The regulation of Bax/Bcl-2, expression of caspase-3 protein and the activation of PI3K/Akt signaling pathway were observed in the cells pretreated with limonin. Treatment of PC12 cells with PI3K inhibitor LY294002 weakened the protective effect of limonin. These results indicated that limonin prevented Aβ25−35-induced neurotoxicity by activating PI3K/Akt, and further inhibiting caspase-3 and up-regulating Bcl-2. This study enables comprehensive utilization of pomelo seed as by-product and offers a theoretical principle for a waste-to-wealth solution, such as potential health benefits of food ingredient and drug.Pomelo seed as a by-product from pomelo consumption is rich in bioactive compounds, however, a huge volume of pomelo seed was disposed as wastes, the comprehensive utilization of pomelo seed could not only generate valued-added products/ingredients, but also decrease the environmental pollution. In this study, the main active substance limonin in pomelo seed was considered as a high-value bioactive compound. The purification of limonin from pomelo seed was investigated, and the neuroprotective and mechanism were characterized. The UPLC-MS/MS results indicated that 29 compounds in pomelo seed were identified, including 14 flavonoids, 3 limonids, 9 phenols and 3 coumarins. Moreover, high purity of limonin was obtained by crystallization and preparative-HPLC. Furthermore, limonin pretreatment can antagonize the cell damage mediated by Aβ Citrus grandis (L.) Osbeck], known as shaddock and pummel, is one of the most important commercially fruit crops. Pomelo belongs to the citrus genus in the Rutaceae family, which is widely cultivated in Asia and some other countries around the world  accounts for 50–70% of all dementia . Currentfunction –21 by cafunction . Aβ25−35 culture . Therefo25−35. This study enables comprehensive utilization of the waste pomelo seed resources and proves the neuroprotective effect of limonin in the current scientific literature.In the present study, the main bioactive compounds in pomelo seed were identified using the UPLC-MSMS system. Then, the neuroprotective effect of limonin isolated from pomelo seed was investigated using an apoptosis model of PC12 cells induced by AβThe pomelo seeds were obtained from Meizhou, Guangdong province, China. Chromatographic grade acetonitrile was obtained from Merck , trifluoroacetic acid, ethanol, formic acid, ethyl acetate, dichloromethane and isopropanol were obtained from Guangzhou Chemical Reagent Factory . RPMI-1640 cell medium, fetal bovine serum and penicillin were all purchased from Sigma-Aldrich . Fluo-3/AM and LY294002 (the inhibitor of PI3K) were acquired from Shanghai Beyotime Biotechnology  and Selleck Chemicals , respectively.Subcritical equipment  was used to remove grease of pomelo seeds with n- butane. The conditions were as follows: material liquid ratio 1: 4 (g/mL), extraction time 50 min, extraction temperature 50°C, and extraction 2 times.*2.1*100 nm, Agilent technologies, USA) at 30°C. The mobile phase contained 0.1% formic acid-water (A) and acetonitrile (B). The flow rate was 0.3 mL/min and injection volume was 2 μL. The gradient conditions were set as follows: 0–5% (B) in 0 to 2 min, 2–30% (B) in 2–6 min, 30% (B) in 6–7 min, 30–78% (B) in 7–12 min, 78% (B) in 12–14 min, 78–95% (B) in 14–17 min, 95% (B) in 17–20 min, 95–5% (B) in 20–21 min, and 5% (B) in 21–25 min.The defatted pomelo seed  was extracted three times with 70% ethanol at 50°C for 4 h. The ethanol extracts were analyzed by the UPLC-MSMS system . Chromatography was carried out on Zorbax Eclipse C18 (1.8 μmThe mass spectra were performed in positive electrospray ionization (ESI) modes, and the optimal conditions were set as follows: heater temperature 325°C; sheath gas flow rate 45 arb; auxiliary gas flow rate 15 arb; purge gas flow rate 1 arb; electrospray voltage 3.5 KV; capillary temperature 330°C; S-Lens RF Level 55%. Scanning mode was primary full scanning  and data-dependent secondary mass spectrometry scanning . Resolution was 120,000 (primary mass spectrometry) and 60,000 (secondary mass spectrometry). Collision mode was high-energy collision dissociation (HCD).The ethanol extracts (118 g) was obtained after drying under vacuum condition at 50°C in a water bath, and then further extracted with ethyl acetate. These extracts were once again concentrated in a vacuum evaporator. The extracts was resolved using dichloromethane and isopropanol (1:2 v/v), then recrystallized at 4°C for 8 h. The recrystallization operation was performed three times until pure white crystals were obtained. The obtained filtrates were concentrated and recrystallized to obtain the mother liquor. The preparative-HPLC  was used later for separation and the analytical-HPLC  for analysis to give compound (101 mg). The preparative-HPLC conditions contained water (A) and acetonitrile (B). The flow rate was 3 mL/min and injection volume was 500 μL. The gradient conditions were set as follows: 0–10% (B) in 0 to 5 min, 10–45% (B) in 5–10 min, 45% (B) in 10–30 min. The purification of limonin was calculated by area normalization method.25−35  stock solution (180 μmol/L) was prepared as follows: Aβ25−35 was dissolved in distilled water, and aliquots were stored at −20°C and incubated at 37°C for 7 days before usage. Highly differentiated rat pheochromocytoma PC12 cells  were cultured at 37°C with 5% CO2 in RPMI-1640 medium with 1% penicillin and 10% fetal bovine serum.Aβ25−35 and limonin for cell treatment. Results showed that treating cells for 48 h with 20-μM of Aβ25−35 significantly inhibited PC12 cell growth. Therefore, 20 μM Aβ25−35 was chosen as the cell injury model concentration.Cell Counting Kit-8 (CCK-8) was applied to determine the final dosage of Aβ25−35, Aβ25−35+12.5 μg/mL limonin, Aβ25−35+25 μg/mL limonin, Aβ25−35+50 μg/mL limonin, Aβ25−35+LY294002, and Aβ25−35+LY294002+50 μg/mL limonin. In the control group, only cell culture medium was added. In Aβ25−35 group, the cells were treated with 20 μM Aβ25−35 for 48 h. In the sample groups, the cells were pre-treated with various concentrations of limonin for 1 h. After that, 20 μM Aβ25−35 was added and cultured for 48 h. In Aβ25−35+LY294002 and Aβ25−35+LY294002+50 μg/mL limonin groups, the cells were treated with LY294002 for 1 h before the addition of Aβ25−35 and limonin, and then incubated for 48 h.Cells were divided into the following groups: control, Aβ4 cells/well and then incubated with Aβ25−35, limonin and LY294002 for 48 h. After incubation, 20 μL of CCK-8 reagent was added and incubated for 2 h. Next, the upper medium was removed and absorbance was measured at 450 nm using a Microplate Reader .CCK-8 assay was used to determine cell viability. The PC12 cells were seeded into 96-well plates at a density of 1 × 10PC12 cells were inoculated into a 6-well plate and cultured for 24 h, and then processed in different groups and incubated for a certain amount of time. After the incubation, cells were collected and washed twice with phosphate buffered saline (PBS). The LDH ratio of each group was measured using a LDH kit .2+ concentration assay was used as previously described method with some modifications .The intracellular Caications . BrieflyIntracellular ROS levels were detected using a reactive oxygen species detection kit . Briefly, after different treatments, cells were collected and 10 μM 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) working solution diluted with serum-free medium was added, mixed well and incubated at 37°C in the dark for 30 min. Then, the cells were washed three times with PBS and analyzed by flow cytometry.Mitochondrial membrane potential was detected using a MMP detection Kit . Briefly, after different treatments, cells were harvested and 1 mL JC-1 solution was added and incubated at 37°C for 20 min. After the incubation, cells were washed three times with PBS, resuspended, and analyzed by flow cytometry.Apoptosis was measured using Annexin V-FITC Apoptosis Detection Kit . Briefly, after different treatments, cells were harvested, binding buffer (300 μL) diluted with PBS was added, and then 5 μL of PI and 5 μL Annexin V-FITC were added and incubated in the dark for 15 min, followed by measurement using flow cytometry: FITC excitation 494 nm, emission 520 nm; PI excitation 493 nm, emission 636 nm.Western blotting assay was used to analyze changes of cell signaling. Briefly, at the end of incubation, the cells were harvested and Protein Extraction Kit  was used to extract total cell proteins by following the manufacturer's instructions. BCA Protein Concentration Determination Kit  was used to determine the total protein concentration. Protein samples were then separated on 12% SDS-PAGE and transferred to PVDF membranes . β-actin served as an internal control. After blocking with 5% skimmed milk at room temperature for 1 h, the blocking solution was removed, and the membranes were washed with TBST buffer. PVDF membranes were then incubated with primary antibodies overnight at 4°C, and then washed three times with TBST buffer, and further incubated with secondary antibodies. The freshly prepared ECL mixed solution (A : B = 1 : 1) was used for membrane exposure in a dark room. The optical densities of the target bands were analyzed using the commercial AlphaEaseFC software.P-value < 0.05 was considered statistically significant.All the data were analyzed using SPSS 25.0 software and are expressed as mean ± standard (SD). The differences between treatments were assessed by one-way ANOVA. R), MS, and MS/MS fragmentation ions are shown in R=0.85) exhibited quasi-molecular ion peaks [M+H]+ at m/z 144.1019 in the MS spectra, using Agilent qualitative analysis software to collect the molecular information of the component ions. The molecular formula of this component was C7H13NO2. Meanwhile, different levels of collision energy were used to obtain appropriate MS/MS information, and the characteristic fragment ions were at m/z 84.08133 (C5H9N) and m/z 98.09681 (C6H11N). Through the secondary mass spectrum, using the Thermo mzCloud online database, the compound was preliminarily identified as DL-Stachydrine. Another substance was identified in the same way; the molecular formula was C26H35O8, ion m/z 471.2009 [M+H]+, and the characteristic fragment ions were at m/z 425.1951 (C25H33O6) and m/z 95.0496 (C5H7O2); this compound was tentatively identified as limonin. The TIC ion chromatogram showed that the response value of limonin was very high.The ethanol extract from the pomelo seed was identified using UPLC-MSMS. The TIC chromatograms of the various compounds of ethanol extract from the pomelo seed was shown in R) in Limonin was principally recovered by isolation and purification. In this study, limonin was obtained with a purity of 99.13% after recrystallization and pre-HPLC in 25−35, the shape of the cells changed significantly, most of the cell protrusions were broken and the edges blurred. While pre-treatment of limonin significantly alleviated the injuries caused by Aβ25−35 and maintained normal cell morphology.The morphological changes of PC12 cells are shown in 25−35 for 48 h resulted in a significant inhibition of cell viability and a significant increase in the release of LDH (P < 0.0001). Pre-treatment of cells with limonin reduced Aβ25−35-mediated cell injury and the release of LDH (p < 0.0001). In the Aβ25−35 + limonin group at doses of 12.5, 25, 50 μg/mL, the release of LDH declined from 192.59% to 85.19%. The results showed that as the concentration of limonin increased so did the protective effect on PC12 cells. The highest protection was observed when 50 μg/mL of limonin was utilized (p < 0.0001).As shown in 25−35, the content of [Ca2+]i increased significantly from 3.26 to 40.37%, indicating that the Ca2+ in the cells were overloaded. The pre-treatment of limonin induced a statistically significant decrease (p < 0.0001) of intracellular Ca2+ concentration when compared to the Aβ25−35 group. In the Aβ25−35 + limonin group at doses of 12.5, 25, 50 μg/mL, the concentration of Ca2+ declined from 25.3% to 6.55%, indicating that limonin could antagonize the release of Ca2+ in Aβ25−35-treated PC12 cells. Additionally, there was a dose-dependent relationship between the decline in the concentration of intracellular Ca2+ and the concentration of limonin utilized for the treatment of cells (p < 0.0001).As shown in p < 0.0001) in PC12 cells that were exposed to Aβ25−35, compared to the control group cells. Pre-treatment with limonin for 24 h prior to the addition of 20 μM of Aβ25−35 to the cells resulted in decreased content of ROS (p < 0.0001). In the Aβ25−35 + limonin group at a dose of 12.5 to 50 μg/mL, the fluorescence intensity of ROS decreased from 2263.67 to 780.00 with increasing concentration of limonin, indicating that limonin can significantly reduce ROS level in Aβ25−35-treated PC12 cells. When the concentration of limonin was at 50 μg/mL, compared to the Aβ25−35 group, the fluorescence intensity of ROS showed a significant decrease (p < 0.0001).25−35 (P < 0.0001), which was confirmed by the increase of red JC-1 staining in the cells of the Q3 area. The pre-treatment of limonin significantly reversed the decrease of MMP caused by Aβ25−35 treatment (P < 0.0001). In the Aβ25−35 + limonin group at low, medium and high doses, the change of MMP was dose-dependent with the concentration of limonin.The change of MMP in PC12 cells was measured by the JC-1 staining assay in 25−35 induced death of PC12 cells is via an apoptotic-like mechanism, Annexin V-FITC/PI double staining method combined with flow cytometry technology was used to analyze the apoptosis of cells. As shown in 25−35 significantly increased the early and late apoptosis of PC12 cells, and the total apoptotic rate reached 42.5% (P < 0.0001). After pre-protecting PC12 cells with different concentrations of limonin , the total apoptotic rate of PC12 cells decreased to 31.7, 20.63, and 9.66%, respectively, compared with the Aβ25−35 group, indicating that limonin could significantly inhibit Aβ25−3-induced apoptosis of PC12 cells in a concentration-dependent manner.To elucidate whether Aβ25−35 for 48 h showed a significant decrease in the expression of Bcl-2, a significant increase in the expression of Bax and an increase in the ratio of Bax/Bcl-2 (P < 0.0001). Meanwhile, cleaved caspase-3 was also up-regulated (P < 0.0001), indicating that PC12 cells were undergoing apoptosis. In the limonin pre-treatment group, the expression of Bax was down-regulated, Bcl-2 was up-regulated, and cleaved caspase-3 was down-regulated, indicating the antagonistic effect of limonin on Aβ25−35-mediated cell growth inhibition. As compared to the LY294002 inhibitor group, limonin was found to antagonize the inhibitor's regulation of apoptotic proteins, thereby preventing the occurrence of apoptosis programs.Bcl-2 and Caspase family proteins are related to cell apoptosis. In the current study, Western blot was used to determine Bax, Bcl-2 and Caspase-3 proteins under different treatment conditions. As shown in 25−35-induced death of PC12 cells, the changes in PI3K/Akt activation were evaluated. As shown in 25−35 -treated PC12 cells were down-regulated and p-Akt/Akt ratio was significantly decreased (P < 0.0001). On the contrary, the expression level of p-Akt protein and the ratio of p-Akt/Akt in limonin treated PC12 cells were increased, indicating that limonin promoted the activation of Akt to form p-Akt, which then stimulated the biological activity of the PI3K/Akt signaling pathway in PC12 cells. LY294002 is a specific inhibitor of this pathway. LY294002 was added 1 h before limonin pre-treatment. It was found that the inhibitor LY294002 reversed the upregulation of p-Akt by limonin, indicating that limonin plays a role in activating the PI3K pathway.In order to explore the potential intracellular signaling mechanism of limonin antagonizing AβIn order to improve the utilization rate of pomelo seed, more high value-added products have to be found and applied. Firstly, the identification of chemical components of pomelo seeds extracts was investigated using UPLC-MS/MS. This study found that pomelo seeds are rich in active substances, such as limonoids, flavonoids, phenolic compounds, coumarin derivatives, among others. The main ingredient limonin was successfully obtained with a purity of 99.13% via using crystallization and preparative-HPLC technologies. This high purity sample was helpful to understand the action mechanism of limonin directly and ensure the correctness of results.in vitro experiment was conducted to explore the role of limonin in antagonizing the neurotoxicity of PC12 cells mediated by Aβ25−35. The experimental data showed that Aβ25−35 significantly inhibited cell viability and increased lactate release. LDH represents the structural integrity of cell membrane to a certain extent , and the Guangdong Province Key Field R&D Program Project (2019B020212002). We thank Zhongkai University of Agricultural Engineering for providing experimental conditions.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.


This study applied peptidomics to investigate potential biomarkers for evaluating pork-meat freshness. The spoilage time points of pork meat stored at −2, 4, 10, and 25 °C were defined by evaluating meat freshness indicators . Peptide MVHMASKE was identified as a potential peptide marker via multivariate analysis. Pearson correlation revealed a negative correlation between intensity of MVHMASKE and total viable count/total volatile basic nitrogen. In addition, the correlation between peptide content and the change in pork-meat freshness was verified using real-life samples, and the content of MVHMASKE showed a significant decline during storage under 4 and 25 °C, correspondingly reflecting the change of pork meat from fresh to spoiled. This study provides favorable evidence to evaluate pork-meat freshness by monitoring the change of peptide MVHMASKE in content based on mass spectrometry-based peptidomics. Pork is a protein-rich food for humans and one of the most highly consumed meats in China . HoweverMytilus galloprovincialis spoilage by a nuclear magnetic resonance (NMR) metabolomics approach. Li et al. [Scophthalmus maximus) muscle during post-mortem storage, and the abundance changes of those proteins were significantly correlated with TVB-N, K value, and TVC.Conventional evaluations of meat freshness include sensory assessments, analysis of physicochemical parameters, and microbiological monitoring ,5. Sensoi et al.  used proSeveral studies have reported that meat spoilage is directly related to the activity of microorganisms and endogenous enzymes on the metabolism of meat proteins . TherefoCrassostrea (C.) gigas during anhydrous preservation. To our knowledge, information on changes in endogenous peptides during pork preservation and their potential as meat-freshness biomarkers is limited.The application of peptidomics to identify biomarkers for assessing meat quality has increased. Gallego et al.  reportedThus, in the present study, peptidomics was applied to provide an evaluation of pork-meat freshness under different storage conditions . UHPLC-LTQ-Orbitrap mass spectrometry combined with orthogonal partial least-squares discriminant analysis (OPLS-DA) was employed to investigate the types and content of endogenous peptides to screen out potential biomarkers that could indicate pork spoilage during storage. This study provides an alternative method with potential to improve the meat-freshness evaluation system, thereby improving meat safety during retail and storage.longissimus lumborum muscles , which were economically fed and slaughtered in a slaughtering workshop at 10–15 °C; 3 h after slaughter, carcasses were chilled at 4 °C, and bone out at 24 h) were sampled at Beijing Hualian Group, Nanjing, China. All samples were transported via insulated chilled boxes to the laboratory within 2 h and aseptically prepared to remove the fat and connective tissues. Subsequently, all longissimus lumborum muscles were cut into 5 × 7 × 3 cm chunks of a similar size (approximately 60 g) on a clean bench and randomly assigned into four groups representing storage conditions of −2, 4, 10, and 25 °C. They were placed onto a plastic tray , sprayed with 95% ethyl alcohol, and then wrapped with two layers of commercial polyethylene (PE) cling wrap . For the 25 °C group, five time points  and 45 pieces  were utilized for analysis. Consistent with the 25 °C group, six time points  and 54 pieces; six time points  and 54 pieces; and seven time points  and 63 pieces were used for the analyses of the −2, 4, and 10 °C groups, respectively. Four individual pieces were randomly selected for the measurements of basic indicators . Five individual pieces per time point were randomly selected for peptidomics analysis.A total of 40 longissimus lumborum muscles (18 from each market). All samples were transported via insulated chilled boxes to the laboratory within 2 h and aseptically prepared to remove the fat and connective tissues. Each muscle in each supermarket was cut into 3 pieces (the same treatment as mentioned above). For each temperature group (4 and 25 °C), 27 pieces (nine pieces per time point) collected at three time points  were utilized for analysis.A new batch of pork samples was sampled from three markets in Nanjing  with 54 L*), redness (a*), and yellowness (b*) of pork surfaces were measured with illuminant D 65, viewing angle 0°, and viewing area diameter 8 mm at three different locations of samples exposed to the air. All measurements were made in quadruplicate (n = 4).The method described by Hunt et al.  was adopn = 4), were expressed as log colony-forming units per gram (log CFU/g).The TVC was determined following the method by Ye et al. . Brieflyn = 4).pH was measured as described by Lan et al.  using a The TVB-N content in samples was determined as described by Bassey et al. . Brieflyg, 30 min, 4 °C), 300 μL of supernatant was diluted with 900 μL ACN. The mixture was incubated for a further 30 min at 4 °C, followed by centrifugation . The resulting supernatant was evaporated and re-dissolved in 0.2% FA containing 0.2% ACN and then centrifuged . Thereafter, 2 and 5 μL of each sample solution were used for peptide and quality control (QC) analyses, respectively.Peptide extraction was performed as described by Azkargorta et al. , with miv/v) FA as follows: 0–5 min, 3–3% B; 5–45 min, 3–35% B; 45–46 min, 35–95% B; 46–56 min, 95–95% B; 56–57 min, 95–3% B; and 57–63 min, 3–3% B. The flow rate was 0.2 mL/min. The temperature of the C18 column (250 mm × 3 mm × 5 μm) was maintained at 35 °C. The column outlet was directly coupled to electron spray ionization (ESI) with an ionization voltage set at 3 kV for the positive scan. Nitrogen was used as the sheath (35 arb) and aux (10 arb) gas. The scanning range of the primary mass spectrum was 200–1200 m/z, and the resolution was 60,000. A data-dependent acquisition (DDA) scan was conducted for collision-induced dissociation (CID) model. The 10 ions with the highest intensity were selected for secondary fragmentation analysis with a collision energy of 35 eV.A UHPLC-LTQ-Orbitrap mass spectrometer  was used to determine the untargeted peptide profiles, as described by Miao et al. and Stefan et al. ,27, withSus scrofa), and filter charge (1–3). The peak area of each identified peptide was integrated using the PEAKS Q module and subjected to logarithmic (log) conversion. The normalized peak area of each peptide after log2 conversion was imported into SIMCA-P 14.0 software  for multivariate analysis. To identify potential markers, variable importance projection parameter value > 1, fold-change > 1.3 or <0.77, and p < 0.05 [The acquired raw data were imported into PEAKS software  for peptide identification and quantification. The parameters were as follows: oxidation of methionine as variable modification, parent mass error tolerance (15.0 ppm), fragment mass error tolerance (0.6 Da), database  at 35 °C. The mobile phases consisted of acetonitrile (A) and water containing 0.1% FA (B), and the elution gradient was set as follows: 3% A (0–0.5 min), 3–35% A (0.5–8.5 min), 35–95% A (8.5–9 min), 95% A (9–14 min), 95–3% A (14–14.1 min), and 3% A (14.1–20 min). The flow rate was fixed at 0.2 mL/min. MS analysis was performed using an LTQ-Orbitrap XL mass spectrometer  equipped with a heated electrospray ionization interface. The ionization voltage was set at 3 kV for the positive scan. The capillary temperature was fixed at 300 °C. Nitrogen served as both the sheath (35 arb) and auxiliary (10 arb) gas. From 0 to 20 min, the results of PEAKS software were imported into Skyline 3.0 software  to select the parent-fragment ion pairs of the potential peptide. The precursor ion was selected using CID model with normalized collision energy of 35%.A total of 5 μL of each sample treated as detailed above was injected. LC analysis was performed using a UHPLC system  equipped with an auto-sampler, a vacuum degasser unit, a quaternary pump, and a column compartment. Samples were separated using a Geminip < 0.05. The results of color, microbiological analysis, pH, and TVB-N are expressed as the mean and standard deviation of four replicates. The peptide MVHMASKE content was analyzed by two-way analysis of variance (ANOVA) with Bonferroni post hoc test, and the results are expressed as the mean and standard deviation of nine replicates.The results of color, microbiological analysis, pH, and TVB-N were analyzed by one-way analysis of variance (ANOVA) using SPSS for Windows version 20 . Duncan’s multiple range test was used to compare significant differences at Peptide sequencing and quantification were performed by PEAKS software . Principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA) were analyzed with SIMCA-P 14.0 software  for overall variable analysis. Pearson correlation coefficients were used to assess the relationship between intensity of peptide MVHMASKE and changes in basic meat-freshness indicators. Results for the selected biomarkers were output and analyzed by Skyline 3.0 software .L*), redness (a*) and yellowness (b*) were markedly affected by storage temperature and sampling time , the a* values slightly increased at the beginning of storage and then declined over time, while b* values trended upwards throughout storage period. Compared with low-temperature (−2 and 4 °C) storage samples, the a* values decreased rapidly after 24 and 5 h in samples stored at 10 and 25 °C, respectively  in b* values were observed from 20 d, 9 d, 48 h, and 10 h in the −2, 4, 10, and 25 °C storage groups, respectively. An increase in b* content is associated with yellow pigment derived from the reaction between lipids and the amines in protein amines or phospholipid head groups [A bright red color was used to denote meat freshness. Except for lightness  in each group during the storage period (p < 0.05), with the results ranging from 5.70 to 6.39 and from 5.71 to 6.00, respectively. Conversely, the values obtained for the −2 and 4 °C groups significantly increased (p < 0.05) after 10 days and 6 days, respectively. Similarly, an upward trend has been observed for rabbit meat stored at 4 °C [pH is associated with meat freshness, as it reflects the degree of protein degradation and accumulation of spoilage metabolites . A previe period E, the 10 at 4 °C . The sli at 4 °C  and lact at 4 °C . Convers at 4 °C .The TVB-N results showed a progressive trend in all groups, with substantially higher values under high-temperature (10 and 25 °C) groups compared with low-temperature −2 and 4 °C) groups F. Throug °C groupConsidering basic indicators, the period of accelerated spoilage ranges from 15 days to 25 days, 9 days to 15 days, 36 h to 72 h, and 10 h to 15 h in the −2, 4, 10, and 25 °C groups, respectively. The results obtained were consistent with the findings of Tang et al. , who repNon-targeted peptidomics was applied to assess changes of peptide profile during pork-storage period. To ensure high reliability and repeatability, the FDR value of the database search algorithm was set to 1%, and the variation coefficient of the peptide response area presented in the QC was <15%. Based on this criterion, 79, 215, 194, and 337 peptides were identified from the −2, 4, 10, and 25 °C groups, respectively . Herein,T2 test, and the aggregation degree of QC (red dots in t (1) axis, while there were obvious separations on day 0, day 9, and day 15 in the 4 °C storage group. In the 10 °C storage group, samples collected from different time points were significantly distinguished from each other. In contrast, samples collected at 0 h in the 25 °C storage group were separated from samples collected at other time points on the t (2) axis. However, the distinction between samples at certain time points remained ambiguous, suggesting the need for further data analysis.SIMCA-P software was used to perform multivariate analyses on the peptide intensity for different pork states. The PCA score plots A indicatR2X (cum), R2Y (cum), and Q2 (cum) revealed satisfactory fitness and predictability of the model. To evaluate the efficacy of the OPLS-DA model, 200 permutation tests were performed  fresh to accelerated spoiled (SS1) and (2) accelerated spoiled to spoiled (SS2) states . Among tA Venn diagram was constructed to identify the peptides presented in each group . Three pC. gigas during anhydrous storage at 4 °C. However, the relationship between peptide content and shelf-life of C. gigas was not determined. To ascertain the relationship between peptide MVHMASKE and meat freshness, Pearson correlation ; the two most intense fragment ions: 351.81 (y62+) and 565.30 (y5+))  . To perf0 (y5+)) . Then, t2 = 0.9993) between the concentration of standard peptide (0.175 to 35 μg/mL) and the peak area of peptide MVHMASKE (According to the results of the representative extracted ion chromatogram of peptide MVHMASKE from pork meat and the synthesized peptide, the peak shape was sharp with little interference and coincided well. The retention time for peptide MVHMASKE from pork meat was consistent to that of the synthesized peptide, being within ±2% , illustrMVHMASKE A, which MVHMASKE  was obseIn this study, basic freshness indicators were monitored to evaluate the pork-meat freshness under different storage temperatures. Our results revealed variations in the spoilage processes of pork meat stored at different temperatures. Hence, by integrating all parameters, time points of fresh, accelerated spoiled, and spoiled states of the samples under different conditions were ascertained. A peptide biomarker MVHMASKE was screened out via multivariate analyses, and the PRM coupled with the external standard quantitative method was established to analyze changes in peptide content using real-life samples. The potential of peptide MVHMASKE was verified; however, the thresholds of this peptide to well define the freshness change of pork meat should be established with a larger size of testing samples and more accurate quantitative analysis in further study. In addition, as the first step, only one peptide was considered as common potential indicator of meat spoilage under all four storage temperatures. To elucidate this matter further, subsequent work needs to be done as a combined strategy to explore more peptides as potential biomarkers at each specific temperature. Overall, this peptidomics-based method for mining biomarkers is a prospective alternative to evaluate pork meat freshness, and the result of this study provides the new theoretical basis for the standardized system in assessing pork quality.


These nanoparticles were characterized with the aid of different methods, including UV, X-ray diffraction (XRD), Fourier transformed infrared spectroscopy (FTIR), transmitted electron microscopy (TEM), and photocatalytic degradation. Y2O3 nanoparticles showed excellent antibacterial activity against Gram-positive Bacillus subtilis and Gram-negative Escherichia coli with a 10 to 15 mm inhibitory zone. Green Y2O3 NPs were released with a 4 h lag time and 80% sustained release rate, indicating that they could be used in drug delivery. In addition, the bioavailability of green Y2O3 NPs was investigated using cell viability in cervical cancer cell lines. These green-synthesized Y2O3 NPs demonstrated photocatalytic degradation, antibacterial, and anticancer properties.Due to their appropriate physicochemical properties, nanoparticles are used in nanomedicine to develop drug delivery systems for anticancer therapy. In biomedical applications, metal oxide nanoparticles are used as powerful and flexible multipurpose agents. This work described a green synthesis of Y Cervical cancer is the 4th majorly diagnosed cancer in women worldwide, with approximately 604,000 new cases and 342,000 global mortalities in 2020 ,2,3,4. AThe most critical components in nanotechnology research are metal oxide nanomaterials. They are significant because of their catalytic, magnetic, electrical, and mechanical properties. Oxidative nanostructures are favorable in a wide range of thermal, photonic, mechanical, power, electrostatic, physicochemical, and gas-sensitive applications due to their enormous surface-to-volume ratios ,16,17. TGreen synthesis is considered the most economical, sustainable, efficient, and reliable approach to nanoparticle synthesis ,26. ThisLantana camara L., commonly called “Unnichedi” (Tamil) and “pulikampa” (Telugu), is a species of flowering plant of the Verbenaceae family that is widely recognized as a significant weed in India. Lantana camara (LC) is an important plant containing alkaloids, terpenoids, phenolics, flavonoids, and other phytochemicals. L. camara is often used to treat various diseases in traditional medicine, including paludism, cancer, hypertension, tetanus, neoplasm, dermatitis, cut-offs, catarrh, abdominal viscera, measles, chickenpox, bronchitis, and fever. L. camara also has antifungal, anti-proliferative, antibacterial, nematicidal, germicidal, anti-ulcerogenic [erogenic ,33, antierogenic , immunoserogenic , anthelmerogenic , anti-inerogenic .2O3), a widespread rare earth metal, is significant for future use due to its thermal stability and chemical and mechanical reliability. Yttrium oxide is used in biomedical images, materials science, synthesis of inorganic compounds, optics, electricity, biology applications [2O3 nanoparticles of different sizes and morphologies were developed through various methods, such as sonochemical, solvothermal, hydrothermal, electrochemical, sol-gel, and thermal decomposition [Yttrium oxide , fetal bovine serum (FBS), and MacConkey agar medium.The following chemicals and reagents were purchased from commercial sources and used as received without further purification: Yttrium (III) acetate hydrate 99.0%, oleylamine of the technical grade 70%, ammonia hydroxide of reagent grade 30%, yttrium (III) nitrate of the hexahydrate 99.8%, anhydrous of the reagent grade of the ammonium hydroxide 99.5%, chloral hydrate 99.8%, Lantana camara L. fresh leaves were collected and washed with tap water and then washed with double distilled water until no impurities remained. The L. camara leaves were dried in the shade for ten days to remove residual moisture. The dried leaves were pulverized in a sterile electric blender to obtain a fine powder and stored in an airtight bottle avoiding sunlight for further use. Then, 10 g of leaf powder was mixed thoroughly with 200 mL of double distilled water and heated for 10 min at 60 °C. This was followed by cooling and filtration through Whatman No.1 filter paper to afford the leaf extract [ extract ,56. The 2O3 were synthesized in the presence of gelatin through precipitation with ammonium hydroxide. In this method, 75 mL of 0.1 M yttrium nitrate hexahydrate (Y(NO3)3·6H2O) aqueous solution was added to 100 mL of ammonium hydroxide, and the solutions were mixed and stirred by REMI Electromagnetic stirrer . The precipitate formed was kept for 4 h at room temperature, washed several times with de-ionized water, centrifuged at 8000 rpm, and finally resuspended in ethanol. The obtained slurry was dried in a hot air oven for 24 h at 70 °C and pulverized with mortar and pestle. The powder was calcined to crystal-clear Y2O3 nanocrystals at 650 °C for four h [2O3 nanoparticles on a Bruker D8 automated multipurpose powder X-Ray Diffractometer with Cu-K radiation of wavelength 1.5406 nm. The Infrared Spectra of the LC Y2O3 NPs were obtained using an FTIR-spectrophotometer . In contrast, a field emission gun was used for TEM analysis at a 200 kV accelerating voltage in a FETEM apparatus  [Nanoparticles of Yr four h ,57. The , Japan) .2O3 NPs. The photodegradation was examined by a 250W Xenon lamp with high pressure, with the liquid approximately 10 cm from the Xenon bulb illumination. In a traditional photodegradation procedure, 20 mg of LC Y2O3 NPs were added to a 20 mL aqueous solution of 10 µM RhB (10 ppm). To obtain dye adsorption-desorption equilibrium on the catalyst, the solution was stirred for 120 min in the dark before being subjected to visible light for different time intervals. Approximately 4mL of the solution was taken every 30 min throughout photocatalytic degradation and centrifuged at 750 rpm for 5 min to remove the excess catalytic nanoparticles. Subsequently, the amount of RhB was instantly observed using a UV-Vis spectrophotometer  to determine the absorption in the spectral region of 500–750 nm [Ci represents the initial concentration of RhB, and Cf represents the final concentration of the dye after a specified reaction time (min).The photo-degradation of Rhodamine B (RhB) revealed the photoelectrocatalytic activity of the LC Y0–750 nm . Under t0–750 nm . The fol2O3 NPs was examined using microbes such as E. coli (MTCC 732) and Bacillus subtilis (MTCC 5981). The bacterial strain was acquired from the MTCC  at IMTECH in Chandigarh, India. The microbiological suspension culture was maintained for 24 h in nutrient broth; the prepared MacConkey agar medium was heated at 120 °C for 15 min, and 20 mL of sterile agar medium was poured into Petri plates. The bacterial culture was spread uniformly over agar plates using a cotton swab stick. Following inoculation, various concentrations  of LC Y2O3 NPs dissolved in DMSO and sonicated for 15 min were placed onto the sterile discs (6 mm). The microbes were then inoculated into discs or plates and kept at 37 °C for 24 h for bacterial species with a triplicate test performed for each microbe. The size of the inhibition zone formed on each disc was determined (mm).The agar diffusion method was used in the microbiological experiment . The ant5 cells/well) and incubated in a 5% CO2 incubator at 37 °C. After the cells were placed in wells and kept in the incubator for 24 h, nanoparticles were added at concentrations of 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 µg/mL. The samples were then removed from the well and washed with DMEM. 3--2,5-diphenyl tetrazolium-bromide (0.5% MTT reagent) was added (10 μL/well) to each well and incubated for 4 h. The spectrophotometer measured the optical density at 540 nm  [Human HeLa cell lines were obtained from the National Centre for Cell Science (NCCS) in Pune, India. DMEM was used to maintain the added cells with 10% FBS. In the median, penicillin (100 μg/mL) and streptomycin (100 μg/mL) were added to avoid bacterial contamination. The HeLa cells were positioned in 24 wells  . A nanopCA, USA) . The foltA = absorption of test compound, bA = absorption of blank, cA = absorption of control.2O3 NPs, and LC Y2O3 NPs dispersion (≈10 mg of NPs) . As recommended to test the dissolution of vagina products, the dialysis tube was placed in 15 mL SVF (pH < 4.2) and kept at 37 °C and 100 rpm. Then, 1 mL sample was removed from the medium at specific time intervals and replaced with a fresh medium  to provide the sample’s persistence. A UV-Visible spectrometer was used to measure the concentration in each sample at 298 nm .The dialysis technology examined drug release rate from synthesized samples . In brie2O3 NPs was studied using UV-Visible spectroscopy. As demonstrated in 2O3-loaded NPs exhibited broad UV absorption with enhanced intensity at 288 nm. Similar results were reported for rare earth oxides [L. camara leaf extract and green synthesized LC Y2O3 NPs were investigated using FTIR. The infrared spectrum of L. camara leaf extract illustrates vibrational bands at 3444, 2963, 1647, and 675 cm−1. The absorption peaks of L. camara leaf extract at 3444 and 2963 cm−1 were attributed to O–H stretching and C–H antisym and sym stretching vibrations, respectively. The absorption peaks of L. camara leaf extract at 1647 and 675 cm−1 bands may be assigned to C=O in ketone esters and C–C–CHO bending in aldehydes, respectively. The absorption band at 1050 cm−1 was obtained by fabricating the green synthesized Y2O3 NPs [−1 is attributed to the C–H stretching of H–bonded alcohols and phenols [−1 band is assigned to the M–O region (Y–O), whereas the 617 cm−1 band confirms the YO NPs’ extending vibration [2O3 NPs revealed 3430 cm−1 is assigned to the NH stretch with primary amines and 2928 cm−1 to C–H antisym and sym stretching vibrations. On the other hand, absorption bands at 1748 cm−1 and 1638 cm−1 are attributed to the C=O in esters and C=O stretch secondary amides, respectively. FTIR spectrum of green synthesized LC Y2O3 NPs showed bands at 1402 cm−1 due to O–H in carboxylic acids, 1022 cm−1 to CH–OH in cyclic alcohols with C=O stretch vibrations. Furthermore, the FTIR peaks observed at 557 cm−1 were ascribed to Y–O band stretching (−1) indicates that the Yttria phase was formed at 600 °C [The effect of green synthesized LC Yh oxides  and orgah oxides , where sY2O3 NPs , whereas phenols ,67. Simiibration . FTIR reretching . The Y–Ot 600 °C .2O3 NPs. Indeed, diffraction peaks of LC Y2O3 NPs at 2θ values of 13.45°, 25.89°, 30.22°, 44.34°, and 52.72° were indexed as 202, 211, 222, 431, and 440 (JCPDS card no. 83-0927) [2O3 NPs could be found as polymorphs. For all samples, the XRD analysis confirms the formation of a single-phase orthorhombic Y2O3 structure, revealing the successful synthesis of Y2O3 NPs. According to the method of Hanawalt (222) and (431), the cubic Y2O3 stage was characterized by sharp peak diffraction angles of 30.22° and 44.34°, respectively [The 2θ values of the green synthesized XRD detected Y83-0927) . The phaectively .2O3 NPs display high crystallinity in the TEM images in 2O3 is very thin in the composite samples, which agrees with the lack of diffraction peaks related to the formation of Y2O3 particles in 2O3 NPs with irregular shapes. The Scherrer equation estimated the crystallite size of green synthesized LC Y2O3 NPs at 30 nm.Meanwhile, TEM images show these particles’ tendency to form agglomerates ranging from 20 nm to 45 nm. The Y2O3 NPs. In the absence of the catalyst, only 18% of the dye degraded after 60 min of exposure, indicating that the catalyst played a significant role in dye photocatalytic degradation [2O3 NPs as a photocatalyst was investigated using LC Y2O3 NPs for photocatalytic degradation of Rhodamine B. Based on these findings, 92% of the dye was degraded within 60 min, as depicted in 2O3 NPs were absorbed, electrons were excited to the conduction band, causing a vacancy in the valence band. The electrons in conduction produce (O2−•) free-radicals by transferring an electron to the oxygen molecule. In contrast, the vacancy in the valence band generates hydroxyl radicals (•OH), which react with water molecules [2O3 NPs.The photodegradation of dyes without a catalyst was evaluated by exposing the blank dye to illumination. For 120 min, absorbance measurements on blank dye were conducted at different time intervals. The degradation of Rhodamine B dye (RhB) was used to illustrate the photocatalytic efficiency of LC Yradation . A reducolecules . These p2O3 NPs produced in LC aqueous leaf extracts (50–200 µL) was studied against gram-positive and negative bacterial strains. The green synthesized LC Y2O3 NPs with typical antibacterial activity (the zone of inhibition) ranged from 10 to 15 mm for the selected bacterial strains. Bacillus was found to have the highest activity (15 mm inhibition). The diameter of the zone of inhibition was 15 and 11 for Bacillus and E. coli (11mm), respectively  of NPs were used to treat the HeLa cells at 24 h. The results showed time- and dose-dependent declines in cell viability decreased by 53 to 98% in the 24 h treatments. These findings suggest that cell viability decreases when the concentration of NPs increases. The green synthesized LC Y2O3 NPs IC50 value against HeLa cell lines was determined after 24 h and was found to be 25 µg/mL.YNanoparticles could be used as anticancer drugs in the treatment. They can penetrate cancer cells for targeted therapy by a smaller number of NPs. Large vascular pores deliver oxygen and nutrients to cancer sites and inflammatory tissues in these locations, allowing NPs to pass through and accumulate ,74. The 2O3 NPs from L. camara aqueous leaf extract with high surface reactivity and biocompatibility. These nanoparticles were characterized using various techniques, such as X-ray diffraction and FTIR, which revealed that they were pure single-phase and crystalline LC Y2O3 NPs with orthorhombic shapes. TEM analysis of green synthetic LC Y2O3 powder confirmed the production of nanoparticles with an average size of 30 nm. The zone of inhibition of LC Y2O3 NPs was found to be Bacillus (15 nm) and E. coli (11 mm). The results of this study revealed that the green synthesized LC Y2O3 NPs are effective at degrading dyes from water and preventing bacterial growth. Thus, these NPs possess potential in water filtration, coatings, and the food-manufacturing sector. The cytotoxicity effects of LC Y2O3 NPs nanoparticles against HeLa cell lines were dose-dependent and can be used to treat cervical cancer in humans. Due to their strong surface reactivity, these NPs could be employed in cancer therapy to deliver targeted drug loading. These characteristics make the green synthesized Y2O3 NPs an appropriate option for use in medicinal applications. Furthermore, detailed in vivo studies are needed to determine the safety and effectiveness of these green synthesized LC Y2O3 nanoparticles.In conclusion, we synthesized Y


AA-HNA and successfully screened a focused library containing 320 natural organic compounds. Furthermore, we applied activity-based protein profiling (ABPP) as an orthogonal method to confirm the inhibitory activity against MAGL in the primary substrate-based screening. Our investigations culminated in the identification of two major compound classes, including quinoid diterpene  and β-carbolines , with significant potency towards MAGL and good selectivity over other 2-AG hydrolases (ABHD6 and ABHD12). Moreover, these compounds also showed antiproliferative activities against multiple cancer cells, including A431, H1975, B16-F10, OVCAR-3, and A549. Remarkably, 23 achieved complete inhibition towards endogenous MAGL in most cancer cells determined by ABPP. Our results demonstrate the potential utility of the substrate-based fluorescence assay in combination with ABPP for rapidly discovering MAGL inhibitors, as well as providing an effective approach to identify potential targets for compounds with significant biological activities.The endocannabinoid 2-arachidonoylglycerol (2-AG) is predominantly metabolized by monoacylglycerol lipase (MAGL) in the brain. Selective inhibitors of MAGL provide valuable insights into the role of 2-AG in a variety of (patho)physiological processes and are potential therapeutics for the treatment of diseases such as neurodegenerative disease and inflammation, pain, as well as cancer. Despite a number of MAGL inhibitors been reported, inhibitors with new chemotypes are still required. Here, we developed a substrate-based fluorescence assay by using a new fluorogenic probe  In gene ABHD12) . As ECS  ABHD12) . However ABHD12) .Among them, MAGL is a membrane-associated soluble enzyme, which hydrolyses the majority of brain 2-AG (∼85%) into arachidonic acid (AA) and glycerol, while other 2-AG hydrolases such as ABHD6 and ABHD12 contribute to less than 20% of 2-AG hydrolysis . StudiesTo date, a variety of MAGL inhibitors were developed, such as carbamates, ureas, maleimides, disulfides, and so on . Among tAA-HNA) by introducing AA as a specific recognition moiety for MAGL into the scaffold of a fluorescent group 6-hydroxy-2-naphthaldehyde (HNA)  and screened the library with the optimal assay condition to identify MAGL inhibitors with new chemotypes.To identify new lead compounds acting as MAGL inhibitors, high-throughput screening is one of the general strategies, and a rapid, effective, as well as the low-cost assay, is crucial for screening a compound library. A common screening method often requires an accurate biochemical readout of enzyme activity and robust assay reproducibility. Therefore, we set out to develop a reliable method to screen and characterize MAGL inhibitors. As fluorescent probes have currently been used for drug candidate screening with a couple of advantages such as easy operation, highly sensitive, and real-time detection , we set de (HNA) . As AA-HDuring the process of MAGL inhibitor discovery, a comprehensive selectivity profile is necessary, particularly the selectivity over enzymes linked in 2-AG and AEA hydrolysis . In addition, as MAGL belongs to serine hydrolases, a superfamily with more than 200 enzymes that use serine as the common active site, a family-wide selectivity profile of MAGL inhibitors over other serine hydrolases is important. Recently, activity-based protein profiling (ABPP) is a highly useful chemical biological technique to assess the activity and selectivity of serine hydrolase inhibitors in a complex native system . Yet, to1H) and 101 (13C) MHz using CDCl3 as solvent. High‐resolution mass spectra (HRMS) were recorded on a Thermo Scientific LTQ Orbitrap XL. HPLC purification was performed on a preparative LC-MS system with an Agilent 6,110 or 6,120 mass spectrometer detector . Graphs and statistics were performed with GraphPad Prism 6, and Bio-Rad Image-lab was used for gel analysis and quantification.The common reagents were purchased from commercial sources. KML29 was purchased from Selleckchem with ≥98% purity. DO264 and KT182 were from MedChemExpress with ≥98% purity. All buffers, as well as solutions, were prepared using analytical grade reagents or solvents, as well as Millipore water . Buffers are prepared at room temperature and stored at 4°C, unless stated otherwise. The antibodies presented in this study, including ABHD6 (#97573), MAGL (#abs77398), and ABHD12 (#abs180944) were purchased from Cell Signaling Technology and Absin, respectively. Electrophoresis reagents were from Bio-Rad Laboratories. Cell reagents and culture plates were purchased from Celbio and BIOFIL, respectively. All fluorescence analyses were conducted on the BioTek microplate reader and Horiba Jobin Yvon-Edison Fluoromax-4. NMR spectra were recorded on a Bruker AV spectrometer at 400 (2Cl2 (10 ml) was added HNA  in CH2Cl2, followed by the dropwise addition of dicyclohexylcarbodiimide  in 10 ml CH2Cl2. The reaction mixture was stirred for 12 h at room temperature. When the reaction was completed and the mixture was diluted with water, and then extracted with ethyl acetate. The combined organic layer was washed with water, and brine, dried over MgSO4, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel to afford AA-HNA . HRMS [ESI+] m/z: calculated for C31H38O3 [M + H]+ 459.2899, found: 459.3555.1H-NMR : δ 10.13 , 8.31 , 8.00 , 7.95 , 7.87 , 7.62 , 7.33 , 5.51–5.30 , 2.88–2.79 , 2.65 , 2.25 , 2.05 , 1.89 , 1.39–1.26 , 0.88 . 13C-NMR  δ 192.37, 172.32, 151.30, 137.49, 134.54, 134.39, 131.47, 130.95, 130.92, 129.70, 129.19, 129.07, 129.04, 128.76, 128.48, 128.22, 127.92, 123.97, 122.94, 119.32, 34.16, 31.94, 29.75, 27.65, 26.93, 26.10, 26.09, 26.07, 25.11, 23.01, and 14.53.The starting material of 6-hydroxy-2-naphthaldehyde (HNA) and arachidonic acid (AA) were commercially available. The fluorogenic substrate AA-HNA was prepared as follows: to the solution of arachidonic acid  and DMAP  in CH2. The medium was refreshed every 2–3 days and the cells were passaged at ∼90% confluence by adding the fresh medium, followed by vigorously pipetting the resuspend cells. Of note, the cells were only washed with PBS when a significant number of dead cells were observed.Various cancer cell lines, including human colon cancer cell line HT-29, ovarian cell line OVCAR-3, melanoma cell line B16-F10, skin squamous cancer cells A431, cervical cancer cells Hela, lung cancer cell lines A549, H1975, and HCC827, were purchased from American type culture collection (ATCC). A431, H1975, B16-F10, OVCAR-3, HT-29, HCC827, Hela, and A549 cells were cultured in Dulbecco’s modified Eagle medium  containing 10% fetal bovine serum , penicillin (100 U/ml) and streptomycin (100 μg/ml). All cell lines were maintained in a humidified 37°C incubator with 5% CO2 in a DMEM medium with stable glutamine and phenolred, containing 10% fetal bovine serum , penicillin, and streptomycin. The cells were passaged every 2–3 days by resuspension in the medium at ∼90% confluence. The membranes used in this study were prepared from transiently transfected HEK293T cells. Briefly, 1 day prior to transfection, ∼107 cells were seeded in 15 cm plates, and then the cells were transfected by the addition of a mixture of polyethyleneimine and plasmid DNA  in 2 ml of medium (without serum). Of note, the empty pcDNA3.1 vector was used in the Mock-control. After 24 h, the medium was removed and the cells were refreshed with fresh medium. After 72 h, the cells were harvested by suspending in 20 ml of medium, and the supernatant was removed by centrifugation for 10 min at 1,000 rpm. The cell pellet was flash frozen in liquid nitrogen and stored at −80°C until use. Subsequently, the cell pellets were suspended in lysis buffer , which was homogenized by sonication (3 × 10 bursts) and incubated for 30 min on ice. The membrane fractions overexpressing MAGL/ABHD6/ABHD12 were separated by ultracentrifuge . The protein concentration was determined with a Qubit protein assay (Invitrogen). The protein samples were quickly frozen in liquid nitrogen and stored at −80 C until use.HEK293T cells were cultured at 37°C under 5% COThe MAGL activity assay is based on the hydrolysis of 2-arachidonoylglycerol-based fluorogenic substrate by MAGL-overexpressing membrane preparations from transiently transfected HEK293T cells. In brief, the reactions were performed in HEPES buffer  in black, flat bottom 96-well plates. The final protein concentration of membrane fractions from overexpressing human MAGL HEK293T cells was 12.5 μg/ml 5 µl of inhibitors were added from×40 concentrated DMSO stocks in the 96-well plate containing 145 µl assay buffer, followed by the addition of 40 µl of the protein (12.5 μg/ml). After the incubation of the inhibitor-protein complex for 30 min, 10 µl of the fluorogenic substrate  was added and fluorescence was measured in 1 min intervals for 30 min on a plate reader (BioTek). Final assay concentrations: 12.5 μg/ml MAGL, 200 μM AA-HNA, and 5% DMSO in the total volume of 200 µl. All the measurements were performed in N = 2, n = 2 for inhibitors and N = 2, n = 4 for controls with Z′ value ≥0.6. The slope is determined in 5–15 min for all the experiments.The ABHD6 activity assay is based on the same principle as the MAGL activity assay described earlier, but with human ABHD6 overexpressing membrane preparations at a final protein concentration of 50 μg/ml and 200 µM AA-HNA. Other conditions are the same as those of the MAGL activity assay.The ABHD12 activity assay is based on competitive activity-based protein profiling (ABPP) with human ABHD12 overexpressing membrane fractions. Briefly, the membrane proteome  was incubated at room temperature with 0.5 µl DMSO (vehicle) or inhibitor for 30 min. Subsequently, the samples were treated with the activity-based probe FP-TAMRA  for 20 min. The reactions were then quenched by 10 µl sample buffer (×4)  SDS, 10% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 0.01% (v/v) bromophenol blue). The samples were directly loaded and resolved on SDS-PAGE gel (10% acrylamide), which were then scanned using a ChemiDoc MP system .2, 25 U/mL benzonase) and incubated on ice for 15 min, followed by low-speed spin  to remove debris. The supernatant was subjected to ultracentrifugation  to generate the cytosolic fraction in the supernatant, as well as the membrane fraction as a pellet. After removal of the soluble supernatant, the pellet was resuspended in storage buffer  by pipetting. Total protein concentrations in membrane and soluble fractions were determined using a Qubit protein assay (Invitrogen). The obtained samples were stored at −80°C until further use.Mouse brains were isolated according to guidelines approved by the ethical committee of Western China Hospital, Sichuan University (No. 2021765A). Mouse brains were dounce homogenized lysis buffer  was preincubated for 30 min with 0.5 µl of DMSO (vehicle) or inhibitor . Subsequently, the proteome-inhibitor complex was treated with the activity-based probe FP-TAMRA  for 20 min. The reactions were then quenched by 10 µl sample buffer (×4). The obtained samples were directly loaded and resolved on SDS-PAGE gel (10% acrylamide). The gels were then scanned and analyzed using a ChemiDoc MP system . All the samples were measured and duplicated.2O2 by ChemiDoc MP (BioRad).Western blot experiments were performed to investigate the expression levels of MAGL, ABHD6, and ABHD12 in various cancer cells according to the previously reported protocols. In brief, cancer cells were washed with phosphate-buffered saline (PBS), harvested by using a cell scraper, and collected by centrifugation. Cell pellets were washed with PBS and lysed with RIPA buffer  on ice for 15 min. After sonication, the protein concentration was determined by Qubit protein assay (Invitrogen) and normalized to 2 mg/ml. Subsequently, 20 µl of protein sample was loaded to each lane in the gel and resolved on an SDS-PAGE gel (10% acrylamide). After that, the protein samples were transferred from gel to a polyvinyl difluoride (PVDF) membrane using a Trans-Blot Turbo (BioRad). The PVDF membranes were then blocked with BSA blocking buffer for 2 h at room temperature, followed by incubation with different primary antibodies , including MAGL (#abs77398), ABHD6 (#97573), and ABHD12 (#abs180944). The membranes were washed with TBST buffer 3 times and then incubated with anti-rabbit IgG  for 1 h at room temperature. In the end, the blots were washed in PBST and immunoreactive proteins were detected using a luminal solution with ECL enhancer and H50 values were determined by plotting a log(inhibitor) v. s normalized response (Variable slope) dose–response curve generated using GraphPad Prism software.The antiproliferation activity of the compounds against the cancer cell lines was evaluated using Cell Counting Kit-8. Briefly, cells  in 100 µl of culture medium were plated in a 96-well plate for 24 h and 100 µl of medium with different concentrations of the tested compounds was added to each well for 48 h. Then 10 μl of CCK8 solution  was added and incubated at 37°C for 2 h. The absorbance of each well was measured with a plate reader (BioTeck) at 450 nm. Cell viability was calculated as follows: cell viability (%) = [(As-Ab)/(Ac-Ab)] × 100. Among them, As represents the absorbance of experimental wells, Ab represents the absorbance of blank wells and Ac represents the absorbance of control wells. The experiments were repeated at least three times and ICTo identify the potential off-targets of the compounds with anti-cancer activities, ABPP was applied. Cancer cells were seeded in the dishes and were washed with PBS, scraped, centrifugated  and pellets snap frozen in liquid in nitrogen. For ABPP experiments, the cell pellets were lysed by probe sonication (3 × 10 bursts) in PBS, followed by the determination of protein concentration of the intact cell lysate by Qubit assay. Subsequently, 20 µl of the cell lysate (1 mg/ml) was preincubated with 0.5 µl of DMSO (vehicle) or inhibitor  for 30 min, followed by the treatment with FP-TAMRA  for 20 min. The reactions were then quenched by 10 µl sample buffer (×4). The obtained samples were directly loaded and resolved on SDS-PAGE gel (10% acrylamide). The gel-based ABPP results were then scanned and analyzed using a ChemiDoc MP system .3i complex  was selected for molecular docking studies. The PDBQT files for the MAGL and compound 23 were prepared using AutodockTools 1.5.6. To prepare the PDBQT files for docking, essential hydrogen atoms and Kollman united atom charges were added using AutoDock Tools . The 2D ligand-protein interaction diagram was generated using the LigPlot+ program  significantly reduced the fluorescent signal to the background level. In a similar fashion, we also observed the fluorescent signal was time-dependent in the ABHD6 assay  and AA-HNA concentration was observed up to 200 µM  were able to dose-dependently reduce MAGL activity with pIC50 values of 4.8 ± 0.1, 4.9 ± 0.1, 5.3 ± 0.1, and 5.8 ± 0.1, respectively  were identified with significant MAGL inhibition (>80% inhibition), but low potency against ABHD6 (<50% inhibition) .Next, we evaluated the selectivity of the compounds against MAGL over ABHD6 and ABHD12. For ABHD6, we used a similar assay protocol as MAGL and assessed the inhibitory activity of all the compounds at 100 µM final concentration. The examination revealed that nine compounds demonstrated higher than 50% inhibition towards ABHD6, whereas the remaining compounds showed minimal activity against ABHD6 . In analibition) , suggestibition) . For ABHibition) . Brieflyibition) . The peribition) . CompariAA-HNA-based substrate assay showed that the compounds were less active in the ABPP assay on endogenous mouse MAGL when compared with the results in the primary screening assay . The results were then analyzed by in-gel-based ABPP and the inhibitory effects on the specific target were calculated and normalized by a DMSO-treated control sample. Of note, Coomassie staining gel was used to correct the protein loading for each sample. ng assay . As showin vitro anti-cancer activity of the compounds in different cancer cells was obtained. Despite most of the compounds showing minimal inhibition of cell viability at concentration of 20 μM, compounds 1, 23, 24, 82, 93, 98, 101, 102, 104, 127, and 154 produced appreciable inhibition against almost all the tested cell lines. Among them, compounds 64, 82, and 93 showed significant potency against the cell viability of A431 with 78, 84, and 80% inhibition, respectively. In particular, 64 selectively inhibited cell proliferation of A431 over other cell lines. Furthermore, compound 95 also showed selective cytotoxic activities against A549 (∼70% inhibition), and was inactive against other cell lines. Besides, compound 9 displayed 54% inhibition of cell viability against B16-F10 and neglectable antiproliferative behavior to other cell lines. To further confirm the anticancer activity, we next measured the IC50 values of the selected compounds with significant antiproliferative activity in cancer cells  towards endogenous MAGL in cancer cells. Competitive ABPP with FP-TAMRA was then performed to assess these compounds across a broad panel of serine hydrolases in intact cell lysates. In brief, compounds at 100 µM were incubated for 30 min with cell lysates, followed by a gel-based ABPP analysis using FP-TAMRA (500 nM). As shown in 23 completely reduced the labeling of endogenous MAGL in A549   .23) and human MAGL. We performed a molecular docking study of 23 with human MAGL (5ZUN) to provide an insight into any potential interactions. After extracting compound 3i (1-(3-Phenoxyphenyl)-4-[4--piperazin-1-yl]pyrrolidin-2-one) from the inhibitor-protein complex, 23 was flexibly docked into MAGL. As presented in 23 could occupy the binding pocket of MAGL. The carbonyl group at the 11-position of 23 could form a direct hydrogen bond with the side chain of Arg57 , which may contain certain components that might cause background fluorescence in the assay. In the end, a single-step enzymatic assay with fluorogenic substrate AA-HNA was set up and optimized in our study.Monoacylglycerol lipases as a serine hydrolase play a crucial role to catalyze the hydrolysis of monoacylglycerol lipids, particularly the endogenous cannabinoid 2-AG in the brain. Selective MAGL inhibitors have been considered as important agents in many therapeutic fields, including anti-nociceptive, anti-inflammatory, and anti-cancer. Despite a number of MAGL inhibitors that have been reported, inhibitors with new chemotypes are still required. In this study, on the basis of the catalytic mechanism of 2-AG hydrolases, we synthesized a new fluorogenic substrate AA-HNA-based fluorogenic assay with recombinant human MAGL, and ABPP was combined as an orthogonal method to confirm the inhibitory activity against MAGL in primary screening. The results demonstrate that four compounds  were able to dose-dependently reduce MAGL activity. Among them, compound 9, known as Apigenin , is a common flavonoid that widely exists in plants. Apigenin has multiple biological activities, including anti-inflammatory, antioxidant, antibacterial, and antiviral activities  has been found to be associated with the inhibition of STAT3 phosphorylation  and trans-isomers (93), have been firstly purified from Codonopsis pilosula by our collaborator. Their chemical structures and biological activities of them will be published elsewhere in detail. The selectivity of the compounds towards ABHD6 and ABHD12 was evaluated specifically, both ABPP and substrate assays suggest that compounds 9, 23, and 93 are potential inhibitors targeting MAGL, but selective over ABHD6 and ABHD12.Activity-based protein profiling (ABPP) is a highly useful chemical biological technique to assess the activity and selectivity of inhibitors in a complex native system . Yet, to the best of our knowledge, there is a limited description of the combination of ABPP with high-throughput screening assays to evaluate inhibitor activity and selectivity in the earliest stage of lead identification. We, therefore, combined ABPP and our substrate-based fluorescent assay to identify MAGL inhibitors with new chemotypes. In this study, a focused library containing 320 natural organic compounds was successfully screened using an tivities . Apigenitivities . Moreove in vivo . A quinotivities . The antrylation . CompounAA-HNA-based substrate assay showed that the compounds were less active in the ABPP assay on endogenous mouse MAGL when compared with the results in the substrate assay. The discrepancy in potency between the ABPP assay and the fluorogenic substrate assay might due to the different species of MAGL . Nevertheless, compounds 23, 82, and 93 demonstrated significant potency towards recombinant human MAGL and maintained a certain activity against endogenous mouse MAGL in ABPP assay (∼50%–60% inhibition), indicating the cross-species inhibition of these compounds against MAGL. In addition, competitive ABPP enables determination of the selectivity profile over a panel of serine hydrolases in a single experiment, including FAAH and ABHD6. The informative competitive ABPP results with mouse membrane proteome revealed broad-spectrum inhibitors  towards serine hydrolases.Next, ABPP was applied to investigate the activity and selectivity of the natural compound library on endogenous MAGL in mouse brain membrane proteome. Analysis of the ABPP assay vs. 23, 102, 104, and 127) produced significant inhibition against all eight cancer cell lines with pIC50 ≥ 5, suggesting these compounds may have broad-spectrum antitumor activities. Moreover, compounds 23, 82, and 93, identified as potential MAGL inhibitors by both ABPP and fluorogenic substrate assay, also showed antiproliferative activities against cancer cells, including A431, H1975, B16-F10, OVCAR-3, and A549. Notably, the selective MAGL inhibitor KML29 only showed antiproliferative activity against B16-F10 and HT-29 cells. These implicated compounds 23, 82, and 93 may have other potential off-targets, thereby contributing to the cytotoxic activities against other cancer cell lines .MAGL is a potential target for cancer diseases such as ovarian cancer and colorectal cancer, we, therefore, evaluated the antiproliferative activity of these compounds and applied ABPP to identify the potential targets. To identify the most sensitive cancer cells, we selected distinct cancer cells, including colon cancer (HT-29), ovarian (OVCAR-3), lung cancer , melanoma (B16-F10), cervical carcinoma  and epidermal carcinoma (A431). According to the results, we found that four compounds  that cannot be detected by FP-TAMRA. Notably, the selectivity evaluation in our study was only limited by serine hydrolases labeled by FP-TAMRA. As compounds derived from natural products often have multiple targets, a comprehensive selectivity profile of these compounds is still required in whole protein classes to elucidate the antiproliferative behavior in cancer cells. The study of proteome-wide selectivity for compounds with antiproliferative potency may also have the potential to discover new anticancer targets, as well as anticancer drug candidates.Comparative ABPP with serine hydrolase probe FP-TAMRA revealed that the activity of MAGL was found in most of the cells, but varied in different cancer cell lines, whereas the activity of ABHD6 was only found in A431 and B16-F10. No obvious ABHD12 activity was found in any of the cell lines using ABPP with probe FP-TAMRA. It might be due to the low sensitivity of FP-TAMRA for ABHD12 in the whole cell lysate proteome when compared with ABHD6 and MAGL. Next, competitive ABPP with FP-TAMRA was then applied to assess the activity and selectivity of the compounds towards endogenous MAGL in these cancer cells. No potential target was found for compounds AA-HNA and developed an AA-HNA-based fluorescence assay to rapidly identify MAGL inhibitors. In combination with ABPP, we screened a focused library containing 320 natural organic compounds with an AA-HNA-based substrate assay. Our investigations culminated in the identification of two major compound classes, including quinoid diterpene  and β-carbolines , with significant potency towards MAGL and good selectivity over other 2-AG hydrolases (ABHD6 and ABHD12). Furthermore, we also found these compounds showed appreciable antiproliferative activities against several cancer cells, including A431, H1975, B16-F10, OVCAR-3, and A549. Importantly, competitive ABPP results revealed that 23, but not 82 and 93, showed nearly complete inhibition against endogenous MAGL in eight cancer cells. In addition, the molecular modeling studies also provide the structural basis for 23 as a potential MAGL inhibitor. Our results demonstrated the potential utility of the fluorogenic substrate assay in combination with ABPP for rapid screening of MAGL inhibitors with therapeutic potential, and the importance of employing ABPP to identify potential targets for compounds with significant biological activities.In summary, we synthesized a new fluorogenic substrate


GPS devices were used to monitor players’ external loads: total distance covered, distance covered at different speeds , peak speed (km·h-1), and the number of accelerations and decelerations . One-way analysis of variance of the magnitude-based inference was used to determine differences between matches. Data indicated that official matches scored statistically higher peak speeds (ES = 1.40–2.20). In modified-sided games more total distance was covered at <13.9 km·h-1 and >14 km·h-1 than in regular matches (ES = 0.72–2.21), but lower distances were covered at >21 km·h-1 and >25 km·h-1 than in official and friendly matches (ES = 0.51–2.53) and at >25 km·h-1 than in training matches (ES = 0.92). Likewise, the modified-side games showed a greater number of accelerations and decelerations than other types of matches (ES = 1.46–2.51). This work shows that friendly and training matches, in conjunction with modified-side games, are suitable tools to prepare soccer players for official matches.The purpose of this study was to compare the external load in competitive  and non-competitive matches  in professional soccer players. Time-motion data for 10 elite male soccer players (age = 20.1 ± 2.1 years; body height = 178.8 ± 5.9; body mass = 71.4 ± 7.3; % body fat = 11.0 ± 1.1 and VO Despite the worldwide popularity of soccer, many uncertainties concerning the game’s multidimensional requirements and consequently uncertainties when planning for teams’ optimal training and conditioning processes still exist . Through2). Since the area of play is one of the most influential factors in the running activity of soccer players  operating at a sampling frequency of 5 Hz and an accelerometer operating at 100 Hz. Players wore a special harness that enabled these devices to be fitted to the upper part of their backs. The GPS device was activated 15 minutes before kick-off, in accordance with the manufacturer’s instructions. After recording, the data were downloaded to a PC and analyzed using a software package . Players were familiarized with the use of these devices and the SSG format used, prior to the commencement of the study. The validity and reliability of the GPS system have been previously reported and widely used with soccer players .The variables used to compare the external loads of matches and MSGs are presented in 2. While official and friendly matches had duration of 90 min (extra time and substituted players were excluded), training matches were played over a period of 30 min with the same official rules as friendly and official matches. At least 72 hours elapsed between each match, opposing teams were always of a similar level, and the match format was kept constant to reduce any variability in the players’ physical performance  to maintain the same relative area per player as that achieved in actual matches (300 m2). This drill was used over periods of 30′ (6 x 5′ with 2′ of passive recovery). During rest periods, players could drink fluids ad libitum. All participants were advised to maintain their normal diet, with special emphasis being placed on high intake of water and carbohydrates  and non-competitive matches . During the first half of the season, 12 official matches, 7 friendly matches, 6 training matches and 3 MSGs were monitored. Official and training matches and 3 friendly matches were recorded in the same stadium, with a dimension of 100 x 60 m, while 4 friendly matches were played on fields with similar characteristics (length: 98.5 ± 0.71 m and width: 59.7 ± 1.5 m), maintaining a relative area per player of around 300 mformance . The teahydrates . To ensuhydrates .Data are presented as mean ± standard deviation (SD). The normality of distribution of the variables was verified using the Shapiro–Wilk test. Differences in official, friendly, training and MSG matches were analyzed using one-way analysis of variance (ANOVA). A post hoc test was conducted with Bonferroni correction for multiple comparisons. In addition, possible differences or within-matches changes were analyzed for practical significance using magnitude-based inferences, pre-specifying 0.2 between-subject SDs as the smallest worthwhile effect . The sta-1 than in training matches (very large and large magnitude), but lower TD, DC <13.9 km·h-1 and DC >14 km·h-1 than in friendly matches (moderate magnitude). When the running activity between friendly and training matches was compared, it was found that friendly matches achieved greater TD, DC <13.9 km·h-1 and peak speed than training matches, but training matches produced more DC >25 km·h-1 (very large and large magnitude). In MSGs, soccer players covered statistically more TD, DC <13.9 km·h-1 and DC >14 km·h-1, but less DC >21 km·h-1 and DC > 25 km·h-1 than in official and friendly matches and less DC >25 km·h-1 than in training matches (very large to moderate magnitude).The running activity in official, friendly, and training matches and MSGs is presented in -2, but fewer decelerations between 4–8 m·s-2 than official and training matches  and performed significantly more decelerations between 1.5–2.5 m·s-2, but fewer accelerations between 2.5–4 m·s-2 than official games (moderate magnitude). In training matches, there were significantly more accelerations between 4–8 m·s-2 than in other studied matches (large magnitude).The acceleration and deceleration demands of official, friendly, and training matches and MSGs are presented in The purpose of this study was to compare external loads in competitive  and noncompetitive matches  in professional soccer players. The main findings of this study were: (1) official matches showed higher peak speeds than other studied scenarios; (2) official, friendly, and training matches had similar requirements at higher speeds among them, and higher than MSGs, but less total distance covered and lower speeds than MSGs, and (3); there were a greater number of accelerations and decelerations in training exercises than in matches, in all studied ranges.-1) than the one in the present study , but similar to our training matches and MSGs . Despite that previous investigations have established that peak speed is related to available space in SSGs, being higher when the area is greater (2). There are numerous reasons which could partly influence this variable, like a playing formation (1-4-4-1-1 vs. 1-4-3-3), the competitive level , the Ligue in which teams compete, etc. Thus, these differences cannot be explained solely by our study, and it strongly suggests the need of more studies to clarify the influence of those possible determinants.In soccer, it is accepted that match status influences teams’ behavior  and that greater , these pvs. 11, rules, and same playing area). Except a large difference in DC >25 km·h-1 between friendly and training matches, all differences in moderate to high intensity running between the different scenarios  were small or moderate. The main finding of the present study shows that the combination of friendly and training matches could be a suitable way to prepare soccer players for official competition because this combination can almost reproduce all the moderate and high intensity running requirements of official matches . To our knowledge, only one study has compared running demands between official and training matches (but not friendly matches) reproducing most of the demands of competition  achieved a greater number of accelerations between 1.0-1.5, 1.5-2.0 and 2.0-2.5 m·s-2, but not >2.5 m·s-2 than matches for semi-professional players  produced ~ 150% difference with respect to the most demanding sections of the match  and 49% higher in 6 vs. 6 (a 184 m2 relative area per player) formats than in official matches (vs. 6 with a lower relative area per player (83 m2) in professional players. Similarly, vs. 5 + 2 goalkeepers (a 124 m2 relative area per player ) than in 6 vs. 6 + 2 goalkeepers, 9 vs. 9 + 2 goalkeepers formats and official matches . All these studies used lower relative areas per player in their training tasks than in matches, confirming that a lower relative area per player increases the number of accelerations and decelerations. In contrast, our MSGs  showed a greater number of changes of speed, indicating that a lower number of players can increase the number of accelerations and decelerations. It is known that a smaller number of players results in more duels, dribbles, and ball touches  in training and official matches, finding higher demands in training matches .-1 between friendly and training matches, all differences in moderate to high intensity running  between official, friendly, and training matches were small or moderate. This suggests that a combination of friendly and training matches could be a suitable means to prepare soccer players for the running requirements of official competition.• Except a large difference in DC >25 km·h• Soccer players in MSGs exhibited more accelerations and decelerations than in official, friendly, and training matches.Understanding that there is clearly a challenge to loading players in a soccer-specific context, especially with high speed running , and tha


Breast cancer stem cells (BCSCs) have a crucial role in breast carcinogenesis, development, and progression. The aim of the current study is to characterize the BCSCs through the genetic profiling of different BCSCs phenotypic subsets to determine their related genetic pathways.+/CD24−/EpCAM− BCSCs, (2) CD44+/CD24− /EpCAM+ BCSCs, (3) mammospheres, and (4) normal breast tissues.Fresh tumor tissue samples were obtained from 31 breast cancer (BC) patients for (1) Mammosphere culture. (2) Magnetic separation of the BCSCs subsets using CD24, CD44, and CD326 Microbeads. (3) Flow cytometry (FCM) assay using CD44, CD24, and EpCAM. (4) RT-PCR profiler Arrays using stem cell (SC) panel of 84 genes for four group of cells (1) CD44+/CD24−/EpCAM−) showed significant downregulation in 13 genes and upregulation in 15, where the CD44, GJB1 and GDF3 showed the maximal expression ; respectively).The BCSCs . The mammospheres showed significant downregulation in 9 genes and a significant upregulation in 35 genes. The maximal overexpression was observed in GJB1 and FGF2 .The CD44CD44, COL9A1, FGF1, FGF2, GDF3, GJA1, GJB1, GJB2, HSPA9, and KRT15. While significant downregulation in BMP2, BMP3, EP300, and KAT8.The genes which achieved significant overexpression in all SC subsets were CCND2, FGF3, CD4, WNT1, KAT2A, NUMB, ACAN, COL2A1, TUBB3, ASCL2, FOXA2, ISL1, DTX1, and DVL1.The genes which were differentially expressed by the mammospheres compared to the other BCSC subsets were BCSCs have specific molecular profiles that differ according to their phenotypes which could affect patients’ prognosis and outcome. Breast cancer (BC) is a major health problem in females worldwide, as it represents the second leading cause of cancer-related deaths in women. It had been estimated that in 2020 there were 2.261 million new cases  and 0.685 million deaths  from BC globally .Despite the variability of the treatment modalities and diagnostic tools available for BC patients, still there is an increased incidence of metastasis and adverse outcomes. Therefore, it is important to understand the underlying molecular mechanisms involved in the carcinogenesis and progression of BC .+/CD24−/low, as well as mammosphere formation  work to confirm the diagnosis.The neoplastic tissues were washed several times in Hanks Balanced Salt Solution  and minced with sterile blades into very small pieces (0.2–0.5 mm each). The single-cell suspension was obtained by enzymatic digestion using collagenase  according to the studied protocol. The cells were incubated for 4–18 h at 37 ℃, and then filtered using a sterile stainless steel or nylon mesh. The cell suspension was washed several times by centrifugation in HBSS, and then the pellets were re-suspended in 500 µl–1 ml of Dulbecco's Modified Eagle Medium (DMEM). Using the haemocytometer, the cells were counted and divided into two parts, one part was used for mammosphere culture, and the other part was used for cellular characterization by FCM using CD44, CD24 and cytokeratin or EpCAM monoclonal antibodies.. [5 viable cells/mL in primary culture and 1000 cells/mL in each passage. The cells were cultured in DMEM/Ham F-12 media (1:1) supplemented with insulin (5 mg/mL), hydrocortisone (0.5 mg/ml), and epidermal growth factor . The cells were then seeded into six-well plates (2.5 ml/per plate) or T25 tissue culture flasks (5 ml per flask). The non-adherent cells were fed weekly; measured using the gridded lens. The mammospheres were enzymatically dissociated every 7 days to 2 weeks by incubation in 0.5% trypsin–EDTA solution (Invitrogen) for about 5–10 min at 37 ℃, then dispersed by pipetting with a 23-gauge needle. During the mammosphere dissociation, a subset of cells from each passage was subjected to subsequent morphologic evaluation by microscopic examination. In addition to immunohistochemistry assessment using primary antibodies for pan CK, CD44, CD24, and CD133 .The mammosphere culture was performed according to the method of Dontu et al.  with mod. , 28. Bri− or EpCAM− cells, CD44 + / CK+ or EpCAM+ cells, CD44−/CK+ or EpCAM + cells, CD44+/CD24−/low cells and CD24 + cells.A portion of the cell suspension was used for flow cytometric characterization of breast cancer stem cells using the Cell Quest program for the following conjugated antibodies: CD45-FITC (lymphocyte marker), CD24-PE , CD44-FITC (cancer stem cell marker), pan cytokeratin or cytokeratin 19-PE  or EpCAM-PE  according to manufacturers' instructions . Appropriate isotype controls were included in all cases to determine the areas of non-specific staining and unstained cells from each sample were also analyzed as a negative control. Accordingly, five subsets of cells were identified in each stained cases: CD44 + / CK+/CD24−/EpCAM− cells; G2: CD44+/CD24−//EpCAM+ cells; and G3: mammospheres,After the preparation of single-cell suspension, magnetic separation of the BCSCs subsets was done using the LS separation column (Miltenyi Biotec B.V. & Co. KG) according to the manufacturers' instructions. The cells were separated by magnetic selection after staining with CD24, CD44 and CD326 (EpCAM) Micro Beads labeled with monoclonal antibodies. Finally, different subsets of cells were collected by magnetic separation and stored for subsequent RNA extraction. Accordingly, the cells were divided into three groups including G1: CD44RNA was extracted and purified from the different groups of cells after magnetic separation using RNeasy Midi Kit  according to manufacturers' instructions. The qRT-PCR was done using the RT2 profiler array . As for the stem cells (SCs) profiling assay, the SABiosciences RT2 qPCR Master Mixes  was used to obtain the most accurate results from the PCR Array.2O. The amplification cycles were formed of an initiation step at 95 ℃ for 10 m, followed by 40 cycles at 95 ℃ for 15 s and, 55 ℃ for 90 s. The cycle threshold (Ct) for each well was determined and the ΔΔCt method was used for data analysis by the instrument's software. The Ct values of the control wells were determined including the Ct value of genomic DNA Control (GDC) and if it was greater than 35, the level of genomic DNA contamination was considered too low to affect gene expression profiling results. The studied genes were illustrated in Table The PCR was performed in the MaxPro3000 real time PCR (Startagen). Regarding the 96 well plate array the following reagents were mixed in a 5-ml tube at the recommended concentrations using a multi-channel pipette: 2X SABiosciences RT2 qPCR Master Mix, diluted First Strand cDNA Synthesis Reaction (using 500 ng RNA) and HData management and analysis were performed using statistical software package SPSS, version 22 . The flow cytometry data were presented as median and interquartile ranges (IQR) according to the performed normality test. Comparison between data were analyzed using Mann–Whitney. The PCR Array Data Analysis Web Portal presents the results in a tabular format, a scatter plot, a three-dimensional profile, and a volcano plot. All tests of hypotheses were performed at the alpha level of 0.05, with a 95% confidence interval.The median age of the recruited BC females was 47 range; 22–68) years, and the mean age was 48.1 ± 11.4 years. There were 15 (48.4%) patients with grade 2 tumor, and 16 (51.6%) patients with grade 3. Lymph node (LN) involvement was encountered in 22 (71%) patients, and capsular invasion was detected in 18 (58.1%) BC patients. The ER and PR were expressed in the tumor tissue of 14 (45.2%) BC females, while HER2 was expressed in the tumor tissue of 7 (22.6%) patients. In addition, there were 8 (25.8%) patients positive for distant metastasis . In these cases, the mammospheres ranged in size from 20 to 180 µm and were successfully cultured past the third passage , CD44 + /CK- or CD44 + /EpCAM- cells (stem cells), CD44 + /CK + or CD44 + /EpCAM + cells (neoplastic breast epithelium).The CD44The neoplastic breast epithelium which expressed both CD44 and EpCAM or CD44 and CK was found to be 39.9% (range: 0.5–92.8%). Cells expressed CD44 only were 23.8% (range: 1.3–74.7%), and those expressed CD24 + only were 12% (range: 0.1–37%). While cells expressed CD44 + /CD24- were 29.9% , compared to 25.3% (range: 0.1–68%) in those who had not metastasize (P = 0.038). Notably, patients with increased CD44 + expression, showed increase incidence of metastasis, ER and PR expression, though it did not reach a significant level , G2: BCSCs (CD44+/CD24low/ /EpCAM+), and G3: cultured mammospheres, compared to G4: the control normal group  showed that there were 13 significantly down-regulated genes which are involved in Cytokines and Growth Factors , Wnt Pathway , Cell Adhesion Molecules (CDH1), Mesenchymal Cell Lineage Markers , and Chromosome and Chromatin Modulators . While there were 15 genes that significantly up-regulated which were involved in cell adhesion , cytokines and growth factors , cell–cell communication , cell cycle regulation (CDK1), self-renewal (HSPA9), and embryonic Cell Lineage markers (KRT15). Where the CD44 showed the maximal expression , followed by GJB1 and GDF3 (fold regulation: 16.56 (P = 0.003) and 11.37 (P = 0.007); respectively) compared to the control group.Out of the 84 studied genes, the BCSCs , embryonic cell lineage markers (MSX1), chromosome and chromatin modulators (KAT8). While there were 28 genes that showed significant differential upregulation compared to the control group. These genes were involved in cell cycle regulation , cell adhesion molecules , cytokines and growth factors , cell–cell communication , self-renewal markers (HSPA9), Wnt pathway (ADAR), Notch pathway , mesenchymal cell lineage markers (ALPI), neural cell lineage markers , hematopoietic cell lineage markers , and embryonic cell lineage markers , Where the CD44 showed the maximal expression , followed by GDF3 and GJB1 (fold regulation: 55.9 (P = 0.001) and 55.8 (P = 0.003); respectively) compared to the control group.Regarding the CD44CDC42, EP300), chromosome and chromatin modulators (KAT8), Wnt Pathway , cytokines and growth factors , Notch pathway . While a significant upregulation in 35 genes which involved in cell cycle regulation , cell adhesion molecules , cytokines and growth factors , cell–cell communication , self-renewal markers (HSPA9), Wnt pathway , Notch pathway , mesenchymal cell lineage markers , neural cell lineage markers , hematopoietic cell lineage markers , embryonic cell lineage markers , and a metabolic marker (FGFR1). The maximal over expression was observed in GJB1 , followed by FGF2, JAG1 and COL9A1 (fold regulation: 94.9 (P = 0.001), 69.7 (P = 0.001) and 64.6 (P = 0.040); respectively) compared to the control group , cell adhesion molecules , cytokines and growth factors , cell–cell communication , self-renewal markers (HSPA9), Wnt pathway , Notch pathway , mesenchymal cell lineage markers , neural cell lineage markers , hematopoietic cell lineage markers (MME), and embryonic cell lineage markers . While the most commonly down regulated genes in all studied groups compared to the control were CDC42 (cell cycle regulation), BMP2, BMP3, (cytokines and growth factors), KAT8 (chromosome and chromatin modulators), PPARD, BTRC (Wnt Pathway), EP300 (Notch pathway), and PPARG .The differentially overexpressed markers in all BCSCs subsets compared to the control group were those mostly involved in cell cycle regulation , (2) BCSCs (CD44+/CD24low/ /EpCAM+), and (3) cultured mammospheres, through molecular profiling of 84 genes involved in the stemness properties of BCSCs, all were compared to normal breast tissue control.Breast cancer is a heterogenous disease characterized by variable genetical and phenotypical subtypes, which accordingly leads to diverse outcomes in BC patients. This heterogeneity of the BC cells is mainly linked to the cell of origin . An incrHSPA9), cell cycle regulation , cell adhesion , cell–cell communication , expression of some cytokines and growth factors , and expression of differentiating cell lineage including mesenchymal markers , neural markers , hematopoietic markers (MME), and embryonic cell lineage markers . In addition, they express some genes required for the stemness properties of the recruited cells included those involved in Wnt pathway  and Notch pathway . However, each type of cells has its own characteristically differential gene expression according to its phenotype, which later will formulate the tumor behavior and patients’ outcomes. In consistent with these data, many published series illustrated the importance of Wnt pathway, hedgehog pathway, and notch pathway for maintain the tumorigenicity, self-renewal and epithelial-mesenchymal transition (EMT) properties of the cancer stem cells [The present data showed that the three subtypes of BCSCs shared over expression of common genes responsible for the self-renewal , cell adhesion molecules , cytokines and growth factors , cell–cell communication , Wnt pathway (ADAR), Notch pathway , mesenchymal cell lineage markers (ALPI), neural cell lineage markers , hematopoietic cell lineage markers , and embryonic cell lineage markers . In agreement with these data, Wu et al. [ITGA6, EPCAM, CCND1, CD44, EGFR, CDH1, and MKI67. Luo et al. [+/CD24−/EpCAM+ BCSCs play an important role in tumor metastasis, as it showed increased expression of genes responsible for EMT. In addition to increased expression of inflammatory cytokines and proteins associated with tumor invasion and metastasis.These genes were those involved in cell cycle regulation  which express EpCAM on their cell surface, were more tumorigenic and had potent invasive properties when transplanted in immunodeficient mice in comparison to those lacking the expression of EpCAM.Our data showed also that CD44o et al. , who repj et al. , concludThus, the increased expression of EpCAM on the surface of BCSCs confers an aggressiveness, metastasis, drug resistance, and tumorigenic properties for the BC, which consequently associated with poorer outcomes in the BC patients.+/CD24−/EpCAM+ and CD44+/CD24−/EpCAM-) showed that the maximal expression was observed in CD44, followed by GDF3 (Growth differentiation factor-3) and GJB1 (Gap junction beta-1 protein). These data are in line with many series reported that CD44 has a fundamental role in EMT as it acts as an adhesion molecule and receptor for extracellular glycosaminoglycan hyaluronic acid, which leads to increased cell motility, and tumorigenicity [Furthermore, both groups of BCSC subsets , were those involved in cell cycle regulation , cell adhesion molecules (CD4), Wnt pathway (WNT1), Notch pathway , mesenchymal cell lineage markers , neural cell lineage markers (TUBB3), and embryonic cell lineage markers . While there was a differential downregulation in DTX1, DVL1 which were involved in the Notch pathway. Theses exclusively upregulated genes in mammosphers confirmed the nature of the mammospher derived cells which allow for the selection of highly undifferentiated and aggressive cells with marked stemness properties. In line with these data, Dontu and his colleagues performed microarray analysis of mammosphers and other differentiated BC cells. They observed a significant upregulation of genes involved in growth hormone receptors, thrombin receptors, and Notch signaling pathway [Regarding the analysis of the gene expression profile of the cultured mammosphers, the current data demonstrated that the genes which were differentially overexpressed by the mammosheres when compared to the other BCSC subsets , cell adhesion molecules , cytokines and growth factors , cell–cell communication , self-renewal markers (HSPA9), mesenchymal cell lineage markers (COL9A1), and embryonic cell lineage markers (KRT15). While those achieved significant downregulation were BMP2, BMP3, (cytokines and growth factors), KAT8 (chromosome and chromatin modulators), and EP300 (Notch pathway). It had been reported that KAT8 (K-lysine acetyltransferase 8), BMP2, BMP3 (Bone morphogenetic protein), and EP300 were significantly downregulated in breast cancer, as they have a major role in controlling tumor progression through histone acetylation, metastasis suppression and inhibition of EMT, respectively [By comparing all studied groups, we identified 10 candidate genes which were significantly overexpressed in all BCSC subsets. These genes were important for cell cycle regulation  which were expressed (up or downregulated) in all the assessed subsets of BCSCs which could serve as molecular markers with a potential diagnostic, prognostic and/or predictive value for breast cancer patients. However, the data of the profiling array should be validated on a larger number of patients, and also it should be correlated to the patients’ clinical courses in the form of distant metastasis, response to treatment and survival rates.The current study provided evidence that BCSCs have specific molecular profiles that differ according to their phenotypes, which could affect patients’ prognosis and outcome. CD44 is an important marker for characterization of BCSCs, and its co-expression with EpCAM provides an aggressive and metastatic properties for the BCSCs rather than those with CD44