, uridine diphosphate glucuronic acid (UDPGA), D-saccharic-1,4-lactone saccharolactone, magnesium chloride (MgCl2), and alamethicin were purchased from Sigma-Aldrich . β-estradiol 17-O-glucuronide sodium salt (E2-17-O-G), estriol 16α-O-glucuronide sodium salt (E3-16-O-G), and zidovudine-glucuronide (AZT-G) were purchased from Toronto Research Chemicals . Four detergents, including Brij35, Brij58, CHAPS, and Triton X-100, and multiple components of XLGB, including icariin, icariside I, icariside II, icaritin, cycloicaritin, epimedin A, epimedin B, epimedin C, magnoflorine, bavachin, isobavachin, bavachalcone, isobavachalcone, neobavaisoflavone, bavachinin, corylifol A, corylin, psoralidin, psoralen, isopsoralen, bakuchiol, asperosaponin VI, sweroside, loganin, salvianolic acid B, tanshinone IIA, 15,16-dihydrotanshinone I, timosaponin B-II, and catalpol, were purchased from Shanghai Winherb Medical Technology Co., Ltd. . The purity of each compound mentioned earlier was more than 98%, and other reagents were of analytical grade or better.Estrone (EPooled human liver microsomes (HLM), pooled human intestine microsomes (HIM), and thirteen expressed human UGT isoforms  were all obtained from Corning Biosciences .1, E2, and E3) and their generated glucuronides by Xevo TQ-XS . UHPLC separation was carried out using an ACQUITY HSS T3 column  at a flow rate of 0.4 ml/min, and the mobile phase consisted of water (A) and acetonitrile (B) (both containing 0.1% formic acid). The gradient elution programs for E2 and its glucuronides were as follows: 10–20% B at 0–2 min, 20–40% B at 2–3 min, 40–50% B at 3–4 min, 40–50% B at 4–4.5 min, 50–90% B at 4.5–5 min, 90–100% B at 5–7 min, maintained 100% B at 7–7.1 min, and 100%–10% B at 7.1–8 min. The gradient systems for E1, E3, and their glucuronides were as follows: 2%–10% B at 0–2 min, 10%–40% B at 2–3 min, 40–50% B at 3–5 min, 50–90% B at 5–6 min, 90–100% B at 6–7 min, maintained 100% B at 7–8 min, and 100–2% B at 8–9 min. The multiple reaction monitoring (MRM) mode was operated in positive ion mode. The detailed parameters were set as follows: source temperature 150°C, desolvation temperature 550°C, capillary voltage 3.3 kV, desolvation gas 1000 L/h, cone gas 150 L/h, the m/z 271.17 → 253.04 transition with a collision energy (CE) of 10 eV for E1, E3, and their glucuronides, and the m/z 273.18 → 106.91 transition with a CE of 25 eV for E2 and its glucuronides. All experimental data were collected in centroid mode and analyzed using MassLynx 4.1 software .LC-MS/MS analysis was performed to quantify three endogenous estrogens . The mobile phase also consisted of water (A) and acetonitrile (B) (both containing 0.1% formic acid). The flow rate was 0.4 ml/min, and the detection wavelength was 316 and 267 nm for 4-MU and AZT, respectively; 4-MU and 4-MU-G were eluted using the following gradient: 5–60% B at 0–3.5 min, 60–65% B at 3.5–3.8 min, 65–100% B at 3.8–4 min, maintained 100% B at 4–5 min, and 100–5% B at 5–6 min. Also, the gradient program for AZT and AZT-G was as follows: maintained 2% B at 0–1 min, 2–5% B at 1–1.5 min, 5–40% B at 1.5–3.5 min, maintained 40% B at 3.5–4 min, 40–100% B at 4–4.5 min, maintained 100% B at 4.5–5 min, and 100–2% B at 5–6 min.In addition, the separation of 4-MU, AZT, and their generated glucuronides was performed by an ACQUITY UHPLC I-Class system equipped with a PDA detector . Chromatographic separation was achieved with a BEH C2 (0.88 mM), alamethicin (22 μg/ml), and D-saccharic-1,4-lactone (4.4 mM) and respective substrate. The volume of organic solvent in incubation mixtures was kept below 1%. After pre-incubation at 37°C for 5 min, the reaction was commenced with the addition of UDPGA (4 mM) and subsequently incubated at 37°C for a period. The reaction was terminated by adding 100 μL of cold acetonitrile. Then, the incubation mixtures were vortexed and centrifuged at 13,800 g for 10 min to collect the supernatant for analysis.The glucuronidation assay was conducted as described previously . In brie1, E2, and E3 (2 and 10 μM) to identify the most important contributors, respectively. A series of E1 solutions (0.625–80 µM) were separately incubated in HLM, HIM, and UGT1A3 for 90 min. In addition, serial E2 solutions (0.5–150 µM) were incubated in HLM, HIM, UGT1A1, and UGT1A10 for 60 min, respectively, while E3 solutions (0.5–150 µM) were incubated in HLM, HIM, and UGT2B7 for 30 min, respectively. All experiments were performed in triplicate.Thirteen recombinant UGT isoforms  were incubated with EGlucuronidation rates were expressed as the amounts of formed metabolites per minute per milligram protein (pmol/min/mg protein) based on Km is the Michaelis–Menten constant; Vg is the formation rate of metabolites; Vmax is the maximal rate of metabolites; Ksi is the substrate inhibition constant; S50 is the substrate concentration resulting in 50% of Vmax. n is the Hill coefficient. The intrinsic clearance (CLint) was derived from Vmax/Km for Michaelis–Menten and substrate inhibition models, and the maximal clearance (CLmax) was obtained using The parameter m values of about 60 μM as described previously (2 (10 μM for UGT1A1), 4-MU (60 μM for UGT1A10), and AZT (800 μM for UGT2B7) were used as the substrates to evaluate their respective metabolic activities. The substrate concentration was close to or below the corresponding Km reported previously (β-estradiol (E2) and zidovudine (AZT) were the recognized probe substrates for UGT1A1 and UGT2B7, respectively . Howevereviously . E2 (10 eviously . Differe2 was incubated in 50 mM Tris buffer (pH 8.0). Furthermore, MgCl2 and UDPGA were also evaluated with final concentrations ranging from 0 to 10 mM and from 0 to 8 mM, respectively. To investigate the effects of β-glucuronidases, 5 mM saccharolactone (a β-glucuronidase inhibitor) was added to the incubation mixtures.In addition, the effects of alamethicin (20 μg/ml) and four detergents  for glucuronidation were estimated; 4-MU and AZT were both conducted in 100 mM Tris buffer (pH 7.4), while EMoreover, for time-dependent assay, the reaction system was expanded to a total volume of 1 ml under the above-optimized conditions. After initiating the reaction, a subsample (100 μL) of the incubation mixtures was immediately removed and terminated by cold acetonitrile (100 μL) at eight time points . For the protein concentration-dependent assay, various concentrations of UGT1A10, UGT1A1, or UGT2B7  were added to the incubation mixtures.50) values were determined by nonlinear regression analysis in the presence of serial concentrations of natural compounds in XLGB. Traditionally, when the IC50 values were less than 10 μM, the inhibitory effects were moderate, and it was worthy of further inhibitory mechanisms  based on optimized incubation conditions. The final volume of solvent in incubations was 1%. In addition, emodin, atazanavir, and diclofenac were considered as positive controls for UGT1A10, UGT1A1, and UGT2B7, respectively. The half-maximal inhibitory concentration (ICchanisms .i) and inhibition type were determined using various concentrations of enzyme-selective substrates  in the presence of different concentrations of natural compounds in XLGB. Competitive inhibition, non-competitive inhibition, and mixed-type inhibition models were used to estimate the Ki values by nonlinear regression based on The inhibition kinetic parameters . αKi is the parameter reflecting the affinity of natural compounds to the complex of enzyme and corresponding specific substrate. When α is far over 1, the binding of herbal compounds could prevent the binding of selective substrate, and the mixed-type inhibition model becomes identical to competitive inhibition.The parameter p < 0.05 (∗), p < 0.01 (∗∗), or p < 0.001 (∗∗∗).All data were presented as mean ± SD (standard deviation). Mean differences between treatment and control groups were analyzed by a two-tailed Student’s t test. The level of significance was set at in vivo; fourth, they were the quantitative markers for individual herbs in XLGB according to the Pharmacopoeia of the Peoples’ Republic of China (2020 edition). Therefore, 30 candidate compounds including prenylated flavanol glycosides from Epimedium brevicornu Maxim., prenylated flavonoids and coumarins from Psoralea corylifolia L., saponins from Dipsacus asper Wall. ex Henry and Anemarrhena asphodeloides Bge., tanshinones from Salvia miltiorrhiza Bge., and other compounds from Rehmannia glutinosa Libosch. were selected for further exploration  of E1 and two glucuronides (E2-3O-G and E2-17O-G) of E2 were obviously detected (O-G and E3-16O-G) and no E3-17O-G were obtained in m values for E1, E2, and E3 ranged from 5.79 to 113.2 μM  by a UDPGA-supplemented incubation system. As shown in 2 concentration, β-glucuronidase inhibitors, UDPGA concentration, protein concentration, and incubation time were separately tested  exhibited moderate inhibitory effects against UGT1A10, UGT1A1, and UGT2B7 with the remaining activity of 52.43, 4.38, and 53.86%, respectively . In addiectively .50 values  were detected in HLM or HIM incubation systems  has higher inhibitory activity on UGT1A1 than icariside I and icariside II, indicating that the number and type of sugar substituent could significantly affect the inhibitory effects . For dihFurther, a previous study has demonstrated that it failed to induce observable liver injury after intragastric administration of XLGB at dosages of 1,000 mg/kg  for 26 weeks in normal rats  or up toPsoralea corylifolia L., an important herb of XLGB, has been widely used for the treatment of bone fracture and osteoporosis , while neobavaisoflavone, isobavachalcone, and bavachinin were identified as moderate inhibitors with Ki values ranging from 1.61 to 9.86 μM  also exhibited different degrees of inhibitory activity toward UGT1A1  usually cause significant clinical safety issues due to the inhibition or activation of drugs  toward clinically important CYP or UGT enzymes . Clinica tissues . In this 9.86 μM . Severald UGT1A1 . These fr injury .i values less than 1.0 μM. These findings suggested that more attention should be paid to avoiding clinical adverse issues  due to DDI after co-administration.Except for endogenous substances, UGT1A1 also mediated the glucuronidation of many therapeutic drugs  . UGT2B7 in vivo levels of E1 and E2 were measured after oral administration of XLGB in OVX-mice serum, because E3 is too low to be detected (in vivo levels of estrogen-related glucuronides in OVX-mice serum and liver were absent, and this also urges us to further investigate the levels of glucuronides and mRNA and protein levels of related UGT enzymes in OVX-mice tissues. It is also necessary to explore the changes in estrogens and their glucuronides after intragastric administration of bavachin, neobavaisoflavone, and corylifol A, respectively. This would benefit the understanding of effective components in XLGB for the increase of estrogens through regulating the glucuronidation of estrogens.Moreover, there were several limitations in this study. For example, except glucuronidation, SULT1E1-mediated sulfation is another important inactivation pathway of estrogen . Hence, detected . Meanwhi50 values ranging from 0.11 to 10.19 μM (Epimedium brevicornu Maxim. and prenylated flavonoids from Psoralea corylifolia L. contributed more to inhibiting the glucuronidation of estrogen. This study provides some new insights into the identification of effective compounds of XLGB.In conclusion, UGT1A10, 1A1, and 2B7 contributed more to the glucuronidation of estrogen. Based on the optimal incubation system, fourteen compounds demonstrated potent inhibitory effects against UGT1A10, 1A1, and 2B7. Among them, icariside II, bavachin, isobavachin, isobavachalcone, neobavaisoflavone, and corylifol A simultaneously exhibited strong inhibitory effects toward UGT1A10, 1A1, and 2B7 with IC10.19 μM . Taken t


Tnfa and Apj in the quadriceps femoris was compared between 4- (young) and 24-month-old (old) C57BL/6J mice (n = 10 each) using qPCR. Additionally, APJ-positive cells and TNF-α protein were analyzed by flow cytometry and Western blotting, respectively. Further, quadricep-derived cells were exposed to 0 (control) or 25 ng/mL TNF-α, and the effect on Apj expression was examined by qRT-PCR. Apj expression and the ratio of APJ-positive cells among quadricep cells were significantly lower in old compared to young mice. In contrast, levels of Tnfa mRNA and TNF-α protein were significantly elevated in old compared to young mice. Exposing young and old derived quadricep cells to TNF-α for 8 and 24 h caused Apj levels to significantly decrease. TNF-α suppresses APJ expression in muscle cells in vitro. The increase in TNF-α observed in SM with age may induce a decrease in APJ expression.Expression of the apelin receptor, APJ, in skeletal muscle (SM) is known to decrease with age, but the underlying mechanism remains unclear. Increased tumor necrosis factor (TNF)-α levels are observed in SM with age and are associated with muscle atrophy. To investigate the possible interconnection between TNF-α elevation and APJ reduction with aging, we investigated the effect of TNF-α on APJ expression in cells derived from the quadriceps femoris of C57BL/6J mice. Expression of  Sarcopenia is a disorder that is characterized by the gradual deterioration of skeletal muscle (SM) mass and strength as a person ages, and leads to a decline in autonomy in older people ,2. Loss The G protein-coupled apelin receptor (APJ) is widely expressed throughout the body. APJ is implicated in various physiological processes, including energy metabolism, cardiovascular function, and angiogenesis. A recent study reported that APJ and its ligand apelin contribute to muscle metabolism and that APJ gene expression is decreased in old mice . HoweverInterestingly, inflammatory cytokine levels are reported to be elevated in the blood and skeletal muscle tissue of sarcopenia patients ,16,17,18To investigate the possible interconnection between TNF-α elevation and APJ reduction with aging, we investigated the effect of TNF-α on APJ expression in cells derived from the quadriceps femoris of C57BL/6J mice.This study was conducted on male C57BL/6J mice . C57BL/6J mice were housed at Jackson Laboratory Japan  under a semibarrier system with controlled temperature (23 ± 2 °C), humidity (55% ± 10%), and light (12 h light/dark cycle). The study protocol was approved by the Kitasato University School of Medicine Animal Care Committee (reference number: 2021-046).n = 10) as the “young” group and 24-month-old mice (n = 10) as the “old” group. Body weight (g) and muscle weight of the quadriceps femoris (mg) were measured and muscle weight (mg)/body weight (n = 10) was calculated. Apelin, Apj and Tnfa mRNA expression in the quadriceps femoris was examined using real-time PCR and compared between the two age groups. In addition, TNF-α protein expression was examined by Western blotting. To determine whether TNF-α affects apelin and APJ expression in SM, quadriceps femoris tissue from young (n = 5) and old mice (n = 5) was digested with collagenase. Muscle cells were subsequently harvested and exposed to 0 ng/mL (control: culture medium only), 2.5 ng/mL or 25 ng/mL TNF-α for 8 and 24 h. mRNA was extracted from the stimulated cells and Apelin and Apj expression was measured using real-time PCR.A previous study showed that muscle/body weight was reduced in 24-month-old C57BL/6 mice compare to 3-month-old C57BL/6 mice . TherefoC57BL/6J mice were sacrificed by inhalation anesthesia with isoflurane. Using a scalpel, the skin and fascia of the upper leg were removed and the quadriceps femoris was harvested. The harvested tissue was then subjected to TRIzol  treatment to extract total RNA based on the manufacturer’s protocol. The total RNA formed the template for cDNA synthesis using SuperScript III RT  in a PCR reaction that comprised cDNA, TB Green Premix Ex Taq  and a specific primer set. Primers in the primer set were fashioned on Primer Blast software and made by Hokkaido System Science Co., Ltd. . Protein levels of TNF-α were measured using Western blotting. After homogenizing muscle cells in sodium dodecyl sulfate (SDS) sample buffer, the homogenates immediately heated at 95 °C for 10 min. Protein concentrations were determined by a bicinchoninic acid (BCA) assay kit . No protein degradation was confirmed in Coomassie Brilliant Blue staining. The homogenates (5 μg/lane) were subjected to SDS-polyacrylamide gel electrophoresis. The separated proteins in the gel were then electrophoretically transferred to a polyvinylidene difluoride membrane in blotting buffer. The membrane was subsequently treated with 10% skim milk in TBST for 30 min at 25 °C to prevent non-specific reactions before incubating with anti-TNF-α antibody  or anti-GAPDH antibody  for 60 min at 25 °C. After further incubating with goat anti-rabbit antibody conjugated to HRP  for 60 min at 25 °C, the membrane was washed a final time. Protein bands were subsequently visualized using enhanced chemiluminescence  and a luminescent image analyzer with a CCD imager . Relative TNF-α expression was normalized to GAPDH.n = 5 each) were treated with a 20 mL solution of 0.1% collagenase  at 37 °C for 1 h. The digested samples were then filtered through a nylon mesh filter  to obtain cell suspensions. The cells were then treated with the following antibodies: anti-CD45-PE/Cy7  and anti-Sca1-APC-Cy7  for 1 h at 4 °C. CD45 is a marker for pan-hematopoietic cells and Sca1 is a marker for mature myocytes. After further treatment with a fixation/permeabilization solution , the cells were exposed to FITC-conjugated anti-APJ antibody, prepared using an FITC conjugation kit  and unlabeled anti-APJ antibody  for 30 min at 4 °C. After washing in wash buffer twice, the labeled cells were used for flow cytometry. The procedure involved acquiring 50,000 total events using a BD FACSVerse system  and analysis of the findings using FlowJo v10.7 . Negative gates were set based on the isotype control.Tissue samples of the quadriceps femoris taken from young and old mice (n = 5), tissue samples were treated with a 20 mL solution of 0.1% type I collagenase for 60 min at 37 °C. The harvested cells (1 × 104 cells/cm2) containing heterogenous populations were cultured in α-minimal essential media  + 10% fetal bovine serum  in six-well plates for 7 days. The cultured cells were then exposed to 0 ng/mL (control: culture medium only), 2.5 ng/mL mouse TNF-α, or 25 ng/mL TNF-α for 8 or 24 h. The gene expression of Apelin and Apj in muscle-derived cells was determined using real-time PCR in the same manner as that described above.To extract mononuclear cells from the quadriceps femoris of young and old mice .Statistical analyses were performed using SPSS version 28.0.0.0 (190) . The Shapiro–Wilk test was used to test for normality, and Levene’s test for the homogeneity of the variance. Mann–Whitney U tests were used to compare body weight and muscle mass between the two age groups. The unpaired t-test was used to compare muscle mass/body weight between the two age groups. As the gene and protein expression data were not normally distributed, the Mann–Whitney U test was used to compare gene and protein expression between the two age groups. Two-way ANOVA with the Bonferroni post-hoc test was used to compare the gene expression among control, 2.5 ng/mL TNF-α-, and 25 ng/mL TNF-α-stimulated cells. p < 0.001; p = 0.481; p < 0.001; Body weight was significantly higher in old mice (32.6 ± 2.9 g) than in young mice  vs. 0.79 ± 0.06 (old), p = 0.253, Apj was significantly decreased in old compared to young mice (1.00 ± 0.06 (young) vs.0.38 ± 0.06 (Old), p < 0.001, p = 0.016, Sca1-negative: 0.72 ± 0.22 (young) vs. 0.11 ± 0.02 (old), p = 0.016, Tnfa was significantly elevated in old compared to young mice (1.00 ± 0.26 (young) vs. 2.47 ± 0.52 (old), p = 0.034, p = 0.006, mRNA expression of Apelin mRNA expression in young mice-derived cells is shown in As our findings indicated that the expression level of APJ differed between young and old mice, we next evaluated whether the response of muscle cells to TNF-α was also different between the two age groups. Relative p < 0.001, control vs. 25 ng/mL TNF-α: p < 0.001, p < 0.001; TNF-α significantly increased Apelin mRNA expression in young mice-derived muscle cells for 8 h (control vs. 2.5 ng/mL TNF-α: Apelin mRNA expression in old mice-derived cells is shown in Relative Apj mRNA expression in young mice-derived cells is shown in Apj mRNA expression in old mice-derived cells is shown in Apj was significantly reduced in both young and old mice-derived muscle cells following stimulation with exogenous TNF-α for 8 h (young, control vs. 25 ng/mL TNF-α, p < 0.001; old, control vs. 2.5 ng/mL TNF-α: p = 0.012, control vs. 25 ng/mL TNF-α: p = 0.009, p < 0.001; old, control vs. 25 ng/mL TNF-α: p = 0.007, Relative Apj expression and a reduced ratio of APJ-positive cells compared to young mice. In contrast, they had significantly higher concentrations of TNF-α than young mice. Further, exposing muscle-derived cells to exogenous TNF-α caused Apj mRNA expression to significantly decrease. Together, our results suggest that the reduction in APJ in old mice may be associated with increased TNF-α.The purpose of this study was to examine interconnection between TNF-α elevation and APJ reduction with aging. We showed that old mice had reduced Sarcopenia has been extensively studied using mouse models. Mice have a lifespan of 2–3 years . The ratAPJ has been previously reported to be associated with age-related muscle atrophy . Pax7-exSeveral studies have shown that TNF-α rises with age in mice and humans ,27,35. PApelin mRNA expression in young mice-derived muscle cells. However, TNF-α failed to stimulate apelin expression in old mice-derived muscle cells. Apelin ameliorates TNF-α mediated physiological changes in hepatocytes [A previous study reported that TNF-α stimulated apelin expression in mice and human adipose tissue . Consistatocytes , suggestatocytes ,37. For atocytes . The musThere were several limitations in this study. First, only two time points were investigated. To better understand the pathogenesis of sarcopenia, further studies should analyze changes across a greater number of time points. Second, we only used muscle mass as an indicator of the pathogenesis of sarcopenia and were unable to examine the pathology of the tissue. Finally, it remains unclear whether TNF-α directly regulates APJ expression.TNF-α suppresses APJ expression in muscle cells in vitro. The increase in TNF-α observed in SM with age may induce a decrease in APJ expression.


Intraoperative anesthesia and postoperative adverse reactions were compared among the 9 groups. Results: the similarity between the Tri-training algorithm optimized by Naive Bayes (NB) classification algorithm and the actual classification (93.49%) was clearly higher than that by decision tree (DT) and K-nearest neighbor (kNN) classification algorithm ; heart rate (HR), systolic blood pressure (SBP), and diastolic blood pressure (DBP) values decreased obviously after Dex used in groups B1, C1, B2, C2, B3, and C3 (P <0.05), but did not change significantly in groups A1, A2, and A3 (P >0.05); the proportion of patients with satisfactory Ramsay score in group A3 was distinctly superior than that in groups A1, A2, B1, B2, B3, C1, C2, and C3 (P <0.05); the incidence of adverse reactions in group A3 was significantly inferior than that in groups A1, A2, B1, B2, B3, C1, C2, and C3 (P <0.05). Conclusion: the optimization effect of NB classification algorithm was the best, and the injection of Dex at the injection rate of 0.8 μg/(kg·h) before induction of anesthesia could apparently improve the fluctuation of HR, SBP, and DBP during perioperative period, and effectively reduce the occurrence of adverse reactions in patients, with better sedative effect on patients.Dexmedetomidine (Dex) is an alpha-2 agonist used for sedation during various procedures. Dex activates 2-adrenoceptors, and causes the decrease of sympathetic tone, with attenuation of the neuroendocrine and hemodynamic responses to anesthesia and surgery; it reduces anesthetic and opioid requirements; and causes sedation and analgesia. Objective: it was to compare the perioperative effects of different doses of Dex at different timing in patients using Dex during the perioperative period adopting a medical data classification algorithm based on optimized semi-supervised collaborative training (Tri-training). Methods: 495 patients requiring surgical treatment in Xingtai People's Hospital were randomly selected as the study subjects. The patients were divided into group A (used before induction), group B (used during induction), and group C (used after induction) according to different induction timing, with 165 cases in each group. Then, groups A, B, and C were divided into groups A1, B1, and C1 (0.4  Anesthesia is one of the necessary operation steps in the surgical treatment of most diseases in clinical practice. Clinical time shows that more sedative drugs are often required to strengthen and maintain the anesthetic effect during surgery, thereby reducing the patient's pain during surgery. However, there are some risks in the use of anesthetic drugs. Through the summary of previous clinical application experience, inappropriate use of anesthetics will lead to certain damage to the central nervous function of patients, resulting in a series of complications, such as increased blood pressure, language impairment, cognitive impairment. In addition, some studies have proposed that the patient's mood before anesthesia has a certain impact on the anesthetic effect, therefore, mood induction during the whole process of anesthesia is relatively important .α2-adrenoceptor agonist that is soluble . All parameters are initialized to obtain w means the upper limit of classification error rate. When t = , Equation  can be obtained. After calculations, the final classifier Because the Tri-training algorithm adopts implicit setting, the accuracy of implicit confidence is lower than that of explicit confidence. It will be optimized in combination with class probability estimation. The class probability estimation algorithms of common classifiers include decision tree (DT) Decision trees use multiple algorithms to decide to split a node into two or more sub-nodes. The creation of sub-nodes increases the homogeneity of resultant sub-nodes , Naive BThe DT classifies the data according to the tree structure, and uses the probability distribution on the leaf nodes to predict the classification of the data. The specific calculation is as below. N represents the total number of training samples in the leaf node; m represents leaf nodes; iC indicates the leaf node category; Cj represents the category of the sample Cj.j refers to order; A refers to characteristics; NB(Ci) refers to the prior probability distribution of the sample; NB means the conditional probability distribution.NB is based on Bayes theorem and classifies data by analyzing the joint probability distribution of data input and output. n probability theory and statistics, Bayes' theorem , and evaluate the similarity between the classification results and the actual situation. 2). Afterwards, intravenous anesthesia was carried out using propofol  and 3 μg/kg of fentanyl. 0.15 mg/kg of atracurium was given when the patient's consciousness disappeared . Patients in group A were injected with 0.4 μg/kg, 0.6 μg/kg, and 0.8 μg/kg of Dex hydrochloride before induction of anesthesia; patients in group B were given during induction of anesthesia; and patients in group C were injected after induction. They were then sent to the anesthesia recovery room after the end of surgery for continued observation until recovery.The patients in group A, group B, and group C were given the same anesthesia method  and monitored by routine monitoring of electrocardiogram (ECG), pulse, and oxygen saturation (SpOThe heart rate (HR), systolic blood pressure (SBP), and diastolic blood pressure (DBP) of patients in each group were recorded before anesthesia, after anesthesia, during intubation, 5 minutes after intubation, and 30 minutes after intubation.Sedation score (Ramsay) after extubation  Table 1Table 1 at-test and (χ2 and percentage (%), and P <0.05 indicated statistical significance.SPSS 23.0 calculation system was used for data analysis, and measurement data were expressed by et al. (2021) [Through calculating the classification results and actual classification of disease types of 2,010 patients with CHD by DT, NB, and kNN classification algorithms, it was concluded that the similarity between Tri-training algorithm optimized by NB classification algorithm and actual classification (93.49%) was significantly higher than that by DT and kNN classification algorithms 76.21%, 74.31%) . The res%, 74.31%P <0.05)  of the nine groups were statistically analyzed by classification algorithm, and the following results were obtained: (1) Gender distribution: 33, 34, 32, 28, 30, 32, 31, 32, and 31 male patients, respectively; 22, 21, 23, 27, 25, 23, 24, 22, and 24 female patients, respectively, in the nine groups. (2) Distribution of mean age: The mean age of the nine groups was (40.01 ± 10.19) years, (42.19 ± 8.22) years, (40.67 ± 9.01) years, (41.09 ± 8.82) years, (42.11 ± 9.05) years, (42.23 ± 9.22) years, (40.09 ± 8.74) years, (40.67 ± 9.42) years, and (41.94 ± 10.92) years, respectively. (3) Average weight distribution: The average weight of patients in the nine groups was (50.01 ± 13.05) kg, (51.12 ± 15.00) kg, (49.07 ± 16.05) kg, (50.22 ± 13.95) kg, (52.03 ± 15.02) kg, (50.11 ± 14.11) kg, (51.22 ± 13.95) kg, (50.29 ± 14.89) kg, and (52.98 ± 14.98) kg, respectively. (4) Distribution of surgical types: 34.56%, 36.36%, 32.72%, 34.56%, 34.56%, 34.56%, 34.56%, 34.56%, and 34.56% of the patients in the nine groups underwent gynecological malignant tumor surgery; 38.18%, 36.36%, 38.18%, 40.00%, 34.55%, 36.36%, 40.00%, 38.18%, and 36.36% of the patients underwent laparoscopic hepatobiliary surgery; and 27.27%, 27.27%, 29.09%, 25.45%, 30.91%, 29.09%, 25.45%, 27.27%, and 25.45% of the patients underwent subtotal thyroidectomy under general anesthesia. The comparison showed that there was no significant difference in the distribution of gender, age, weight, type of surgery among groups A1, A2, A3, B1, B2, B3, C1, C2, and C3 (P <0.05) .μg/(kg · h), the HR, SBP, and DBP results of patients in groups A1, B1, and C1 before anesthesia, after anesthesia, during extubation, 5 min after extubation, and 30 min after extubation. According to the results, there was no significant difference in HR, SBP, and DBP among the three groups before anesthesia (P >0.05), but the HR, SBP, and DBP values of patients in groups B1 and C1 after application of Dex decreased (P <0.05), while those of patients in group A1 were no obvious change (P >0.05), suggesting that the perioperative hemodynamic changes in group A1 were relatively stable.The effects of groups A1, A2, A3, B1, B2, B3, C1, C2, and C3 on hemodynamics HR, SBP, and DBP were compared before anesthesia, after anesthesia, during intubation, 5 min after intubation, and 30 min after intubation. μg/(kg · h), the results of HR, SBP and DBP of patients in groups A2, B2 and C2 before anesthesia, after anesthesia, during extubation, 5 min after extubation, and 30 min after extubation were compared. μg/(kg · h), HR, SBP, and DBP results of patients in groups A3, B3, and C3 before anesthesia, after anesthesia, during extubation, 5 min after extubation, and 30 min after extubation. Through analysis, the change trend of HR, SBP, and DBP results in Figures et al. (2020) [From the above results, Dex injection before induction of anesthesia could improve the changes in blood pressure and HR caused by anesthesia in patients. The purpose of induction of surgical anesthesia was to improve the depth of anesthesia and reduce the stress response caused by tracheal intubation or surgical stimulation (non-specific defense response caused by strong stimulation), improving the adverse hemodynamic fluctuation of patients . Before . (2020)  found thP values of group A3 were more stable than those of group A1 and A2 at different time points. The results showed that Dex had the best sedative effect when the injection dose was 0.8 μg/(kg · h). Shamim et al. (2017) [μg/kg · h than at 0.4 ~ 0.6 μg/kg · h at the same injection timing. It was also in line with the findings of Ramachandran et al. (2021) [μg/(kg · h) of Dex had the best sedative effect.The HR, SBP, and DBP of groups A1, A2, and A3 before anesthesia, after anesthesia, during intubation, 5 min after intubation, and 30 min after intubation were statistically compared. The results were given in . (2017)  proposed. (2021) . TherefoP <0.05). Ramsay sedation score scale is an effective and reliable evaluation scale for sedation monitoring of invasive operation under deep sedation, which has been widely used in related clinical studies. Lozano et al. (2021) [. (2021)  pointed P <0.05). Okello et al. (2018) [The statistical results of adverse reactions such as severe bradycardia, hypotension, hypertension, dry mouth, nausea, and vomiting in each group during the perioperative period are shown in . (2018)  raised t. (2018)  presenteP <0.05) (μg/(kg·h) before induction of anesthesia was better than that at 0.4 and 0.6 μg/(kg·h). This was consistent with research results of Jiang et al. (2017) [et al. (2021) [According to further statistics, the number of patients with adverse reactions was 12 in group A1, 12 in group A2, 7 in group A3, 18 in group B1, 19 in group B2, 18 in group B3, 17 in group C1, 15 in group C2, and 16 in group C3. It was concluded that the incidence of adverse reactions in group A3 was significantly lower than that in groups A1, A2, B1, B2, B3, C1, C2, and C3 (P <0.05) . It sugg. (2017) . Pan et . (2021)  presenteμg/(kg · h) before induction of anesthesia can significantly improve the fluctuation of HR, SBP, and DBP during perioperative period, and can effectively reduce the occurrence of adverse reactions in patients, which has a better sedation effect on patients. However, the formulation of Dex injection dose was not detailed enough, and there was a 0.2 μg/(kg · h) difference among groups, which made the dose assessment inaccurate and needed further exploration. Nowadays, anesthesia is a necessary technique for various clinical operations, and its sedative effect has a great influence on the surgical effect and prognosis of patients. Therefore, the best application method of sedative drugs during anesthesia will be a hot topic to better benefit patients.It was to understand when to inject what dose of Dex for perioperative patients with the best sedative effect. In addition, many study subjects were selected nationwide, and the optimized Tri-training algorithm was adopted to classify and count the index data of the samples. The results show that NB classification algorithm has the best optimization effect; the injection of Dex at the injection rate of 0.8


Furthermore, we examined the preventive role of IAP in IR induced by a high-fat diet in mice. We found that an IAP + IAP enhancer significantly ameliorated blood glucose, insulin, low-density lipoprotein, gut barrier function, inflammatory markers, and lipopolysaccharide (LPS) in serum. IAP could dephosphorylate LPS and nucleoside triphosphate in a pH-dependent manner in vitro. Firstly, LPS is inactivated by IAP and IAP reduces LPS-induced inflammation. Secondly, adenosine, a dephosphorylated product of adenosine triphosphate, elicited anti-inflammatory effects by binding to the A2A receptor, which inhibits NF-κB, TNF, and PI3K-Akt signalling pathways. Hence, IAP can be used as a natural anti-inflammatory agent to reduce intestinal inflammation-induced IR.In this study, we investigated the effects of intestinal alkaline phosphatase (IAP) in controlled intestinal inflammation and alleviated associated insulin resistance (IR). We also explored the possible underlying molecular mechanisms, showed the preventive effect of IAP on IR  Type 2 diabetes (T2DM) is a major global health problem, and its incidence increases every year. In 2019, the latest epidemiological data showed that the number of adults with T2DM reached 463 million , which induces an exacerbated inflammatory response . Neverthrylation . Lipid Arylation . Howeverrylation .1, A2A, A2B, and A3  and pyrimidines (UDP and UTP), which are released from host cells including nerve termini, immune cells, injured or dead cells, and the commensal bacteria that reside in the gut lumen, stimulate purinergic receptors in autocrine and paracrine ways . Once re, and A3 . M. Esta, and A3  reported, and A3 . Therefo, and A3 .IAP is a phosphomonoesterase that catalyses the hydrolysis of non-specific phosphate ester bonds, whose activity is positively correlated with pH . We aimeHowever, studies have yet to explore how IAP deficiency is linked to these diseases. Therefore, this study was designed to investigate the protective role of IAP during intestinal inflammation and reveal the underlying molecular mechanisms of action that would delay the further development of inflammatory bowel disease and IR. This study also aimed to explore the safety and initial efficacy of IAP and provide a basis for using IAP as a potential biological treatment for IR caused by intestinal inflammation.2PO4, and its volume was fixed to 1,000 ml in distilled water. Then, the juice was sterilised  and added to 3 g/l pepsin . Buffer solution broth was prepared by adjusting the pH to 2.5 with 1 mol/l HCl. Simulated intestinal juice was prepared by supplementing 10 g/l trypsin  and 3.0 g/l bile salt  added into an artificial gastric fluid, and pH was adjusted to 6.5 with 1 mol/l NaHCO3.Simulated gastric juice was prepared by supplementing 2 g NaCl, 1.12 g KCl, 0.11 g CaCl, and 0.4 g KHHydrotalcite, oat flour, and IAP enhancer  were used as raw materials  was added to 0.9 ml buffer solution with different pH levels. The IAP activity was measured using an alkaline phosphatase detection kit  (Tris–HCl buffer (50 mmol/l) was adjusted to pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 by using HCl (1 mol/l). Then, 0.1 ml IAP  .E. coli 0111: B4, Sigma, USA) was diluted to 0.1 mg/ml with Tris–HCl buffer . Then, 10 μl of the diluted LPS solution (pH = 8.0) was added to 990 µl of IAP solutions at pH 5.0–8.0. and incubated at 37°C for 180 min. IAP was diluted to 1, 5, 10, 20, and 40 U/ml by using Tris–HCl buffer . Afterwards, 10 µl of LPS (0.1 mg/ml) was added to 990 µl of IAP solution with different IAP activities to obtain an LPS concentration of 1 µg/ml. The mixtures were incubated at 37°C for 180 min. LPS activity was determined using a quantitative Limulus endotoxin detection kit  via the chromogenic matrix method  was added to Tris–HCl buffer , and the final pH was adjusted to 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0. LPS  and 0.40 U/ml (pH 8.0) at different pH levels was used in this experiment. Then, 50 µl of a solution containing ATP, adenosine diphosphate (ADP), adenosine monophosphate (AMP), uridine triphosphate (UTP), uridine diphosphate (UDP), uridine monophosphate (UMP), guanosine triphosphate (GTP), guanosine diphosphate (GDP), guanosine monophosphate (GMP), cytidine triphosphate (CTP), cytidine diphosphate (CDP), cytidine monophosphate (CMP), thymidine triphosphate (TTP), thymidine diphosphate (TDP), thymidine monophosphate (TMP)  with the same pH was added. The mixture was incubated at 37°C for 60 min. Afterwards, 10 µmol/l CD39 and 10 µmol/l CD73  were prepared with Tris–HCl buffer  and mixed at equal volumes. Subsequently, 950 µl of CD39 and CD73 mixture was added, and 50 µl of ATP, ADP, and AMP solutions was added. The solution was incubated at 37°C for 60 min. Free phosphate was assessed using a phosphate assay kit  in accordance with the manufacturer’s protocol.Twelve-week-old male C57BL/6J mice  were maintained in filter-top cages in a temperature-controlled room (22°C–24°C) with a 12-h light/12-h dark diurnal cycle and given food and water ad libitum in accordance with the regulations designated and approved by the Northeast Agricultural University Committee on Animal Resources (UCAR), which adheres to FDA and NIH animal care guidelines and reviews all animal protocols prior to approval (NEAUEC20220231).The mice were placed on a high-fat diet . After 14 weeks of HFD treatment, those that exhibited obesity and IR, as measured by fasting blood glucose ≥11.1 mmol/l, were used in the study (n = 32). HFD-fed mice were randomly divided into four groups. 1) In the control group, mice (n = 8) were administered with a control vehicle; 2) in the IAP group, mice (n = 8) were given 100 U/100 g/day IAP; 3) in the IAP-enhancer group, mice (n = 8) were provided with 5 g/1,000 g/day IAP enhancer; and 4) in the IAP + IAP enhancer group, mice (n = 8) were treated with 100 U/100 g/day IAP + 5 g/1,000 g/day IAP enhancer. The treatments were administered by gavage at 9 a.m. for 4 weeks –21. As avia bladder massage at 3 p.m. (6 h after the experiment started) and after 24 h to measure pH. Urine samples were collected by gentle abdominal pressure and analysed with a BW-901 urine analyser . Blood samples were drawn from the tip of the incised tail after the mice fasted for 8 h, and analysis was completed within 24 h. GTT was performed after overnight (8:00 p.m.–8:00 a.m.) fasting. The glucose levels of the tail vein blood samples were measured 0, 15, 30, 60, and 120 min after oral administration of glucose . Fasting and postprandial blood glucose, triglyceride, cholesterol, low-density lipoprotein, and high-density lipoprotein were measured using a BS-280 automatic biochemical analyser . Fasting and postprandial insulin levels were determined via radioimmunoassay by using UniCel DxI800 automatic chemiluminescence immunoassay .Body weight was measured using a scale . Faecal samples were collected at baseline and 4 weeks after treatments. Urine was collected In this procedure, 0.1 g of fresh faecal sample was taken from the mice; each sample was added with 5 ml of Tris–HCl buffer , mixed, and centrifuged at 12,000 r/min and 4°C for 10 min. The supernatant was diluted to 100-fold, and faecal IAP levels were determined using an alkaline phosphatase detection kit . The result was reported as units per gram .Plasma was collected from the LFD control group. Then, the plasma was added to a processing solution  at a ratio of 1:9, and the mixture was incubated in a water bath at 70°C for 10 min and cooled for 3 min. LPS at final concentrations of 0.00, 2.50, 6.25, and 12.50 pg/ml was used to generate a standard curve for plasma LPS measurements. The plasma samples to be tested were added to the same processing solution at a ratio of 1:9, and the mixture was incubated in a water bath at 70°C for 10 min and then cooled for 3 min. Plasma LPS was measured using a Limulus endotoxin detection kit . Every obtained result was multiplied by 10-fold because of sample dilution .The collected plasma and the levels of CRP, GLP-1, TNF-α, IL-6, IL-17, IFNγ, and IL-22 were measured using ELISA kits .Six-centimetre segments of the intestine were removed, rinsed with ice-cold saline, everted, filled with 700 ml of PBS, and ligated at both ends. The filled jejunum segments were incubated in PBS containing 1 mol/l FITC-dextran  or 0.4 mg/ml HRP . The jejunum sacs were removed after 45 min, and the contents of each sac were collected carefully using a 1-ml syringe. The amount of FITC-dextran transversing the jejunum was quantified by using a fluorescence plate reader at 521 nm. The HRP activity in the contents of each sac was determined spectrophotometrically based on the rate of pyrogallol oxidation .via the tail vein for 2 h. After being anesthetised with 1% pentobarbital, the mice were perfused with normal saline to wash residual dye out of the blood vessels. Intestinal samples were then collected to evaluate intestinal barrier disruption. Evans blue was extracted from tissue homogenates incubated in formamide at 60°C for 24 h. Then, the infiltrated Evans blue was quantified using a spectrophotometer at 620 nm. The content of the dye was calculated with a standard curve method  was administered at 4 ml/kg The collected jejunum tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Paraffin-embedded jejunum tissues were sectioned (4 μm in thickness), quickly stained with haematoxylin and eosin (HE), mounted on slides, and examined using a US Moticam 3000 photomicrography imaging system for observations and photography.in vitro model to study absorption, transport, and secretion by intestinal cells, were inoculated in McCoy’s 5A medium  containing 1% penicillin–streptomycin dual antibodies  and 10% FBS  in T-75 culture bottles. They were cultured in a humidified 5% CO2 incubator at 37°C. On the experimental day, the cells were incubated in RPMI-1640 medium  containing 10% FBS. Then, cells with a density of 2.0 × 104 cells/well were added to a well plate and cultured at 37°C for 24 h. In accordance with the manufacturer’s protocol, human leucocytes from healthy participants were freshly prepared using a human peripheral blood leucocyte separation solution kit . Freshly extracted human leucocytes were resuspended in RPMI-1640 medium supplemented with 10% FBS. They were added onto the top of a confluent HT-29 cell monolayer at a density of 1.0 × 105 cells/well. Physical examinations, electrocardiography, and routine laboratory studies on all healthy participants before the start of the experiment showed normal results. The healthy participants were not taking any prescription medications and tested negative for hepatitis B surface antigen and human immunodeficiency virus infection. This study was reviewed and approved by the Institutional Changchun Jiahe Surgical Hospital  and complied with the Declaration of Helsinki including current revisions and the Good Clinical Practice guidelines. Written informed consent was obtained from all study participants.All the cell lines passed the mycoplasma contamination detection. HT-29 cells , which were used as an An LPS-induced intestinal inflammation model of HT-29 cells + freshly extracted human leucocytes was established by adding 100 μl of LPS to each well, and the final concentrations were 0.10, 0.20, 1.00, and 2.00 ng/ml. Then, 100 μl of IAP (40 U/ml), 100 μl of LPS (1.0 ng/ml), 50 μl of IAP  + 50 μl of LPS (2.0 ng/ml), 50 μl of IAP (80 U/ml), and 50 μl of LPS (2.0 ng/ml) were incubated for 3 h in advance or at the same time; IAP (80 U/ml) was inactivated in a water bath at 65°C for 60 min, as well as the following: 50 μl of inactive IAP and 50 μl of LPS (2.0 ng/ml); 50 μl of IAP (40 U/ml) + 50 μl of ATP/UTP/GTP/CTP/TTP (4.0 mmol/l); 50 μl of IAP (2/10/20/40 U/ml), and 50 μl of ATP (4.0 mmol/L); 100 μl of ATP, ADP, AMP, and adenosine  at final concentrations of 0.10, 0.25, 0.50, and 1.00 mmol/l; 50 μl of IAP (40 U/ml) and 50 μl of IAP (40 U/ml) + 50 μl of CCPA, DPCPX, CGS21680, DMPX, NECA, MRS1754, Cl-IB-MECA, and MRS1220 ; and 100 μl of uridine, guanosine, cytidine, thymidine . Additionally, 100 μl of adenosine (2.0 mmol/L) and 50 μl of adenosine (2.0 mmol/l) + 50 μl of uridine, guanosine, cytidine, and thymidine (2.0 mmol/l) were added at final concentrations of 0.10, 0.25, 0.50, and 1.00 mmol/l. All groups were added to the same cell line and cultured at 37°C for 24 h.Supernatants were collected, and TNF-α and IL-6 levels were determined using ELISA .3PO4 solution containing 1% sulfa) and Griess B (1% N-1-naphthyl-ethylenediamine-hydrochloric acid solution) were added, and the OD value of each well was measured at 570 nm. PGE2 was determined using a prostaglandin E2 assay kit   .The control group was treated with 100 μl of RPMI-1640 complete medium, and the experimental group was treated with 100 μl of LPS (1.0 ng/ml), 100 μl of IAP (40 U/ml), and 50 μl IAP (4/20/40/80 U/ml) + 50 μl LPS (2.0 ng/ml). Additionally, 100 μl of adenosine (2.0 mmol/l) and 50 μl adenosine (2.0 mmol/l) + uridine, guanosine, cytidine, and thymidine (2.0 mmol/l) were added to the cell lines and cultured at 37°C for 24 h. The supernatant was discarded, and adherent cells were cleaned with PBS. Each well was added with 180 μl of serum-free medium and 20 μl of CCK8  and cultured for 4 h. The OD of each well was measured at 450 nm. Survival rate (%) was calculated as follows: survival rate = (OD test group/OD blank group) × 100% .Control, LPS, IAP, IAP + LPS, adenosine, and adenosine + uridine, guanosine, cytidine, and thymidine were added in a 24-well plate in triplicates. The morphological characteristics of the cells were observed under an inverted phase-contrast microscope  at 0, 24, and 36 h.Control, LPS, IAP, IAP+LPS, adenosine, and adenosine + uridine, guanosine, cytidine, and thymidine were seeded in a six-well plate. Scratches were generated using a 1-ml micropipette tip when the cells reached 100% confluence. The cells were then washed twice with PBS and incubated in a complete medium at 37°C. Images were observed under an inverted phase-contrast microscope  after 0, 48, and 72 h. Cell areas were quantified using ImageJ, and cell migration was defined as [Cell area (48/72 h)–Cell area (0 h)/Cell area (0 h)] .The supernatant was collected 20 min after LPS administration, with or without IAP pretreatment. ATP, ADP, AMP, and adenosine production were directly measured using the Bioluminescence Assay Kit CLS II .4 cells/well) in the presence of 5% CO2 at 37°C for 24 h. The supernatant was removed, and 1 ml of freshly extracted leucocytes (2 × 105 cells/well) was added. The control group was supplemented with 1 ml of R1640 medium, and the LPS group was added with 1 ml of LPS (1.0 ng/ml). The IAP + LPS group was added with 500 μl of IAP (40 U/ml) and 500 μl of LPS (0.5 ng/ml). The adenosine group was added with 1 ml of adenosine (2 mmol/l). Plates were then centrifuged, and supernatants were discarded. Total RNA was extracted from the mycelia by using TRIzol reagent . The quantity and purity of the RNA were assessed via 1% agarose gel electrophoresis and NanoDrop 2000 . RNA was quantified using a Qubit3.0, Agilent 2100, StepOnePlus™ Real-Time PCR System . RNA integrity was examined with an RNA Nano 6000 assay kit of the Bioanalyser 2100 system .HT-29 cells were inoculated on six-well plates  in the IAP + LPS vs. LPS. Of the total differential KEGG pathways between IAP + LPS and LPS groups, 17 signalling pathways that were closely related to the immune system and signal transduction were selected (P < 0.05), and eight of these pathways were associated with intestinal inflammation, TNF, chemokine, Toll-like receptor, PI3K-Akt, JAK-STAT, IL-17, NOD-like receptor, and NF-κB signalling pathways  and CX3CIn comparison with the control group, 72 signalling pathways were identified after adenosine treatment (P < 0.05). Furthermore, 13 immune systems were screened in signal transduction pathways (P < 0.05) of which seven were closely related to intestinal inflammation: TNF, chemokine, NOD-like receptor, PI3K-Akt, Jak-STAT, IL-17, and MAPK signalling pathways , CXCR3 , CX3CL1 in vivo , and approved the manuscript.Funding from this article was from Talent Introduction Program of Northeast Agricultural University, Study on the production and mechanism of antiinflammatory food B-HA/AP products.We are grateful to the family described in this manuscript for their willingness to participate and interest in this research. We thank Dr. Pei for reading and editing the manuscript. We acknowledge Pro Han and members of the Food college laboratory for help with physicochemical experiments. We thank Shaoxing Huihui Biotechnology Co. for help with cell experiments. We thank the Laboratory of Molecular Nutrition and Immunity for valuable comments and discussion.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 strain was identified as Enterococcus faecium through the morphological, physiological, biochemical and genetic tests. UA was produced at the beginning of the stationary phase and its levels peaked at 50 h, with the highest concentration being 10.80 μM. The strain Enterococcus faecium FUA027 is the first isolated strain of Enterococcus sp. producing urolithin A from ellagic acid, which may be developed as probiotics and used to explore molecular mechanisms underlying the biotransformation of ellagic acid into UA.Urolithin A (UA) has received considerable research attention because of its health benefits. However, only a few strains have been reported to produce UA from ellagic acid (EA), and the molecular mechanisms underlying the gut microbiota-mediated transformation of ellagic acid into urolithin A is limited. In the present study, a single strain FUA027 capable of converting ellagic acid into UA  A healthy diet not only supports the energy and physiological needs of the body even in the absence of excess food intake but also prevents diseases and positively influences health , the rolCaenorhabditis elegans  and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS)  , 27.L-Cysteine hydrochloride, urolithin A, urolithin B, urolithin C, isourolithin A, urolithin M6, and urolithin D were purchased from Dalton Pharma Services . Ellagic acid was purchased from Sigma-Aldrich . MS- or LC-grade solvents, namely methanol, acetonitrile, formic acid, and ethanol were purchased from Merck .All 7 volunteers (ages 22–27) with no history of gastrointestinal diseases signed an experimental informed consent form at the Jiangsu Ocean University . The volunteers had not used antibiotics 3 months before the study. After took 0.7 g food/kg body weight/day for 2 consecutive weeks, feces samples were collected at 6:30 AM on the 15th day before they ate any food. Within 10 min, the samples were used for the fermentation experiments described below , 28.2/H2/CO2 (80:10:10) at 37°C. Ellagic acid was first dissolved in polyethylene glycol (PEG) 300. The resulting solution was added to ABB medium 20.00 μM. In addition, media without fecal suspension and those without ellagic acid were used as controls. Samples (5 ml) were collected once per day from each culture, extracted with 5 ml C2H3N:H2O:CH2O2 (80:19.9:0.1), and further analyzed using High Performance Liquid Chromatography (HPLC), as explained below. If urolithin A was detected in the broth, it was used to screen urolithin A-producing bacteria. ABB medium (g/L): Peptone 16.0, Yeast extract 7.0, NaCl 5.0, Starch 1.0, Glucose 1.0, C3H3NaO3 1.0, Arginine 1.0, C4H4Na2O4 0.5, L-Cysteine HCl 0.5, NaHCO3 0.4, Fe4(O7P2)3 0.5, C34H32ClFeN4O4 0.005,Vitamin K 0.0005, C2H3NaO2S 0.5, C4H10O2S2 1.0,pH6.8 ± 0.2.In total, 10 g of fecal samples were diluted 1/10 (w/v) with anaerobe basal broth  and homogenized using a stomacher in filter bags , 30. 1 m–3 to 10–6 with ABB media. An aliquot of the diluted suspension (0.1 ml) was spread on the ABB agar plate, and the plate was incubated at 37°C in an anaerobic chamber under the condition of N2/H2/CO2 . Selected single colonies were inoculated into 50 ml of ABB containing 20 μM ellagic acid  and further analyzed through HPLC and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses, explained below. The urolithin A-producing strains were stored in ABB media with 20% (v/v) glycerol at –80°C for further experiments.The urolithin A-containing fermentation broth was diluted to 10gic acid . After aThe morphological, physiological, and biochemical properties of strain FUA027 were investigated using previously described methods . GenomicEnterococcus faecium FUA027was streaked to single colonies on an ABB agar plate at 37°C for 6 days. A liquid culture for the inoculum was prepared by transferring a single colony to a tube containing 5 ml ABB by using a sterile inoculation loop. Two milliliters of the inoculum were transferred to 100 ml ABB media containing 20 μM ellagic acid and incubated under anaerobic conditions consisting of N2/H2/CO2  at 37°C for 7 days. During incubation, 5-ml aliquots were taken and extracted with 5 ml of C2H3N:H2O:CH2O2  for HPLC and UPLC-MS analyses as described below. Similarly, culture samples were collected every 2 h on all 7 days of incubation . A 0.22-μm cellulose acetate filter  was used to filter the extract. The HPLC analysis was performed on a ZORBAX SB-C18 column  at 25°C. The gradient started at 20% A and 80% B, followed by 70% A and 30% B at 15 min, 95% A and 5% B at 20 min, 100% A and 0% B at 21 min, 100% A and 0% B at 23 min, 20% A and 80% B at 24 min, and 20%A and 80% B at 25 min. The flow rate was 1 ml/min, and the column temperature was 25°C. Injection volume was 5 μl for each sample. UV chromatograms were recorded at 305 nm.The extracted samples were evaporated to dryness under a N® BEH C18, 2.1 × 50 mm, 1.7 μm; 0.5 ml/min] and HR-ESI-MS scan range from 150.0 to 2,000.0 Da. Sheath gas flow rate, 45 arb; aux gas flow rate; 15 arb; Capillary temp:320°C; S-Lens RF Level 50 T. chromatographed using LC-MS/MS at 30°C. The mobile phase consisted of solvent A  and solvent B (acetonitrile). Separation was performed at a flow rate of 0.5 ml/min with the following gradient elution: 0–10.5 min, a linear gradient runs from 10 to 100% solvent B; 10.5–12.5 min, 100% solvent B; 12.5–12.6 min, a linear gradient runs from 100 to 10% solvent B; 12.6–16.5 min 10% solvent B for re-equilibration. The limits of detection (LODs) were determined based on a signal-to-noise ratio (S/N) of 3 and of 10 for the limit of quantification (LOQ). The LODs: 0.5 μM (EA and urolithin A), 0.2 μM (urolithin C), the dynamic range: 0.5–20 μM. Urolithin A and urolithin C in the fermentation broth were first identified through HPLC and further identified by comparing the molecular mass of the compound obtained with that of a pure standard by using LC-MS/MS (The Q-Exactive LC-MS/MS (Thermo Fisher) was used to analyze urolithin A using a C18 column [ ACQUITY UPLCLC-MS/MS , 36.Urolithins were identified through a direct comparison of the retention times with those of pure standards . The retA total of 395 strains were screened for urolithin A production. According to the results of HPLC analysis  and LC-ME. faecium (OM670243). The phylogenetic tree, which was constructed for comparing the 16S rDNA sequences, suggested that strain FUA027 belongs to the genus Enterococcus . Therefore, strain FUA027 was identified as E. faecium.The cells of FUA027 strain were gram-positive and rod-shaped . Simulta faecium . The 16Srococcus . The cloE. faecium is a part of the normal flora of human and animal intestines. When E. faecium, harmful intestinal bacteria in humans (Escherichia coli), and human intestinal probiotics (Bifidobacterium) were cultured, E. faecium was found to inhibit these harmful bacteria along with Bifidobacterium. E. faecium rapidly adheres to the intestinal mucosa after entering the intestine, forming an intestinal barrier , which was observed at 28 h and whose levels peaked at 40 h, with the maximum concentration being 1.54 μM. Then, the urolithin C levels declined progressively as urolithin A was formed  reported in the literature is less than 2% (E. faecium FUA027) is 32.2%, the conversion rate was higher than the reported strain significantly. FUA027 is the great candidate strain to study the mechanism underlying the biotransformation of ellagic acid into urolithin A.To date, four urolithin-producing strains have been reported; however, they differ from one another in ellagic acid biotransformation processes.  strains , 24. Elltabolism . The enddetected . Similardetected . The con than 2% . CompareE. faecium strain capable of converting ellagic acid into urolithin A in vitro. The strain E. faecium FUA027 could potentially be useful for the development of next-generation probiotics, functional foods, and food complements and be used to explore the regulation of their transcription.The transformation of ellagic acid into urolithin A by a single bacterial strain is described herein. This is the first description of an The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.XZ: conceptualization, methodology, software, and writing – original draft. YF: resources and supervision. GY: conceptualization and resources. XH: data curation and supervision. YH: software and validation. MX and YZ: investigation. FH: formal analysis. SL: methodology, supervision, and project administration. All authors contributed to the article and approved the submitted version.


Synthesis of MMs by chemical surfactants is not ecofriendly. This study focused on the biological synthesis of a MM by sol–gel method, using a Bacillus subtilis BBK006-mediated surfactant (template) and a precursor (TEOS). The biologically synthesized mesoporous silica nanoparticles (BMSN) were formed at calcination temperatures of 450–600 °C. The BMSN comprise Si and O elements with specific weights of 56.09% and 42.13% respectively, where the atomic% was detected to be 41.79% and 55.10%, respectively. The phase identity of the synthesized particles  was confirmed with wide-angle XRD (10°–81°). A typical type IV isotherm was exhibited (BET curves) following IUPAC nomenclature and confirmed the mesoporous nature. The green-synthesized biosurfactant-mediated BMSN is an environmentally promising material to apply in biomedical science  and oil spill management.Mesoporous materials (MMs) have recently been applied as advanced nanomaterials in different fields (separation, catalysis, adsorption  Synthesis of a novel BMSN material using bacteria-mediated biosurfactant and their characterization. CTAB) still exhibit toxicity to living organisms at higher concentrations (>1 μM), such as cytotoxicity  and chronic and subacute toxicity .12–14In particular, the sol–gel method is popular as it is easy, less time-consuming (compared to other methods), has a low operational temperature, and is a more expedient process for synthesizing MMs.etc.) due to their low toxicity,15–18 ecofriendly nature, low cost and easy synthesis. Microbiologically, biosurfactants are produced from microorganisms with different principal carbon sources .19,20 Currently, a significant percentage of biosurfactant production takes place in Europe , as well as India, China, Japan, Thailand, Brazil, and the USA (Jeneil Biotech) in the global market,21 where biosurfactants are mainly utilized in personal care, agriculture, the food industry, detergents, etc. The production of biosurfactants is now easy and effective due to advances with analytical instruments in industry that allow biosurfactants to be conveniently extracted from various microbes.15,16,22,23 Apart from these advantages, biosurfactants are biodegradable, environmentally friendly, economical, and renewable substrates.24 Moreover, the physical characteristics of biosurfactants are mainly dependent on the temperature and pH.24 Thus, in nanobiotechnology, natural substrates are used as precursors for synthesizing microbial-based nanoparticles to minimize the hindrance (e.g. reduce the toxicity and increase the ecofriendliness).In recent years, biosurfactants have received increasing interest in different research arenas , which is a key objective for improving the economic and environmental aspects of nanobiotechnology. A sol–gel process (bottom-up approach) with different calcining temperatures was used to synthesize the BMSN. The physicochemical characteristics of the BMSN were investigated in terms of crystal structure, chemical bonding, surface adsorption, absorptivity, morphology, pore shape and size, etc.In the present study, a 2.2.1Bacillus subtilis BBK006 .15,16,22,25,26 The chemical reagents Na2HPO4 (98% purity), KH2PO4 (99.5% assay), NaCl (99.9% assay), NH4Cl (99.5% purity), MgSO4·7H2O (99.8% assay), CaCl2 (76.07% assay) and glucose (99.9% purity) were used to prepare the M9 medium for bacterial growth.15,16,22,25,26 The material was synthesized using 25 wt% of aqueous ammonia, ethyl alcohol (99.5% assay), tetraethyl orthosilicate (TEOS)  and methanol. All chemical reagents were used without further purification.The biosurfactant was extracted from 2.2Bacillus subtilis BBK006 was grown in M9 broth media with the following composition: 6.78 g L−1 of Na2HPO4, 3.0 g L−1 of KH2PO4, 0.5 g L−1 of NaCl, 1.0 g L−1 of NH4Cl, 0.493 g L−1 of MgSO4·7H2O, and 0.011 g L−1 of CaCl2.15,16,22,25,26 All of these chemicals were entirely dissolved in the double deionized water separately to avoid the formation of precipitation on the surface of the transparent glass reagent bottle during the sterilization process. The dissolved components were transferred to a 1 L transparent glass reagent bottle and made up to the mark to maintain the balanced concentration. The prepared broth media was wrapped with aluminium foil for sterilization (autoclave at 121 °C for 20 min at 15 atm). The sterilized media was cooled down and placed in laminar flow hood for inoculation with Bacillus subtilis BBK006. 0.4% glucose was added to the sterilized broth media in laminar flow hood . A small amount (2 μL at ∼1.2 OD) of bacteria was inoculated for growth.The −1 at 600 nm wavelength) by UV-vis-spectrophotometer , and was used for biosurfactant production. The scan rate of the ultraviolet-visible spectrophotometer is 2500 nm min−1 with accuracy of ±1.2 nm over the wavelength range of 320–1100 nm using a halogen bulb as the light source.The flask containing the inoculated bacterial media was placed in an orbital shaking incubator (JSL-530) at 37 °C (200 rpm) and incubated for 24 h to increase the bacterial count per volume. The highest growth of bacteria was estimated as ∼1.2 OD  was centrifuged at 10 000 rpm (12 000 × g) for 15 min, at 25 °C  to collect the supernatant . The collected supernatant was adjusted to pH < 2 using 5 M hydrochloric acid (HCl) to precipitate the biosurfactant properly by incubating overnight (12 h). After precipitation, the biosurfactant was collected by centrifugation at 5000 rpm for 12 min at room temperature and the precipitated biosurfactant was then dried to a powder using a precision oven (JA-72) set to 65 °C for 24 h. Finally, the biosurfactant was obtained as a solid light brown powder, which was used to synthesize the MM.Bacterial population growth plays an important role in producing biosurfactants since the production rate is proportional to the amount/number of bacteria. The biosurfactant was produced following the procedure of Maity 2.4The MM was synthesized following a novel procedure. Initially, 0.57 g of biosurfactant was dissolved in 50 mL of double deionized water, and stirred continuously until a clear solution was obtained. 11 mL of 25 wt% ammonia and 76 mL of ethanol were added to the clear solution simultaneously. At the same time, 10 mL of TEOS was also added dropwise to the mixture whilst stirring the solution. The mixed solution turned into a milky gel form due to the hydrolysis of TEOS. Gradually, the mixed solution was uninterruptedly stirred for ∼1 h 45 min to complete the hydrolysis of TEOS. A white precipitate was observed in the mixture and was separated by centrifugation at 5000 rpm for 10 min, . The precipitate was washed with double deionized water and methanol thrice, and then dried at 70 °C for 24 h. The dried material was finally calcined at different temperatures  in a muffle furnace to produce the MMs.2.52.5.1et al.27 The small-angle powder X-ray diffraction pattern was recorded using a Vantec-2000 detector with an exposure time of 3600 s per frame, with Ni-filtered KFL Cu Kα radiation , an operating current of 0.65 mA and a voltage of 45 kV with a 2θ range of 0.1 to 5°. The wide-angle X-ray diffraction patterns were recorded using Ni-filtered Cu Kα radiation with an operating current of 10 mA, voltage of 30 kV and a scintillation counter detector . The data were collected in 2θ angle with the range of 10–81° at elevated temperature (3 °C per minute) and a step size of 0.020135°.The crystallographic structure, phase composition, and physical properties of the materials were determined by XRD following the procedure of Maity 2.5.2et al.15 The functional groups were determined with KBr powder (KBr/sample weight ratio = 20 : 1) in the range of 4000–400 cm−1.The functional groups of the biosurfactant-mediated raw material and the synthesized/calcinated MMs were determined using Fourier transform infrared spectroscopy  following the procedure of Maity 2.5.3High-resolution X-ray photoelectron spectroscopy  was used for the elemental quantification of the synthesized materials. Al Kα radiation was used in the XPS as a monochromatic X-ray beam of light with 49.3 W, 45.0°, and 280.00 eV.2.5.4λ range = 200–2600 nm) the reflectance spectra of the samples with BaSO4 as a reference sample and constant temperature using cooling Pbs Gain detector.The absorption of the synthesized materials was determined by ultraviolet-visible/near-infrared spectrometry . The UV-vis-NIR spectrometer recorded .2.5.6et al. using a field emission-scanning electron microscope (FE-SEM)  with a coating of Pt in the operating range of 0.1–30 kV.16 The particle size and morphology of the synthesized materials were determined using a high-resolution transmission electron microscope (HR-TEM)  with an acceleration voltage of 200 kV. HR-TEM is carried the electron gun filament LaB6. The elemental analysis was also carried out by HR-TEM/EDX with the same analytical instrument.The surface morphology of the materials was determined following the procedure of Maity 2.5.716The pore volume, pore diameter and specific surface area were studied using the Brunauer, Emmett, and Teller method with a surface area analyzer instrument . The surface analysis was performed in the presence of liquid nitrogen at 77 K.3.3.13.1.1d100 and d003 planes in the low-angle XRD spectrum  were found to be almost the same, which is similar to the findings of Wu et al.28 The unit cell diameter of BMSN was observed to be higher (unit cell diameter 62.18 Å) than the chemical surfactant-based synthesized material (unit cell diameter < 50 Å) owing to efficient collaboration between the precursor and the biosurfactants.5,29 The phase composition and crystallinity of the synthesized materials (BMSN) were determined using wide-angle XRD (θ of 21.72 (d100), 21.95 (d100), 22.59 (d100), and 21.03 (d100) for the calcination temperatures of 450 °C, 500 °C, 550 °C, and 600 °C, respectively  were determined using both small-angle and wide-angle XRD . The twospectrum  are ascrngle XRD . In the ectively , which in angles  as compa3.1.2−1 , which represents the stretching vibration of a hydroxyl group (2OH).33The FTIR spectral bands of the functional groups were revealed within the range of 4000–400 cm−1 . In the −1 were assigned as –CH2 group stretching vibrations, which are comparable with the findings of Huo et al.33 The multiple bands at 2361 and 2343 cm−1 appeared due to N–H group stretching vibration. Strong, medium and numerous very weak intensity bands in the range of 1635–1558 cm−1 were assigned to N–H, C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O, NO2 and O–H groups, which indicates the appearance of the biosurfactant in the synthesized material. Weak and medium intense peaks were also observed in the BMSN at 1402 and 1456 cm−1 for O–H and ring groups. A less intense band at 870 cm−1 represents the C–H and C–O–C functional groups with different modes of vibration , which is comparable with the findings of Salam et al.34 Spectral bands at 1102, 1105, 1113, 1115, and 1120 cm−1 were observed, representing the existence of asymmetrical vibration of a siloxane group (Si–O–Si) in the materials , which is comparable with previous literature.35 Moreover, characteristic peaks at 957, 968, 966, 960 cm−1 representing the bending vibrations of a –Si–OH functional group were observed for both the non-calcinated and calcinated materials (except 600 °C), which is similar to previous results.36 Interestingly, the spectral band of the Si–OH functional group disappeared at 600 °C calcination temperature due to the loss of hydrogen. A peak at 808 cm−1 was observed for the symmetrical stretching vibration of Si–O–Si in the non-calcinated material, whereas the peak shifted to 801, 808, 803, 802, and 808 cm−1 for the materials calcinated at 450 °C, 500 °C, 550 °C, and 600 °C, respectively. Furthermore, in the present study, the peaks at 450 cm−1 to 810 cm−1 were found to be free silica as the Si–O–Si group of synthesized particles due to bending vibrations, which is comparable with the previous results of Salam et al.34 Thus, in the calcinated synthesized material (450–600 °C), it was clearly indicated that the functional groups –H, CO, NO2 and ring had vanished due to the eradication of the biosurfactants from synthesized material, nevertheless, Si–OH still remained and the synthesized material was designated as BMSN .A spectral band at ∼1635 cm3.1.33/2, C 1s and O 1s atoms . The B.E. of the Si 2p3/2 orbital increased with increasing calcination temperature, with 102.05 and 102.11 eV at 500 °C and 550 °C, respectively. However, the B.E. of the Si 2p3/2 orbital at 600 °C (102.03 eV) was smaller than that at 550 °C due to less interaction of Si 2p3/2 with oxygen, where the B.E. of the O 1s orbital was detected 531.26, 531.42, 531.57, and 531.47 eV at calcination temperatures of 450 °C, 500 °C, 550 °C, and 600 °C, respectively, which indicated the coupling of silicon with oxygen to generate SiO44− by Si–O linkage, and this result is comparable with the findings of Sterczyńska et al.37 Likewise, the B.E. of oxygen (O 1s) are intense peaks (38 A single highly intense peak of silicon and oxygen was identified at 550 °C with the maximum B.E. and atomic percentage (%), where the highest peak intensity designates strong bonding between silicon and oxygen atoms .28,39,40X-ray photoelectron spectroscopy (XPS) provides information about the surface composition, electronic interactions and bonding between two elements. In the BMSN, the detected XPS spectra  clearly 1s atoms . As per se peaks  in BMSN,en atoms . The bin3.1.4e.g. MCM-41, SBA-15, KIT-6 etc.).41,42The spectral properties of a material are a fundamental key to distinguish the coordination environment of the molecules by interpreting the absorption of ultraviolet radiation, where the radiation propagates to transitions flanked by their electronic energy levels. The variation in the absorption spectral band (by UV-vis-NIR) of the material at the various calcination temperatures is shown in 3.1.543 which reported a total weight loss of 14.5–16.5% after the formation of a material calcinated at 600–700 °C. Thus, in the present study, a higher production rate of biologically synthesized mesoporous silica nanoparticles (BMSN) was observed.The weight loss (%) by exclusion (organic and inorganic constituents) of surface water, interlayered water molecules, bio-templates, and the establishment of a novel BMSN material at the optimum temperature from the synthesized material are shown in 3.1.644 which indicates that the BMSN particle sizes are smaller/analogous (61–300 nm). Interestingly, the diameter of the BMSN particles was observed to be slightly increased (average particle diameter 264 nm) at 600 °C temperature  of silicon increased with increasing calcination temperature from 450 °C to 550 °C, then declined at the calcination temperature of 600 °C. This occurred due to the enhancement of the particle diameter at the calcination temperature of 600 °C and this phenomenon was illustrated by FE-SEM and HR-TEM. Hence, the elemental composition (silicon and oxygen) of the synthesized material demonstrated the formation of a MM.3.1.746 due to the mesoporosity of BMSN (3 g−1 consecutively at STP (standard temperature and pressure) for calcination temperatures of 450 °C, 500 °C, 550 °C and 600 °C, respectively (P/P0) of multilayered adsorption process (P/P0) was observed in the range of 0.1–0.2 due to capillary condensation of particles. A large amount of adsorption was observed with a sharp hysteresis loop over the relative pressure (P/P0) range of 0.8–1.0 due to uniform distribution of pore size in BMSN particles, where the desorbed quantity was accompanied by the adsorbed quantity. The pore diameter gradually decreased from 29.7070 to 20.2166, 14.8516 and 16.5246 nm with increasing calcination temperature from 450 °C to 600 °C  as compared to those in previous reports on chemical template-based MMs (SBA-15 and MCM-41) .1,47According to IUPAC nomenclature, typical type IV isotherm adsorption was observed in the BET curves of BMSN . The spe of BMSN . The BJHectively . In case process . The firo 600 °C . Thus, i3.1.84OH combines with H2O in the presence of C2H5OH to form NH4+ and OH− ions by dissociation. The anionic hydroxide further reacts with tetraethyl orthosilicate to produce orthosilicic acid,48 where the production of materials takes more than an hour to complete the hydrolysis through a condensation method n], forming a highly complexed ordered BMSN mesoporous Si–OH material  influenced the physical and chemical characteristics of the synthesized particles. At the optimum temperature of 550 °C, the crystallographic properties of BMSN confirmed that it was an amorphous silica material with mesoporous character. In the synthesis process, the biosurfactant combines with TEOS  and forms Si–O–surfactin complexed molecules, which later bind with each other and produce a spherical molecule with numerous silica–O–surfactin-based complex through a condensation process. Highly complexed ordered BMSN mesoporous Si–OH material was formed to remove the biosurfactants from the silica–O–surfactin-based complex [(Si–O–surfactin)n] by calcination. BMSN mesoporous nanoparticles consist of Si–OH, where Si–O–Si linkage is observed. The absorption of BMSN was higher at the wavelength of 256.72 nm in the material calcinated at 550 °C. Effectively, 87.87% yield of BMSN was obtained (weight loss: 12.13%), using the calcination temperature range of 550–600 °C. Perfectly spherical shaped particles with regular arrangement were observed with an average particle size of 280 nm (diameter) (range: 61–300 nm), where the surface area, pore volume and pore diameter were found to be 8.2616 m3 g−1, 0.020407 cm3 g−1, and 14.8516 nm, respectively. Thus, this study suggests that materials prepared using biotemplate-based synthesis could be highly efficient and worthwhile for numerous industrial practices with very low toxicity, reduced synthesis time and cost, as well as applications in catalysis, drug delivery systems, gas separation, cosmetics, diagnostics, bio-separation, etc.A novel BMSN MM was successfully synthesized by an ecofriendly and green process using R. K. S. prepared the manuscript. J. P. M., J. B., P. G. H., and Y. H. H. revised and gave necessary inputs. J. P. M., C. Y. C., S. C. W., H. C. C., and T. H. C. revised and gave suggestions for the manuscript. J. P. M., G. D., P. B., and S. C. W. revised the manuscript, tables, and figures.There are no conflicts of interest.


The results show that Al toxicity was higher in ZH2 than in 99-1507 and higher under LD than under SD. Root length decreased by 30, 37.5, and 50% in ZH2 and decreased by 26.08, 34.78, and 47.82% in 99-1507 under SD, ND, and LD, respectively, under Al stress. Photoperiod and Al induced cell death and ROS production. MDA content, PME activity, and LOX activity increased under SD, ND, and LD, respectively, under Al stress both in ZH2 and 99-1507. APX, SOD, CAT, and POD activities were higher under SD, ND, and LD, respectively. Al stress increased the level of AhLHY expression under SD and ND but decreased it under LD in both ZH2 and 99-1507. Contrastingly, AhSTS expression levels increased exponentially and were higher under SD, LD, and ND, respectively, under Al stress. Our results will be a useful platform to research PCD induced by Al and gain new insights into the genetic manipulation of the circadian clock for plant stress response.Peanut is among the most important oil crops in the world. In the southern part of China, peanut is highly produced; however, the arable land is acidic. In acidic soils, aluminum (Al) inhibits plant growth and development by changing the properties of the cell wall and causing the disorder of the intracellular metabolic process. Circadian rhythm is an internal mechanism that occurs about every 24 h and enables plants to maintain internal biological processes with a daily cycle. To investigate the effect of photoperiod and Al stress on the Al-induced programmed cell death (PCD), two peanut varieties were treated with 100 μM AlCl It has been shown that about 50% of the total arable land around the world is acidic . Al can Circadian clock is an intrinsic timekeeping mechanism that synchronizes with the periodic environment through daily entrainment, especially light and temperature to adjust the internal rhythm ,26,27. TArabidopsis thaliana ) was calculated using the following formula: To analyze the lipid peroxidation (LPO) levels, the malondialdehyde (MDA) content was assayed following the method of  with litw/v) polyvinylpyrrolidone (PVP) then centrifuged at 13,000× g for 20 min, 4 °C. The enzyme activity was analyzed by mixing 1 ml 0.5% (w/v) pectin (pH 7.5), 0.4 mL 0.01% (w/v) bromothymol blue (pH 7.5), 1.55 mL distilled water (pH 7.5), and 50 µL of the root tip extract; then, the mixture was measured with a spectrophotometer at 620 nm. To determine the pectin methylesterase (PME) activity, 0.2 g of the root tips was grounded and homogenized with 2 mL 1 M sodium chloride (NaCl) containing 1%  titanic sulfonate (Ti(SO4)2) and 0.1 mL 25% ammonia, and was centrifuged at 3000× g for 10 min at 4 °C; the pellet was resuspended in 4 mL of 2 N sulfuric acid (H2SO4). H2O2 content was spectrophotometrically determined at 415 nm. The Hethod of . Briefly2.− content was measured as described by [g for 10 min at 4 °C. The aliquot of 1 mL from the supernatant was mixed with 0.9 mL of 65 mM phosphate buffer (pH 7.8) and 0.1 mL of 10 mM hydroxylamine hydrochloride (HONH2·HCl), then the mixture was incubated at 25 °C for 20 min. A total of 1 ml of the mixture was extracted and added to 1 mL of 17 mM anhydrous aminobenzene sulfonic acid (H3NC6H4SO3), as well as 1 mL of 17 mM 1-naphthylamine (C10H9N). The mixture was incubated at 25 °C for 20 min, then 3 ml of n-butanol was added. The O2.− content was measured at 530 nm.Oribed by . Root tiw/v) PVP, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), and 0.05% (v/v) Triton X-100 and centrifuged the homogenate at 13,000× g for 15 min at 4 °C. Lipoxygenase (LOX) activity was assayed following the method of [v/v) Tween 20, and then measured at 234 nm. CAT, APX, and SOD activities were assayed following the method of [2O2 solution and CAT activity was spectrophotometrically measured as H2O2 decomposition at 240 nm. To detect APX activity, the root tip extract was mixed with 0.25 mM ascorbic acid, 0.5 mM H2O2 and measured at 290 nm. SOD activity was determined, as the root tips were homogenized in 1 ml cold 100 mM potassium phosphate buffer pH 7.8 containing 0.1 mM EDTA, 1% PVP, and 0.5% v/v triton x-100, and it was measured at 560 nm, as enzyme amount required us to inhibit the reduction of 50% of NBT at 560 nm. To determine the antioxidant enzyme activity, root tips (0.2 g) were ground with mortar and pestle in liquid nitrogen and then homogenized with 50 mM sodium phosphate buffer (pH 7.8) containing 1 mM ethylenediaminetetraacetic acid (EDTA), 2% . After the homogenization, the solution was centrifuged at 1000× g for 8 min. The supernatant was extracted into a 10 mL centrifuge tube and kept on ice. The pellet was resuspended with 2.5 mL 0.5 M CaCl2 and centrifuged again (repeated twice). Before POD assay, buffer solutions (A and B) were brought to 25 °C with a water bath. We mixed 1.4 mL of solution A(Phenol and 4-aminoantipyrene), 1.5 mL of solution B , and 200 µL roots extract. Then, we measured the absorbance at 510 nm. All activities were measured with a spectrophotometer .POD activity was assayed following the “standard operating procedures” of scientific engineering response and analytical services . Frozen root tips (1 g) were ground using mortar and pestle with liquid nitrogen; then, they were homogenized into ice-cold 0.5 M calcium chloride solution  following the manufacturer’s instructions. The quality of RNA was proved by the ThermoFisher Scientific NanoDrop 2000c, Germany (RNA concentration 1.9–2.2 µg was used). cDNA was synthesized through RNA reverse transcription using PrimeScriptTM RT reagent Kit with gDNA eraser  Cat#RR047A, Takara bio, China. qRT-PCR was performed with CF × 96 TM Real-Time System, Bio-Rad Laboratories, Hong Kong, using SYBR Green qPCR super mix. The actin was used as the normalization control. The following primers were used:Total RNA was extracted using EastepGene NameGene IDPrimerAhLHY-forwardAH02G04520.15′-ATTGACTCTAGTAATCGTCGTA-3′AhLHY-reverse5′-CTTTGTGGCAACACCTCT-3′AhSTS-forwardAH04G08660.15′-CCCAAGCGTCAAGAGGTA-3′AhSTS-reverse5′-TTGCCCACAAGACTATCCA-3′Actin-forwardAH03G02610.15′-ACCTTCTACAACGAGCTTCGTGTG-3′Actin-reverse5′-GAAAGAACAGCCTGAATGGCAAC-3′t-test to test the differences between groups. The data are presented as mean ± standard deviation (SD). p-value ≤ 0.05 was considered statistically significant.The experiments were independently replicated three times and their mean values were subjected to sata processing and statistical analysis with Excel 2007 and SPSS 12.0 . Statistical analysis was performed with Student’s paired 2O2 and O2.−), and the acceleration of lipid peroxidation; increased MDA concentration; accelerated PME activity; and positively induced the expression of LHY and STS genes. Prolongation of the light period resulted in photoperiod stress, and it has an important influence on Al stress and tolerance. Peanut showed a strong resistance to Al toxicity under SD, moderate under ND, and low under LD. The molecular mechanism and pathways of AhLHY and AhSTS in Al tolerance under the circadian rhythms should be studied next, including the metabolic engineering of stilbene biosynthesis as a strategy to directly demonstrate the role of this phytoalexin in plant stress resistance.Different light–dark conditions showed different levels of Al toxicity, which could reveal the influence of photoperiod in the regulation of Al stress. The induction of ROS by Al stress led to the deterioration of the cell wall, the release of free radicals (H


TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. Its N-terminal domain (NTD) can dimerise/oligomerise with the head-to-tail arrangement, which is essential for function but also favours liquid-liquid phase separation and inclusion formation of full-length TDP-43. Using various biophysical approaches, we identified an alternative conformational state of NTD in the presence of Sulfobetaine 3-10 (SB3-10), with higher content of α-helical structure and tryptophan solvent exposure. NMR shows a highly mobile structure, with partially folded regions and β-sheet content decrease, with a concomitant increase of α-helical structure. It is monomeric and reverts to native oligomeric NTD upon SB3-10 dilution. The equilibrium GdnHCl-induced denaturation shows a cooperative folding and a somewhat lower conformational stability. When the aggregation processes were compared with and without pre-incubation with SB3-10, but at the identical final SB3-10 concentration, a slower aggregation was found in the former case, despite the reversible attainment of the native conformation in both cases. This was attributed to protein monomerization and oligomeric seeds disruption by the conditions promoting the alternative conformation. Overall, the results show a high plasticity of TDP-43 NTD and identify strategies to monomerise TDP-43 NTD for methodological and biomedical applications. Amyotrophic lateral sclerosis (ALS) is a fatal disorder characterized by a progressive degeneration of the upper and lower motor neurons of the brain, brainstem, and spinal cord, but also in the frontotemporal cortex and hippocampus in a fraction of patients ,2,3, leaTARDBP is located on chromosome 1. It comprises an N-terminal domain , a nuclear localization signal , two RNA recognition motifs , a nuclear export signal , and a C-terminal domain , which encompasses a prion-like glutamine/asparagine-rich (Q/N) region (aa 345–366) and a glycine-rich region (aa 366–414)  is the mean residue ellipticity in deg cm2 dmol−1, θ is the ellipticity in mdeg, the optical path is in cm, concentration is in g/L, and the molecular weight is in g/mol.The TDP-43 NTD sample was centrifuged at 18,000× g for 15 min, at 4 °C, and two samples containing 0.5 mg/mL (45µM) NTD, in 5 mM sodium phosphate buffer, 50 mM NaCl and 1 mM DTT, pH 7.4, were prepared, one in the absence and the other in the presence of 3.0% SB3-10. Fluorescence spectra were acquired at 25 °C from 290 to 500 nm (excitation at 280 nm) using a 3 × 3 mm black wall quartz cell cuvette on an Agilent Cary Eclipse spectrofluorimeter  equipped with a thermostated cell holder attached to an Agilent PCB 1500 water Peltier system . Excitation and emission slits were 5 nm. Spectra were then blank subtracted.The TDP-43 NTD sample was centrifuged at 18,000× g for 15 min, at 4 °C, and two 1 mL samples containing 0.05 mg/mL (4.5 µM) NTD, in 5 mM sodium phosphate buffer, 50 mM NaCl and 1 mM DTT, pH 7.4, were prepared, one in the absence and the other in the presence of 3.0% SB3-10. For each sample, a first fluorescence spectrum was acquired at 25 °C from 300 to 420 nm (excitation at 280 nm) using a 10 × 4 mm quartz cuvette under magnetic stirring on an Agilent Cary Eclipse spectrofluorimeter  equipped with a thermostated cell holder attached to an Agilent PCB 1500 water Peltier system. Excitation and emission slits were 5 nm. Then, 5 µL of 1 M acrylamide was added directly to the cuvette and another spectrum was recorded. This step was repeated 15 times. For each recorded spectrum, the total fluorescence was corrected to take account of the dilution with the acrylamide solution. The quenching of acrylamide in the 0.0% SB3-10 sample was analyzed with the Stern-Volmer equationF0 and F are the integrated fluorescence intensity areas at 300–400 nm in the absence and presence of acrylamide, respectively, SVK is the Stern-Volmer constant and [Q] is the concentration of the quencher (acrylamide) in the cuvette. The quenching of acrylamide in the 3.0% SB3-10 sample was instead analysed usingSTK is a constant that considers the static quenching caused by the binding of acrylamide to tryptophan residues.The TDP-43 NTD sample was centrifuged at 18,000× 13C, 15N uniformly labelled TDP-43 NTD samples (250 µM) in 5 mM sodium phosphate, 50 mM NaCl, 3% (w/v) SB3-10, 1 mM DTT, 5% (v/v) D2O. For reference purposes, trimethylsilylpropanoic acid (TSP) was added  was obtained with the 6 standard data points and fitted to a linear regression curve. This calibration curve was then used to interpolate the experimental eV (y axis) of NTD samples and to obtain the corresponding MW (x axis) and, thus, their oligomeric states. The MW values determined with this approach are valid only for globular proteins. For the NTD sample in 3.0% SB3-10, the calibration curve was re-determined mathematically to account for the differences between the hydrodynamic radii of folded and pre-molten globule states ). Their fluorescence spectra were acquired at 25 °C from 290 to 500 nm (excitation at 280 nm) using a 10 × 2 mm quartz cuvette on an Agilent Cary Eclipse spectrofluorimeter , equipped with a thermostated cell holder attached to an Agilent PCB 1500 water Peltier system. Excitation and emission slits were 5 nm. Spectra were then blank subtracted.The TDP-43 NTD sample was centrifuged at 18,000× F0/F was then calculated for each sample, where F and F0 are the blank-subtracted fluorescence values at 485 nm of ThT+protein and free ThT, respectively. An over 5-fold ThT fluorescence increase was considered to be diagnostic for amyloid [TDP-43 NTD samples were prepared at various SB3-10 concentrations, as described in the previous subsection and then incubated for 6 h at 25 °C, while the sample in 3.0% SB3-10 was incubated also for 24, 48, 72 and 96 h. 100 µL of buffer or protein sample were then mixed with 400 µL of 25 µM ThT. Fluorescence spectra were acquired at 25 °C from 450 to 600 nm (excitation at 440 nm) using a 10 × 2 mm quartz cuvette on an Agilent Cary Eclipse spectrofluorimeter  equipped with a thermostated cell holder attached to an Agilent PCB 1500 water Peltier system. Excitation and emissions slits were 5 and 10 nm, respectively. All spectra were blank subtracted (using only PBS as a blank). The ratio  amyloid ,34,43,44t-test and the resulting p values were indicated in the text for each comparison. A p value < 0.05 was considered to be statistically significant.Data were expressed as means ± SEM. Data pairs were compared using the Student’s