With fast rising incidence, papillary thyroid carcinoma (PTC) is the most common head and neck cancer. Parthenolide, isolated from traditional Chinese medicine, inhibits various cancer cells, including PTC cells. The aim was to investigate the lipid profile and lipid changes of PTC cells when treated with parthenolide.Comprehensive lipidomic analysis of parthenolide treated PTC cells was conducted using a UHPLC/Q-TOF–MS platform, and the changed lipid profile and specific altered lipid species were explored. Network pharmacology and molecular docking were performed to show the associations among parthenolide, changed lipid species, and potential target genes.With high stability and reproducibility, a total of 34 lipid classes and 1736 lipid species were identified. Lipid class analysis indicated that parthenolide treated PTC cells contained higher levels of fatty acid (FA), cholesterol ester (ChE), simple glc series 3 (CerG3) and lysophosphatidylglycerol (LPG), lower levels of zymosterol (ZyE) and Monogalactosyldiacylglycerol (MGDG) than controlled ones, but with no significant differences. Several specific lipid species were changed significantly in PTC cells treated by parthenolide, including the increasing of phosphatidylcholine (PC) (12:0e/16:0), PC (18:0/20:4), CerG3 (d18:1/24:1), lysophosphatidylethanolamine (LPE) (18:0), phosphatidylinositol (PI) (19:0/20:4), lysophosphatidylcholine (LPC) (28:0), ChE (22:6), and the decreasing of phosphatidylethanolamine (PE) (16:1/17:0), PC (34:1) and PC (16:0p/18:0). Four key targets  were discovered when combining network pharmacology and lipidomics. Among them, PLA2G2A and PLA2G4A were able to bind with parthenolide confirmed by molecular docking.The changed lipid profile and several significantly altered lipid species of parthenolide treated PTC cells were observed. These altered lipid species, such as PC (34:1), and PC (16:0p/18:0), may be involved in the antitumor mechanisms of parthenolide. PLA2G2A and PLA2G4A may play key roles when parthenolide treated PTC cells.The online version contains supplementary material available at 10.1186/s12906-023-03944-7. Thyroid carcinoma is the most frequent type of head and neck cancer, with an incidence rate among women that is three times greater than that among men, accounting for around 5% of cancer cases reported among women . PapillaTanacetum parthenium). It has been widely utilized for its anti-inflammatory and antioxidant properties, with many studies demonstrating its effectiveness in treating headaches, fever, and rheumatoid arthritis [Parthenolide is a sesquiterpene lactone, which is isolated and purified from traditional herbal medicine feverfew , and human PTC cell line BCPAP was purchased from Shanghai Institutes for Biological Sciences, China. RPMI 1640 medium was purchased from Corning, USA. Fetal bovine serum was purchased from Shuangru Biology ScienceandTechnology Co.Ltd. HPLC-grade formic acid and HPLC-grade ammonium formates were purchased from Sigma. MS-grade acetonitrile, MS-grade methanol, and HPLC-grade 2-propanol were purchased from Thermo Fisher.2, 37 °C. Sufficient cell samples were divided into 12 groups. According to the appropriate concentration (IC50) explored in the preliminary experiment [n = 6) for 24 h, and 6 control groups were added with the same amount of complete culture medium.BCPAP containing 10% fetal bovine serum, 100 U/mL streptomycin, and 100 U/ml penicillin, was maintained in a complete RPMI 1640 medium. BCPAP were cultured in an environment of 5% COperiment , parthenThe samples were homogenized with 200 µL water and 240 µL methanol. Then 800 µL of MTBE was added and the mixture was managed by ultrasound at 4 degree centigrade for 30 min at room temperature. Finally, the solution was centrifuged at 14,000 rpm for 15 min at 10 degree centigrade to obtain the supernatants and dried with nitrogen.Reverse phase chromatography was used for liquid chromatography separation with a column . The lipid extracts were re-dissolved in 200 µL 90% isopropanol /acetonitrile, then centrifuged at 14,000 rpm at 10 degree centigrade for 15 min, at last 3 µL of each sample was injected onto the CSH C18 column. Solvent A contained acetonitrile–water  with 0.1 mm ammonium formate and 0.1% formic acid. Solvent B contained acetonitrile–isopropanol  with 0.1 mm ammonium formate and 0.1% formic acid. With a flow rate of 300 μL/min, 30% solvent B was maintained for 2 min. After that, solvent B increases to 100% in 23 min, and then it was balanced at 5% for 10 min. Mass spectra was performed on a Q-Exactive Plus in positive and negative mode, respectively. ESI parameters were adopted for all measurements as follows: heater temperature, 300 degree centigrade; sweep gas flow rate 1 arb; aux gas flow rate 15 arb; sheath gas flow rate 45 arb; spray voltage 3.0 kV and 2.5 kV for positive and negative electrospray ionization mode, respectively; S-Lens RF Level 50% and 60% for positive and negative, respectively; and the scan ranges 200–1800 m/z and 250–1800 m/z for positive and negative, respectively.Based on MS/MS math, LipidSearch was used for the identification of lipid species. LipidSearch contains more than 1,500,000 fragment ions and more than 30 lipid classes in the database. Both mass tolerance for precursor and fragment were set to 5 ppm.http://omim.org/), GeneCards Database (http://www.genecards.org/), and DrugBank (http://go.drugbank.com/). In addition, the targets of parthenolide were retrieved from SEA search server (http://sea.cbkslab.org/), STITCH (http://stitch.embl.de/), SwissTargetPrediction (http://swisstargetprediction.ch/), and PharmMapper server (http://www.lilab-ecust.cn/pharmmapper/). The intersection of the above targets was considered the potential targets of parthenolide against PTC. Afterward, the standard target names were obtained from UniProtKB (http://www.uniprot.org/). The lipid targets were generated from the differential lipid metabolites by using MetScape in Cytoscape 3.8.0 software. Then, a protein–protein interaction (PPI) network was presented by STRING 11.5 (http://cn.string-db.org/) to show the link between these predicted targets and lipid targets. Hub targets were obtained via CytoHubba in Cytoscape. As a result, the compound-targets-metabolites network was constructed containing relationships among parthenolide, relevant target genes, and lipid metabolites.In order to discover the related disease targets, the keyword ‘‘papillary thyroid carcinoma” was searched from OMIM Database (http://www.rcsb.org/). Four protein targets were studied: PLA2G4A (PDB ID: 1RLW), LCAT (PDB ID: 4X90), LRAT (PDB ID: 4DPZ), and PLA2G2A (PDB ID: 3U8I). Then, the molecular docking was performed using LibDock with the default docking parameters, and the results were sequenced according to the LibDockScore of each protein.The three-dimensional structure of parthenolide was acquired from PubChem Compound . The structure was preprocessed by adding hydrogen atoms and extracting water molecules. The protein structures of the hub targets were obtained from PDB database , supervised partial least squares discrimination analysis (PLS-DA), as well as orthogonal partial least squares discrimination analysis (OPLS-DA). Discriminant lipids were determined by the variable importance in the projection (VIP) parameter  and p value . Lipid Pathway Enrichment Analysis (LIPEA) software was used to perform the pathway enrichment analysis of metabolites [The data extracted by LipidSearch were analyzed, including univariate statistical analysis, multivariate statistical analysis, as well as hierarchical clustering and correlation analysis. Student's t-test and multiple of variation analysis were used for univariate statistical analysis. The lipid profiles showing differences with lower than 0.67 fold decrease or more than 1.5 fold increase along with abolites .To evaluate the stability and repeatability of the experiment, each group of samples was equally mixed into the quality control sample (QC). The results of QC UHPLC-obitrap MS base peak were compared with overlapping spectra, which showed that the response intensity overlapped significantly, as well as the retention time of chromatographic peaks, indicating that this experiment was highly reproducible. The results of pearson correlation analysis showed that the coefficient between QC samples is above 0.9, indicating good reproducibility. All QC and experimental samples were extracted and PCA analysis was performed after Pareto-scaling. The QC samples were closely clustered, indicating that lipidomic analysis was highly reproducible , cholesterol ester (ChE), simple glc series 3 (CerG3) and lysophosphatidylglycerol (LPG), lower levels of zymosterol (ZyE) and monogalactosyldiacylglycerol (MGDG) than controlled ones, but with no significant differences. The levels of other lipid classes were similar in both groups. . A total of 10 lipid species were selected and listed in Table The VIP obtained by the OPLS-DA model can be used to measure the impact strength of various lipid species on the discrimination of each group of samples. Univariate analysis can be used to visualize the significance of lipid species changes between the two groups Fig. A, thus hIn order to assess the rationality of different lipid species, and to show the association between the samples and the expression patterns of lipid species in different samples more comprehensively, a hierarchical clustering  was performed to demonstrate the 10 lipid species that have changed significantly. As shown in Fig. P < 0.01, |r|> 0.5, Fig. P < 0.01, |r|> 0.5, Fig. The correlation analysis was performed to obtain the correlation degree between the significant lipid species, and help measure the closeness of differences between lipid classes and species in the lipid metabolic process, further understand the relationship between lipid species in the process of biological changes. In this study, the correlation analysis of selected ten lipid species showed significant positive correlations between LPE (18:0) and PC (12:0e/16:0), ChE(22:6), and LPC (28:0), respectively, between LPC (28:0) and PC (12:0e/16:0), and between CerG3d18:1/24:1) and PI (19:0/20:4)  and it was closely related to a group of significant lipid species identified in this study after PTC cells treated with parthenolide. Other pathways identified included ferroptosis 27%), glycosylphosphatidylinositol—anchor biosynthesis (18%), autophagy—other (18%), and autophagy—animal (18%)  were identified, and PLA2G2A and PLA2G4A were further confirmed to be able to bind with parthenolide through molecular docking.A previous study demonstrated that parthenolide could promote apoptosis of PTC cells . MetabolFormer studies have shown that abnormal PC distribution may alter the microenvironment of the cellular lipid membrane, resulting in the variation of membrane fluidity and function , 28. As In recent years, the lipid composition of thyroid cancer patients has undergone considerable changes, which may play a key role in the pathogenesis of the disease. . IshikawIn our research, the significant difference of lipid class was not found from PTC cells treated by parthenolide, but several lipid species were changed significantly in PTC cells, such as increased levels of LPE (18:0), LPC (28:0), PC (12:0e/16:0) and PC (18:0/20:4), as well as decreased levels of PE (16:1/17:0), PC (34:1) and PC (16:0p/18:0). One of the mechanisms of parthenolide for treating thyroid cancer may be achieved by lowering the levels of PC (34:1) and PC (16:0), which have been found to be diagnostic markers for thyroid cancer , 32, 33.The two key targets, PLA2G2A and PLA2G4A, which were discovered through network pharmacology and lipidomics and were subsequently confirmed via molecular docking, may play a crucial role in the treatment of parthenolide against PTC. As family members of phospholipase A2 (PLA2), PLA2G2A and PLA2G4A have a variety of biological functions, including involvement in cell signaling and inflammatory response , 37. FurThis study has some limitations. First, due to limited time and funds, the roles of PLA2G2A and PLA2G4A in the parthenolide-induced antitumor effect have not yet been validated through more experiments. Second, the sample size is relatively small, which may be related to the unstable results of PLA and insignificant difference in lipid classes between two groups. As we found altered lipid profile and changes of PTC cells treated with parthenolide, as well as the key targets from network pharmacology and molecular docking, it suggests that further research should use a larger sample size and more in vitro experiments.This study is the first of its kind to report the lipid profile and changes of PTC cells following treatment with parthenolide. We observed alteration in lipid species, such as PC (34:1), and PC (16:0p/18:0). To better elucidate the mechanisms by which this occurs, network pharmacology and molecular docking were employed to identify two key targets (PLA2G4A and PLA2G2A) that are able to bind parthenolide. This research provides novel insights into the underlying mechanisms of PTC, and further exploration is warranted.Additional file 1. A. UHPLC-Obitrap MS BPC of quality control samples shows high precision. B. Pearson correlation analysis of QC samples shows good reproducibility. C. Principal component analysis (PCA) analysis shows a good experimental repeatability.Additional file 2. Lipid profiling of papillary thyroid carcinoma cell between two groups.Additional file 3. Lipid class analysis between two groups.Additional file 4. The correlation analysis between the significant lipid species.


Results: A total of 618 teeth among 31 individuals were analyzed, 475 (76.8%) GC were detected by CBCT in 23/31 patients and of these, 6 belonged to G2. G2 had a decreased GC detection rate (n = 180–37.9%) and the most common tooth with GC detected was the mandibular 1st molar (21 GC/25 teeth—84%) and the absence of GC was most frequently observed in impacted and delayed/unerupted teeth of Ds individuals. Conclusion: We concluded that GC absence was higher among Ds individuals, explaining the increased rates of unerupted or impacted teeth in Ds individuals.Background: The gubernacular canal (GC) is an important dental structure that enables the alveolar bone ridge cohesion of permanent teeth, although GC absence may indicate a dental eruption that might be associated with certain syndromes such as Down’s syndrome. This study aims to correlate the eruptive delay of permanent teeth in individuals with Down’s syndrome (Ds) and the gubernacular canal (GC) through cone-beam computed tomography (CBCT). Methods and Results: This cross-sectional study was conducted between January and July 2022 with a total of 31 individuals (G1 = 16 nonsyndromic and G2 = 15 Down’s syndrome) who went through imaging evaluation using CBCT with the following acquisition parameters: tube voltage of 95 kVp, tube current of 7 mA, exposure time of 5.9 s and voxel sizes and field of view 0.15 mm and 0.30 mm, respectively. The imaging evaluation was to assess whether all teeth analyzed had the presence of GC and/or teeth eruption disturbance, with a descriptive statistical analysis of relative frequencies and quantitative variables as well as the  Mixed dentition is a stage of growth and development that begins with the dental eruption of the first permanent teeth at about 6 years old and continues until 12 years old . In thisAs mentioned previously, both the gubernacular canal and cord are intraoral structures very important to the tooth-eruption process, although a small number of studies in the literature about GC and cord demonstrated their influence in permanent teeth eruption physiologic processes, especially in the follicular theory ,6. The gThe gubernacular cord formation begins with the remnants of cells from the dental lamina that are assembled as a fibrous cord through the enamel organ’s reduced epithelium toward the oral mucosa ,11,12,13GC obstruction might occur for various reasons such as impacted teeth, abnormal teeth orientation, maxillary atresia, overly dense bone, excessively soft tissue, craniofacial abnormalities, genetic abnormalities such as Down’s syndrome (DS), supernumerary teeth, and odontogenic tumors such as odontoma ,14. Ds, GC obstruction among individuals with Ds has not yet been reported in the literature. A few studies demonstrated oral cavity abnormalities in individuals with Ds; however, this does not correlate with GC obstruction. De Moraes et al.  evaluateCuoghi et al.  evaluaten = 15 individuals previously diagnosed with Ds who were spontaneously undergoing routine Ds treatment and management at the Integrated Service for Oral Diagnosis and Dental Care for Special Patients (SIDOPE).This observational, cross-sectional population-based study was conducted between January and July 2022 in Belém, the capital city of Pará State, in northern Brazil, and it included The study complied with resolution 196/96 of the National Health Council and the Ethics and Research Committee of the Health of Science Institute from the Federal University of Pará (UFPA) and was approved under protocol number 3.405.106. Written informed consent was obtained from the parents or legal guardians of all participants prior to study enrollment.The sample consisted of underaged patients registered and treated at SIDOPE, 40 individuals and their parents or legal guardians were informed about the purpose of the study and invited to participate, although only 31 agreed to sign a written consent form before data collection and evaluation or met inclusion criteria. The study eligibility criteria were (i) individuals with a confirmed diagnosis of Ds; (ii) attended a monthly follow-up at SIDOPE in Pará State; (iii) at least 10 teeth in the mouth and without periodontal disease or cavities for about 1 year; (iv) aged between 10 to 15 years old; (v) resident in Pará State; (vi) both genders; and (vii) teeth completely encased within the alveolar bone.The exclusion criteria were (i) individuals who were moved to other states; (ii) individuals who started follow-up at SIDOPE but no longer returned to monthly follow-up; (iii) medical records not filled out correctly; (iv) low-quality CBCT images; and (v) CBCT images with motion blurring or metal artifacts. Each participant was physically and orally evaluated in a private SIDOPE location. Clinical data were collected by a single researcher, a specialist in oral pathology, previously calibrated by Kappa test and with previous experience in clinical studies, and the intraoral clinical examination was performed in a dental office, dental chair, under indirect and artificial light, using a dental mirror, Williams periodontal probe  and clinical tweezers, all sterile.After clinical exams, demographic and epidemiological data were obtained through a pretested standardized semi-structured questionnaire and medical records. The individuals were divided into two groups according to the presence or absence of Ds. The groups were formed and organized by nonsyndromic patients as the control group and Ds patients, with the nonsyndromic patients presenting with the GC open: G1 group: 16 patients ; G2 group: 15 (possible GC obstructed).The study consisted of CBCT examinations for all 31 patients with impacted/unerupted permanent teeth in mixed dentition, for dental planning of orthodontic treatment, periodontal nonsurgical or surgical treatment, impacted teeth location, and identifying dental anomalies. All CBCT images were taken and collected from the Department of Oral and Maxillofacial Radiology of UFPA, the tomography machine was a Pax-i 3D smart , and acquisition parameters were a tube voltage of 95 kVp, a tube current of 7 mA, an exposure time of 5.9 s (Green Protocol) and voxel sizes and a field of view (FOV) of 0.15 mm (8 × 8 cm and 6 × 6 cm) and 0.30 mm (8 × 8 cm), respectively .After the CBCT images were acquired, they were converted to the digital imaging communication in medicine (DICOM) file format, uploaded to an independent storage software , and then opened with open-source DICOM viewer software OsiriX 12.0 . All DICOM file evaluations were carried out by a single examiner, an oral radiologist with twenty years of experience, and a standardized CBCT view procedure  was established by the examiner .All CBCT teeth images were visualized in axial, coronal, sagittal, cross-sectional, and panoramic views in the X, Y, and Z axes. The parameters analyzed included the presence or absence of GC, permanent eruption stages, the presence of physical obstructions in the eruption path or pathological conditions, root formation stage, angulation, and dilaceration . Radiogrp-value (p < 0.005) by G Test for the selected groups; additionally, the odds ratio (OR) and associated 95% confidence interval (CI) were used as measures of the strength of dependent association between GC absence (outcome) and Ds individuals.The statistical analysis was performed using JAMOVI Statistics for Windows, Version 3  statistical software. A descriptive analysis of the data was performed, with a distribution of absolute and relative frequencies, using the minimum and maximum value, mean and standard deviation as well as the A total of 40 individuals were invited, although only 31 participated in the present study due to not satisfying the inclusion criteria or not agreeing to sign the consent term; therefore, the final analyzed sample consisted of 15 individuals diagnosed with Ds and 16 nonsyndromic individuals.n = 17—54.9%) were more prevalent than females (n = 14—45.1%); as self-declared ethnicity, white race was the most prevalent with 16 (51.6%) in both groups (G1: n = 6—37.5%)/G2: n = 10—66.6%); most of the individuals were from Belém city, capital of Pará State (n = 15—48.3%), in both groups (G1: n = 8—50%/ n = 7—46.6%); and regarding the type of education, most of the individuals attended private school (n = 21—67.7%) in both groups (G1: n = 10—62.5%/ n = 11—73.3%).All clinical, demographic, and epidemiological characteristics are shown in n = 8—33.3%), especially among Ds individuals (n = 7—46.6%), otitis media was the most prevalent hearing impairment (n = 12—50%), mainly in Ds patients (n = 10—66.6%), dacryocystitis and refractive errors were the most prevalent visual impairments (n = 18—75%), mostly in Ds patients (n = 12—80%), atrioventricular septal defect and systemic arterial hypertension were the most prevalent cardiovascular disorders (n = 20—83.3%), especially in Ds individuals (n = 14—93.3%), and prevalent endocrine disorders were the least common systemic conditions, although, among our sample, the most prevalent endocrine disorders were hypothyroidism (n = 7—29.2%) and mostly in Ds patients (n = 4—26.6%).Regarding clinical disorders, the most common were blood disorders, hearing impairment, visual impairment, cardiovascular disorders, and endocrine disorders. Among blood disorders, the most prevalent was anemia (n = 13/31—42%) in both groups (G1: n = 6/13—46.2%/ G2: n = 7/13—53.8%) followed by mouth breathing (n = 8/31–25.8%), digit sucking (n = 4/31—13%), tongue thrusting and obstructive sleep apnea (n = 3/31—9.6%), and during the analysis, no cases of nail biting were presented  among the individuals in this study, the most recurrent were digit sucking, tongue thrusting, bruxism, mouth breathing, nail biting, and obstructive sleep apnea. Bruxism was the most prevalent POH (resented .n = 17/70—24.2%) in both groups (G1: n = 6/17—35.5%/G2: n = 11/17—64.7%), followed by periodontal disease (n = 13/70—18.5%) in both groups (G1: n = 3/13—23%/G2: n = 10/13—77%), and maxillary atresia (n = 10/70—14.2%) in both groups (G1: n = 2/10—20%/G2: n = 8/10—80%), which are, according to the literature, common OMs for individuals with Ds.Regarding oral manifestations (OM), hypodontia or missing teeth  was the most prevalent OM for individuals (n = 280—45.3%), impacted (n = 203—32.8%), or delayed/unerupted (n = 135—21.9%). Among normal, impacted, or delayed/unerupted teeth eruption states, there were a total of 618 teeth in 31 individuals, G1 had 351 (56.8%) teeth and G2 had 267 (43.2%). For dental prevalence, for both nonsyndromic and Ds patients, the most prevalent permanent teeth were maxillary central incisors (n = 85—13.7%), mandibular central incisors (n = 70—11.3%), maxillary lateral incisors (n = 63—10.2%), maxillary canines (n = 55—8.9%), and maxillary 1st molars (n = 54—10.8%). Regarding teeth eruption state, teeth with normal eruption were 280/618 (45.3%), G1 had 181/280 (64.6%) and G2 had 99/280 (35.4%), impacted teeth were 203/618 (32.8%), G1 had 64/203 (31.5%) and G2 had 139/203 (68.5%), and delayed/unerupted teeth were 135/618 (21.9%), G1 had 65/135 (48%) and G2 had 70/135 (52%).Through CBCT analysis, n = 34—48.5%), maxillary canines (n = 28—50.9%), maxillary lateral incisors (n = 26—41.2%), mandibular lateral incisors (n = 24—47%), and maxillary central incisors (n = 22—25.8%). Regarding delayed/unerupted teeth, the most prevalent types were maxillary lateral incisors (n = 18—28.7%), maxillary canines (n = 16—29.1%), mandibular 1st molars (n = 14—26%), mandibular canines (n = 12—24.6%), maxillary 2nd molars (n = 11—44%), and maxillary central incisors (n = 11—12.5%). Evaluating both groups, G2 had the most prevalence of impacted or delayed/unerupted teeth among all individuals, and Ds craniofacial characteristics such as brachycephaly and OM such as maxillary atresia might influence the dental eruption state. Finally, a significant association between teeth eruption state and Ds was established for maxillary central incisors, mandibular central incisors, mandibular canines, and mandibular 2nd premolars (p < 0.0001).Among teeth eruption state, the most common type of impacted permanent teeth were mandibular central incisors (n = 475), as shown in n = 295—62.1%) prevalence of GC was higher than G2 (n = 180—37.9%), therefore, we could suggest that Ds or the craniofacial characteristics associated with Ds might be a risk factor for GC obstruction. The detection rates of the GC in G1 permanent dentition were maxillary central incisors (38 GC/40 teeth—95%), mandibular central incisors (33 GC/33 teeth—100%), maxillary lateral incisors (30 GC/31 teeth—96.7%), maxillary canines (28 GC/29 teeth—96.5%), and mandibular canines (26 GC/26 teeth—100%); maxillary 1st premolars, maxillary 2nd premolars, mandibular lateral incisors, mandibular 1st premolars, and mandibular 2nd premolars had 100% GC rates, although compared with the previous teeth, there were fewer teeth with which to diagnose GC.Among 618 teeth, the GC prevalence was 76.8% , mandibular central incisors (29 GC/37 teeth—78.3%), maxillary 1st molars (25 GC/32 teeth—78.1%), maxillary 2nd molars (10 GC/13 teeth—76.9%), and mandibular lateral incisors (23 GC/31 teeth—74.2%). Therefore, in analyzing the GC rates between groups, it appears that G2 had a decreased prevalence over G1, showing that in our sample, Ds itself, or Ds craniofacial characteristics, or OM could influence GC formation because in G1, there were 56 nondetected GCs and G2 had 87 nondetected GCs. Regarding a possible correlation between Ds individuals and lower GC detection rates, a bivariate logistic analysis identified a significant association in mainly mandibular canines (GC absence 44 times more likely in G2), maxillary canines (GC absence 27 times more likely in G2), mandibular central incisors (GC absence 22 times more likely in G2), and maxillary 1st premolars (GC absence 21 times more likely in G2).Regarding possible eruption disturbances (213/338—63%) or pathological conditions (125/338—37%) leading to GC detection, Concerning a viable correlation between eruption disturbances or pathological conditions related to Ds and lower GC detection rates, a bivariate logistic analysis identified a significant association between eruption disturbances and pathological conditions. According to our analysis, G2 was 0.5 times more likely to have eruption disturbance than G1, and G2 was twice more likely to have pathological conditions than G1. In pathological conditions, no significant association among each pathology was found, although, in eruption disturbances, there was a significant association between maxillary or mandibular atresia (2.4 times more likely to show in G2), teeth ectopic position (2.2 times more likely to show in G2), and follicle space widening (0.03 times more likely to show in G2). These results could demonstrate that Ds OM or craniofacial characteristics might delay or impact dental eruption state and also prejudice GC formation.To the best of the authors’ knowledge, the present study is one of the few studies that have focused on the use of CBCT imaging to study GC characteristics and its role in teeth eruption associated with pathological conditions among Ds individuals and is the first study evaluating this population in northern Brazil. According to Kaplan et al. , the firThrough the years, studies demonstrated that GC structures can be visualized on panoramic and dental radiographs, although some diagnoses propose that the two-dimensional characteristics of dental radiographs would make it difficult to visualize or measure some GC aspects such as length, diameter, pathway, deformation, and/or obliteration. Therefore, recent studies introduced CBCT as a justified imaging examination to evaluate GC presence and its possible disturbances such as impacted or delayed/unerupted teeth that will influence clinical pediatric dentistry, orthodontics planning, and oral and maxillofacial surgical treatment due to its specificity in different images ,22,23.n = 209/338—61.9%), which had, as major pathological conditions (n = 125/338—37%), odontoma in 72 (57.6%) cases, and supernumerary teeth in 40 (32%) cases. Additionally, we had a Ds individuals group in our study for which we evaluated common eruption disturbances (n = 213/338—63%) associated with this syndrome, which had a major influence on our 338 impacted or delayed/unerupted teeth [In our study, there were 143 nondetected GC among 618 teeth, and of these, there were 338 impacted or delayed/unerupted, mainly in G2  had a higher prevalence of OM than G1 (n = 21/70—30%), and the main OM were hypodontia or missing teeth, periodontal disease and maxillary atresia, all common OM in Ds. Another interesting fact in our results that corroborated the literature [n = 5/70—7.1%) in G2 (n = 1/5—20%) than in G1 (n = 4/5—80%), whereas periodontal disease (13/70—18.5%) had a higher prevalence in G2 (n = 10/13—77%) than in G1 (n = 3/13—23%). Some parafunctional habits including bruxism, mouth breathing, digit sucking, tongue thrusting, and obstructive sleep apnea were observed; in total, there were 31 abnormal habits among both groups which are more prevalent in G2 (n = 18/31—58%) than in G1 (n = 13/31—42%).In our study, OM and parafunctional habits in both groups were evaluated and as seen in our results, a total of 70 OM were found, G2 (terature  is that n = 139/203—52%) had a higher prevalence of impacted teeth than G1 (64/203—18.2%), and the most common impacted permanent teeth were mandibular central incisors, maxillary canines, and maxillary lateral incisors. Oddly, delayed/unerupted teeth were most prevalent in G1 (n = 106/135—30%) compared to G2 (n = 29/135—11%), and the most common delayed/unerupted permanent teeth were maxillary lateral incisors, maxillary canines, and mandibular 1st molars. Furthermore, the only tooth which was statistically significant was the mandibular 2nd premolar [Regarding the dental eruption state, there were 280 (45.3%) normal positioned teeth, 203 (32.8%) impacted teeth and 135 (21.9%) delayed/unerupted in a total of 618 teeth. In our results, G2 (o G2 n = /135—11%,premolar . This stBased on the findings of our study, we concluded that GC was more common among nonsyndromic individuals than Ds individuals, the prevalence rates between the two groups presented a possible influence of Ds and its oral manifestations or craniofacial characteristics, which might increase the delay or impaction of teeth or dental eruption state, which will also prejudice GC formation, as demonstrated in our results. Therefore, the higher GC absence among Ds individuals may be a new disturbance eruption or impaction for these patients, so Ds might be a prevalence parameter for GC.


The elevated loaded partial nitritation MBBR system achieves a TAN surface area removal rate (SARR) of 2.01 ± 0.07 g TAN/m2.d and NO2−-N: NH4+-N stoichiometric ratio of 1.15:1, which is appropriate for downstream anammox treatment. The elevated TAN SALR design strategy promotes nitrite-oxidizing bacteria (NOB) activity suppression rather than a reduction in NOB population as the reason for the suppression of nitrite oxidation in the mainstream elevated loaded partial nitritation MBBR system. NOB activity is limited at an elevated TAN SALR likely due to thick biofilm embedding the NOB population and competition for dissolved oxygen (DO) with ammonia-oxidizing bacteria for TAN oxidation to nitrite within the biofilm structure, which ultimately limits the uptake of DO by NOB in the system. Therefore, this design strategy offers a cost-effective and efficient alternative for mainstream partial nitritation MBBR systems at water resource recovery facilities.There is a current need for a low operational intensity, effective and small footprint system to achieve stable partial nitritation for subsequent anammox treatment at mainstream municipal wastewaters. This research identifies a unique design strategy using an elevated total ammonia nitrogen (TAN) surface area loading rate (SALR) of 5 g TAN/m Compared to conventional nitrification and denitrification, PN/A is more cost-effective and energy-efficient and could significantly reduce the energy consumption of water resource recovery facilities (WRRFs) by 20–40% . Over thLong-term nitrite oxidation suppression in mainstream PN/A systems has been investigated using a one-stage system, where partial nitritation and anammox occur in the same reactor, or a two-stage system with partial nitritation and anammox occurring in separate reactors . While tIn a two-stage configured system, optimization of the partial nitritation process requires effective operational control strategies to ensure that the nitrite-oxidizing bacteria (NOB) population or activity is continuously suppressed within the system . In thisSeveral operational control strategies have been applied using the MBBR technology to selectively inhibit NOB population or activity . Some of2.d in an MBBR system fed with TAN concentration of 125mg TAN/L, simulating TAN concentrations observed in industrial wastewaters. In this system, stable partial nitritation was attributed to the morphological impacts resulting from operating at an elevated TAN loading rate that could allow for NOB activity to be effectively suppressed  characterize the performance of a two-reactor in series designed mainstream elevated loaded partial nitritation MBBR system; (ii) determine the effects of elevated TAN loading rate on biofilm thickness, biofilm mass, biofilm density, and embedded cells, and how these characteristics influence the performance of the mainstream partial nitritation MBBR system; and (iii) quantitate the AOB and NOB population counts within the attached growth, biofilm community and identify whether NOB population suppression or NOB activity suppression is responsible for partial nitritation.2.2.1.2/m3. The AnoxK™5 were seeded carriers harvested from a biological oxygen demand (BOD) removal municipal IFAS wastewater treatment system located in Hawkesbury, Ontario, Canada, and were operated in a single bench lab partial nitritation MBBR system operated at elevated TAN concentrations prior to this study. The reactors were designed at a target TAN SALR of 5 g TAN/m2.d, recommended as the optimum for the partial nitritation MBBR system based on the previous findings, as it demonstrated stable and steady performance as well as high partial nitritation rate  influent TAN concentrations were 30 (32.16 ± 0.46) mg TAN/L for PN1 and 20 (20.4 ± 1.54) mg TAN/L for PN2, all within the conventional limits of mainstream municipal TAN concentrations. The operational conditions for both reactors were the same with the following target (measured) values: SALR of 5 (5.07 ± 0.22) g TAN/m2.d, HRT of 2 (2.02 ± 0.02) h, DO of 6.5 (6.84 ± 0.05) mg O2 /L, pH of 7.5 (7.76 ± 0.05), and temperature 20 (19.9 ± 0.10)oC.P nitrate . Moreove2.2.4+)2SO4 (corresponding to a concentration of approximately 30 mg NH4+-N/L), 0.39 g NaHCO3, 0.06 g MgSO4· 7H2O, 0.02 g CaCl2·2H2O, 0.06 g KH2PO4, and 0.004 g FeSO4·7H2O. Trace nutrients (per L of SWW) included: MnCl2·4H2O: 0.10 mg, Na2MoO4·2H2O: 0.03 mg, CuSO4·5H2O: 0.10 mg, CoCl2·6H2O: 0.001 mg, and ZnSO4·7H2O: 0.03 mg. The carbon source composition (per L of synthetic wastewater): glucose 4.86 mg, sodium acetate 2.59 mg, and peptone 4.86 mg resulted in a sCOD concentration of 25 mg sCOD/L, thereby mimicking post-carbon effluent (Synthetic wastewater (SWW) was prepared based on a recipe from previous studies . The SWWeffluent .2.3.N1 and PN2. Wastewater samples were collected three times a week from both reactors for analyses, and the samples were tested in triplicate for the following parameters: TAN, nitrite, nitrate, DO, sCOD, pH, temperature, alkalinity, total suspended solids (TSS), and volatile suspended solids (VSS). To quantify the kinetics of the reactors, the following standard methods were used: Nessler-4500C-NH3, 4500B-NO2− and nitrate 4500A-NO3− for TAN, nitrite, and nitrate, respectively. DO and temperature measurements were acquired using a HACH Flexi HQ30d DO/temperature probe  and pH was measured using a SympHony VWR pH probe . sCOD was quantified using a HACH 8000 , and TSS and VSS were measured using standard methods 2540D .Stereomicroscopy was used to acquire images of N1 and PN2 and dried at 105°C overnight. Dried carriers were cooled in a desiccator for a minimum of 30 min, after which their weights were recorded as W1. The dried carriers were then thoroughly cleaned with a stiff-bristled brush and warm water and were dried again at 105°C overnight. The dried carriers were cooled for another 30 min in a desiccator, and their weights were recorded as W2. The biofilm mass was calculated as the difference between W1 and W2. Prior testing was done to ensure that biofilm removal and heating did not cause any significant change in the mass of the biofilm carrier.Biofilm mass was measured using a protocol described and modified by 2.5.N1 and PN2 and cut into sections to expose the inner biofilm surfaces. The sections were stained using a Film Tracer LIVE/DEAD biofilm viability kit , which comprises SYTO 9 stain (a green nucleic acid stain that illuminates the intact cell membrane) and propidium iodide, which only stains cells with damaged cell membranes . Calcofluor white stain  was used to fluoresce biofilm extracellular polymeric substances (EPS) (Biofilm carriers were harvested from each reactor Pes (EPS) . Viable es (EPS) .2.6.2.6.1.N1 and PN2, were collected, and genomic DNA from their biofilms was extracted using a FastDNA*Spin Kit for Soil . The DNA concentrations were analyzed using an Invitrogen Qubit™ 3.0 Fluorometer. Extracted DNA was stored at –80°C until ddPCR analysis.Ammonia-oxidizing bacteria and NOB cell counts were determined using droplet digital PCR (ddPCR). Two carriers each, from reactors P2.6.2.amoA-1f/amoA-2r) targeted a stretch of conserved regions of the known amoA gene sequence of Nitrosomonas europaea. Also, two coupled primers, FGPS872f/FGPS1269r and NSR1113f/NSR1264r, were used to amplify the NOB, Nitrobacter, and Nitrospria, respectively (AOB cell counts were quantified by targeting ammonia monooxygenase subunit A (amoA). The set of amoA primers .Statistical analyses of wastewater constituents, biofilm thickness, mass and viable/non-viable cell percentages were determined using the student’s 3.3.1.N1 and PN2, was operated at a target elevated TAN SALR of 5 g TAN/m2.d and showed steady and stable performance over a 90-day period of operation  of the first reactor, PN1, is 2.25 ± 0.08 g TAN/m2.d, corresponding to a TAN oxidation efficiency of 45.3 ± 1.1%. The resultant TAN SARR of 2.01 ± 0.07 g TAN/m2.d from the complete partial nitritation MBBR in series system, PN1-N2,  in series to the first reactor (PN1) in this study, the average effluent TAN, nitrite, and nitrate concentrations of 12.9 ± 1.84 mg TAN/L, 13.9 ± 1.56 mg NO2−-N/L and 3.11 ± 0.29 mg NO3−-N/L, were measured respectively. These PN2 effluent concentrations correspond to a metabolic ratio of 1.15:1. As such, this ratio provides suitable nitrogen speciation for the subsequent downstream anammox treatment, and the two-reactors in series partial nitritation MBBR system configuration in this study herein provides a stable effluent quality for subsequent downstream anammox operation.Finally, the average effluent TAN, nitrite, and nitrate concentrations from reactor Pf 0.36:1 . This stR system . Therefo3.2.3 in the PN1 and PN2 reactors, respectively. The average biofilm thickness of 547 ± 65 μm in the partial nitritation MBBR system (PN1 and PN2) is higher than the 300 μm reported in the mainstream partial nitritation MBBR system , builds an increased mass transfer resistance that possibly limits the mass transfer of oxygen from the bulk solution into the biofilm, resulting in limited DO uptake by NOB and subsequently suppressing their activity. Hence, by maintaining significant AOB activity and suppressing NOB activity, the MBBR system achieves 59.7% TAN oxidation efficiency and, thus, a NO3.6.The study shows that an elevated loading design strategy, as opposed to operational control strategies, is able to achieve stable, steady, and robust partial nitritation in an MBBR system under mainstream municipal conditions. The finding from this study provides a feasible design strategy that does not require multiple and complex operational control measures or monitoring, to achieve stable and steady partial nitritation. The operation at elevated TAN SALR and the resulting impact on biofilm morphology essentially restricts DO uptake by NOB, which is beneficial to the long-term suppression of their activity. Therefore, elevated TAN SALR allows for effective NOB suppression that could result in long-term process stability and stable effluent quality for the subsequent downstream anammox treatment at mainstream municipal wastewater. The design strategy is herein demonstrated at the laboratory scale with synthetic wastewater; hence, the elevated TAN SALR strategy requires further investigation at the pilot scale using real wastewater with the potential to guide future implementation of a high-rate, compact, and low-operational intensive partial nitritation system at WRRFs.2−-N:NH4+-N metabolic ratio. According to the results from this study, the elevated loaded partial nitritation MBBR system is robust, effective, stable, steady and can maintain appropriate effluent ratios. As such, anammox enrichment and operation under mainstream conditions is feasible with the possibility of achieving high nitrogen removal rates and long-term process stability. Also, designing the partial nitritation MBBR system at an elevated TAN loading rate allows for high rate partial nitritation and, consequently, a small land footprint. Therefore, the introduction of the combined two-stage PN/A system can be achieved where insufficient land availability exists. On this note, further studies are proposed exploring the elevated loaded partial nitritation MBBR system as a two-stage PN/A system for enhanced nitrogen removal from mainstream municipal wastewaters.In addition, regarding the future of the elevated loaded partial nitritation system following an anammox system as a two-stage configuration for effective nitrogen removal at mainstream municipal wastewater. Successful anammox operation as a two-stage PN/A system is achievable if operated with stable and steady partial nitritation effluent as well as an appropriate NO4.2−-N:NH4+-N concentration corresponding to a molar stoichiometric ratio of 1.15:1. Hence, demonstrating possible suitable nitrogen speciation for the subsequent downstream anammox treatment. The biofilm thickness and morphology in the reactors show thick biofilms with the embedded biomass showing no viability constraint, as the embedded cells remained viable, supporting the observed stable and steady partial nitritation performance. The thick biofilm morphology likely reduced the diffusive transport of DO into the biofilm, that limits the DO uptake by NOB population. The AOB and NOB gene copy numbers of the MBBR biofilm show that rather than NOB population suppression, NOB activity suppression is the likely mechanism responsible for the stable and steady partial nitritation performance in the mainstream elevated loaded partial nitritation MBBR system.The two-reactor in series elevated loaded mainstream partial nitritation MBBR system achieves stable and steady partial nitritation with an effluent NOThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.JI contributed to the conception and design of the study, performed the research, analyzed the data, and wrote the manuscript. HC contributed to data acquisition and analysis. RD developed the research questions and experimental design, supervised, and coordinated the research, reviewed the manuscript, and acquired funding for the research. All authors contributed to the article and approved the submitted version.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.


MPO-dependent oxidant formation initiates bioactive oxidation and chlorination products and induces oxidative post-translational modifications (oxPTMs) on proteins and lipid oxidation. Besides HOCl and H2O2, further reactive species such as singlet oxygen and nitric oxide are generated in inflammation, leading to modified proteins, potentially resulting in their altered bioactivity. So far, knowledge about multiple free radical-induced modifications of MPO and its effects on HOCl generation is lacking. To mimic this multi-oxidant microenvironment, human MPO was exposed to several reactive species produced simultaneously via argon plasma operated at body temperature. Several molecular gas admixes were used to modify the reactive species type profiles generated. MPO was investigated by studying its oxPTMs, changes in protein structure, and enzymatic activity. MPO activity was significantly reduced after treatment with all five tested plasma gas conditions. Dynamic light scattering and CD-spectroscopy revealed altered MPO protein morphology indicative of oligomerization. Using mass spectrometry, various oxPTMs, such as +1O, +2O, and +3O, were determined on methionine and cysteine (Cys), and -1H-1N+1O was detected in asparagine (Asp). The modification types identified differed between argon-oxygen and argon-nitrogen plasmas. However, all plasma gas conditions led to the deamidation of Asp and oxidation of Cys residues, suggesting an inactivation of MPO due to oxPTM-mediated conformational changes.Tissue inflammation drives the infiltration of innate immune cells that generate reactive species to kill bacteria and recruit adaptive immune cells. Neutrophil activation fosters the release of myeloperoxidase (MPO) enzyme, a heme-containing protein generating hypochlorous acid (HOCl) from hydrogen peroxide (H Neutrophils secrete myeloid peroxidase (MPO) during the oxidative burst, where aggressively lytic oxygen-centered radicals are secreted by these innate immune cells, supporting the first line of unspecific antimicrobial immune defense. MPO is known to generate hypobromous acid (HOBr), hypochlorous acid (HOCl), and hypothiocyanous acid (HOSCN) ,2,3,4, w2−), tyrosyl radicals, and polyphenols. This formation of compound II contains a ferryl iron-oxygen moiety, and superoxide anion radicals can convert ferric MPO into compound III, a mixed ferric-superoxide/ferrous-dioxygen complex. Interestingly, a negative feedback loop of H2O2 on MPO was shown in vitro [2O2, is present [The 150 kDa enzyme MPO forms a dimer from two light chains (15 kDa) each and two heavy chains with different sizes depending on the isomer . Different oxidized states of MPO are known for generating reactive species and are described in detail elsewhere . In briein vitro , and simin vitro ,15. Duri present ,17, supp2−), nitrate (NO3−) [.O [2 [Cold physical plasma  simultaneously generates short-lived reactive oxygen and nitrogen species ,19. By ce (NO3−) ,21 or hyO3−) [.O . PlasmasO3−) [.O  with proO3−) [.O ,25. ReceO3−) [.O ,27 and i) [.O [2 . However2O (concentration of 1 mg mL−1). The MPO supplier’s datasheet can be found as MPO  was reconstituted following the manufacturer’s recommendations in dH2) was attached around the jet for the 0.5% oxygen admixture setting, causing a near-laminar curtain gas flow around the jet effluent, suppressing its interaction with the ambient atmosphere. During all treatment conditions, the plasma effluent was in direct contact with the target surface (conducting mode) [2O (or HPLC water), and protein concentration was determined to ensure constant protein concentrations. Scavengers were freshly prepared in potassium phosphate buffer, added to the MPO solutions before the plasma treatment, and incubated for at least 10 min at room temperature. The final concentration of the scavenger was 2 mM 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide , 20 mM L-ergothioneine , 20 mM histidine , and 100 mM mannitol .The kINPen  is an atmospheric pressure plasma jet operating at radiofrequency at a sinusoidal voltage waveform, ranging from 2 to 3 kV amplitude peak at a frequency of 1 MHz. In the jet, a ceramic capillary with an inner diameter of 1.6 mm has mounted a pin-type electrode at its center with a diameter of 1.0 mm. The cold plasma is generated at the tip of the central electrode, and the effluent expands into the ambient air. The kINPen was operated at flow rates of 1.0 to 1.5 standard liters per minute (slm) with argon or helium gas with admixtures of oxygen  and/or nitrogen (0.5%). All gases were 99.999% pure . A homemade gas shielding device made of glass  (flushedng mode)  or at th2O2 and chloride ions (2O2 (0.75 mM) and 110 µL TMB solution . The solution was incubated in a 96-well plate for 3 min at 37 °C, and then the reaction was stopped by adding 50 μL of 2 M sulfuric acid. To assess the impact of plasma treatment on the enzyme activity, MPO solutions were plasma-treated at least in duplicate, and each of the duplicates was then measured in triplicate. The APF assay was performed in a 96-well plate with 90 µL PBS containing 5 µg/mL MPO per well. Then, 5 µL of 100 µM APF solution and 5 µL H2O2 were added. A kinetic experiment was started measuring the fluorescence at an λex 485 nm and λem 535 nm using a microplate reader .Enzymatic activity was measured based on the formation of HOCl by the enzymatically active MPO  from Hide ions a, assaye2, Ar/N2) for 30 s. Subsequently, the protein was digested by trypsin V5111   and subjected to nano-liquid chromatography mass spectrometry (nLC-MS). Peptides (100 ng) were loaded onto a PepMap C18 trap column. A Dionex UltiMate 3000 RSLCnano HPLC was connected to an Exploris 480 mass spectrometer using a Nanospray Flex ion source . Peptides were eluted from the trap column and separated on a 150 mm × 75 µm ID Acclaim PepMap C18 column using buffer A (0.1% v/v acetic acid) and buffer B (95:5 acetonitrile: 0.1% v/v acetic acid) at a flow rate of 300 nL/min with an elution gradient of 4–40% buffer B over 50 min. The column temperature was set to 40 °C. The mass spectrometer was run in positive polarity mode with a transfer capillary temperature of 250 °C and a spray voltage of 2 kV. Spectra were recorded in DDA acquisition mode (Top 15), and peptides were analyzed in full scan  with a target of 5 × 103 ions, followed by 15 data-dependent MS/MS scans with higher energy collisional dissociation  50 ms, isolation width 1.0 m/z, NCE 30%), detected in orbitrap . Dynamic exclusion was enabled and set to 30 s.MPO was diluted in ammonium bicarbonate buffer (10 µg/mL) and exposed to plasma . MS/MS spectra were extracted from the raw files, and search was performed using custom FASTA files, each containing a single protein sequence. MPO was unambiguously identified . PMI-Byonic  was used for the identification and quantification of PSM and PTM. The search engine parameters for PMI-Byonic were set as follows: peptide mass tolerance = 10 ppm, fragment mass tolerance = 10 ppm, cleavage specificity = trypsin, missed cleavages = 2, and total common modifications = 2. A custom modification list was used based on previous experiments  Figure . The rawg and 4 °C for 2.5 h. DLS measurements were carried out at a set angle of 90° and attenuator at 11. Size was measured at 22 °C with an equilibration time of 120 s and a cuvette position at 3 mm. Backscatter-angled detection was performed at 173° with a scattering collection angle of 147.7°. The measurement was repeated three times, and each replicate was measured in several replicates with minimal time between repeats.Particle size analysis of MPO and plasma-treated MPO was carried out using a ZS90 dynamic light scattering (DLS) device  equipped with a helium-neon laser light source (632 nm). Proteins  in a buffer  were measured in low-volume cuvettes (70 µL). All identical samples and controls were concentrated to twice the concentration (200 µg/mL) using a vacuum concentrator, pooled, and centrifuged at 21,000× MPO and oxidized MPO (1 mg/mL) measurements were performed in 1 mm cuvettes using a Chirascan CD spectrometer  coupled to a temperature controller. The CD spectra were recorded at 25 °C from 190 to 250 nm. The bandwidth was 1 nm, and the scan time per point was 1.5 s. CD analysis was measured in four replicates, each containing two pooled technical replicates. All spectra were blank-corrected.Denaturated  proteins (35 µg) were loaded with 4× sample buffer on a 10% polyacrylamide gel. Then, 10 µL of a prestained protein ladder with a 10 to 250 kDa size range was used as a protein standard . The loaded gels were run in a gel electrophoresis chamber with a running buffer, first at 50 V for 20 min and then at 120 V for 60 min. MPO and plasma-treated MPO were visualized using the ready-to-use Silver Stain Plus Kit according to the manufacturer’s instructions .p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).Graphing and statistical analysis were performed using GraphPad Prism 9.5.1 . Statistical comparisons and the tests employed are referenced in the figure legend. Levels of significance are indicated as follows: 2O2 (2O2 was observed (grey line)  showed best signal-to-noise ratios after 4 h  b. When a control c. Higher of HOCl d. Howevefter 4 h .2O2 was added (2O2 concentration (10 µM) resulted in reduced MPO activity within the first 50 min but reached similar HOCl signals to untreated MPO after 60 min , or Ar/N2 (blue) plasmas. OxPTMs that occurred in multiple plasma conditions were highlighted in yellow and labeled separately.As previous studies revealed, protein-bound amino acids show dramatically different rate constants for chemical reactions with reactive species, yielding to a variety of oxPTMs types and their abundances ,32. MPO  plasmas , which i2 plasma. On the contrary, Ar/O2 plasma did not result in any observed Tyr modifications, albeit Tyr was modified by the exposure to Ar and Ar/N2 plasmas  and Ar (20%) plasmas but not after Ar/O2 plasma exposure (8%). Those proportions were also reflected in the total number of modifications  that clustered together, while oxidized MPO treated with the different plasma conditions differed strongly from each other and the controls  or in the liquid bulk via the reaction of H2O2 with NO. Since this reaction is driven only in strongly acidic conditions (pH 3.3) [2− can also yield peroxynitrite (.NO + O2− -> ONOO−) [On the other hand, we find rescuing effects of NO scavenger cPTIO on argon plasma-treated MPO pointing to NO as one major contributor for protein oxidation by plasma. However, NO itself is not very reactive, and few oxPTMs can be attributed to its activity. Among these is the nitrosylation of free thiol groups that has been observed in a minute extent after plasma treatment of cysteine . Accordi of note . Having (pH 3.3) , it can > ONOO−) . Superox> ONOO−) . This is2O2 triggers autocatalytic reactions [2. Therefore, we conclude that plasma oxidation does not influence hem binding and probably does not promote autocatalytic reactions. We mainly identified oxidation on Met with all tested conditions, with slight variations. While Ar/O2 plasma modified 11 Met residues , the other plasmas modified only 8 Met. We further determined dioxidized and trioxidized cysteines, indicating the cleavage of disulfide bonds. Disulfide bridges are critical stabilizing motifs in maintaining the secondary structure of proteins, and previous studies have reported structural changes due to oxidative damage [In mature MPO, the amino acids Asp94, His95, Glu242, Met243, and His336 are major features of the MPO heme-binding pocket , and H2Oeactions . Due to e damage ,49 and pe damage . Importae damage , so brea2O2 and NO2− [2Tyr) and 3-chlorotyrosine [2Tyr and MPO [Moreover, DLS measurement revealed a substantial increase in the size after plasma treatment, suggesting MPO homo-multimerization that can be reversed under denaturing and reducing conditions during SDS page. However, no differences in MPO’s chlorinating activity have yet been found in monomeric and dimeric forms of MPO , suggestand NO2− . Howeverand NO2−  and is sand NO2− . Hence, tyrosine , and Bal and MPO . Other s and MPO . However2, albeit the enzymatic activity was disrupted. Since tyrosin modifications were found in all other conditions, it can be assumed that the Ar/O2 treatment led to unidentified modifications that were not captured by the software algorithm. Another reason for dimerization might be the deamidation on Asn 133, as occurs in MPO after exposure to all plasma conditions. Deamidation would suggest a hydrogen bonding shift by removing NH residue and adding O (−1H−1N+1O), leading to acidic residue formation and protein dimerization. Interestingly, conversions of Asn residues to aspartic acid through deamidation and aspartate isomerization (IsoAsp) have been reported to affect protein stability [The hypothesis of oxidative dimerization of phenols, including protein Tyr residues, would support the results of previous preclinical and clinical studies where co-localization of MPO and Tyr-modification was found. Interestingly, nitrotyrosine formation by plasma treatment was identified previously and prevented by cPTIO , supporttability ,62. Aspatability ,64.Further aspects that would help to understand the inhibitory effects of multiple reactive species on MPO are the analysis of structural changes, multimerization, and identification of specific radicals. For instance, investigating protein melting points due to the modifications would give insights into the folding. However, we can rule out that plasma fully denatures the protein since (1) kINPen plasma operates at body temperature and (2) the heat-denatured protein did not show any comparable restrictions in activity. The identification of dimerization in combination with reactive species would clarify the inactivation through a Western blot, which is more sensitive than silver staining. However, a Western blot would still require that the samples run in SDS gel, and unstable dimerization can break down due to the reducing conditions. A compound for the controlled multimerizing of MPO has not yet been described in the literature, and a similar situation is true for individual radicals as positive controls for protein oxidation. Since our study aims at mimicking an inflammatory response and not the effect of a single reactive species, their use is limited anyway. During inflammation, a mixture of several species is present simultaneously, and physical plasma technology can generate various short-lived species. Altering the gas fed into the plasma jet used in this study even changes the reactive species composition produced with this plasma.− and OCl− are more important for MPO inactivation compared to OH, and our data suggest that amino acid oxidation at multiple sites may be linked to conformational changes and reduced activity observed after MPO plasma treatment.Altogether, our study provides evidence that NO/ONOO


We hypothesize that different PET/CT scanners with distinct crystal properties will require different optimal settings for the Q.Clear algorithm. Many studies have investigated the improvement of the Q.Clear reconstruction algorithm on PET/CT scanner with LYSO crystals and SiPM detectors. We propose an optimum penalization factor (18F-FDG PET-CT scans were acquired from NEMA phantom with lesion-to-background ratio (LBR) of 2:1, 4:1, 8:1, and 15 patients with rectal cancer. Clinical lesions were classified into two size groups. OSEM and Q.Clear  reconstruction was applied. In Q.Clear, background variability (BV), contrast recovery (CR), signal-to-noise ratio (SNR), SUVmax, and signal-to-background ratio (SBR) were evaluated and compared to OSEM.β value of 500. Conversely, RC from OSEM to Q.Clear using β value of 500 decreased by 3.3–7.7% for a sphere with a diameter of 10 mm and 2.5–5.1% for a sphere with a diameter of 37 mm. Furthermore, the increment of contrast using a β value of 500 was 5.2–8.1% in the smallest spheres compared to OSEM. When the β value was increased from 100 to 500, the SNR increased by 49.1% and 30.8% in the smallest and largest spheres at LBR 2:1, respectively. At LBR of 8:1, the relative difference of SNR between β value of 100 and 500 was 43.7% and 44.0% in the smallest and largest spheres, respectively. In the clinical study, as β increased from 100 to 500, the SUVmax decreased by 47.7% in small and 31.1% in large lesions. OSEM demonstrated the least SUVmax, SBR, and contrast. The decrement of SBR and contrast using OSEM were 13.6% and 12.9% in small and 4.2% and 3.4%, respectively, in large lesions.OSEM had 11.5–18.6% higher BV than Q.Clear using β value for small lesions ranges from 200 for LBR 2:1 to 300 for LBR 8:1. For large lesions, the optimal β value is between 400 for LBR 2:1 and 500 for LBR 8:1. We recommended β value of 300 for small lesions and β value of 500 for large lesions in clinical study.Implementing Q.Clear enhances quantitative accuracies through a fully convergent voxel-based image approach, employing a penalization factor. In the BGO-based scanner, the optimal  The combination of positron emission tomography (PET) with computed tomography (CT) is commonly employed as a powerful imaging tool in the clinical assessment of oncological issues . There iβ value), which governs the overall degree of noise suppression in Q.Clear, thereby allowing for numerous iterations without a corresponding increase in noise [The block sequential regularized expectation maximization (BSREM) is a Bayesian penalized likelihood (BPL) reconstruction algorithm, marketed under the name Q.Clear  and lutetium yttrium oxyorthosilicate (LYSO) detectors are currently favored in commercial time of flight (TOF) systems due to their quicker decay times and shorter coincidence timing windows. Bismuth germanium oxide (BGO) detectors possess a broader coincidence window, rendering the incorporation of TOF technology into a PET system utilizing BGO crystals unfeasible. The BGO timing resolution is lower than that for LYSO and LSO, due to its longer decay time and lower light output. This deficiency contributes to higher scatter and elevated noise levels within the BGO scanner. Despite this, BGO crystals still offer specific advantages, such as a higher effective atomic number that results in increased detection efficiency, lower intrinsic radiation, and reduced production costs. However, their utilization necessitates reconstruction enhancement procedures . A PET schnology –23.n = 13), 18F-fluoride (n = 10), and 11C-acetate (n = 13). The data were acquired using a GE Discovery MI equipped with LYSO crystals, and reconstructed images were generated through both OSEM and Q.Clear methods, using β values of 133, 267, 400, and 533 . In tracer-specific ranges of β values, Q.Clear reconstruction yielded higher SNR (a minimum of 25%) and SBR (a maximum of 23%) compared to conventional OSEM reconstruction. Comparable levels of SNR, SBR, and noise could be achieved with BSREM in shorter acquisition times or with lower administered dosages, contrary to what OSEM could achieve[For instance, Lindström et al. analyzed data from a NEMA image quality phantom and clinical whole-body scans, including 68Ga-DOTATOC  to evaluate Q.Clear in 68Ga-PSMA PET-CT examinations. We concluded that at LBR 4:1, Q.Clear with a β value of 400 is most effective for reconstructing small lesions, while larger lesions in the phantom benefit from β values of 600 [18F-FDG solution in spheres. The data were acquired using a 3-ring DMI scanner and were reconstructed using Q.Clear with β values ranging from 100 to 500 at 100-point intervals, in contrast to OSEM. The target-to-background ratio (TBR) and contrast noise ratio (CNR) exhibited an increase corresponding to the higher beta value, whereas the noise demonstrated an inverse trend, except for the smallest sphere (10 mm diameter). For the smallest hot sphere, the TBR reached a plateau when the beta value reached 300, while the CNR gradually declined thereafter [18F-FDG radiopharmaceutical studies, the lesion-to-background ratio emerges as a crucial parameter for consideration. We incorporated three different lesion-to-background ratios in our phantom study, facilitating the evaluation of diverse lesion sizes while simulating various focal point uptakes.To the best of our knowledge, Q.Clear's performance using different s of 600 . Chen etereafter . This st18F-FDG PET-CT images using a non-time-of-flight PET/CT system based on BGO crystals. The quantitative evaluation encompassed different penalization factors (β) for both phantom and clinical data across various LBRs.Discovery IQ PET/CT scanners, which utilize BGO crystals, have gained worldwide recognition and acceptance among clinicians. The next generation of Discovery IQ has been meticulously engineered and designed to function as a scalable, high-performance diagnostic system capable of delivering exceptional image quality while employing a reduced dose. However, due to the widespread adoption of BGO crystals, specialized studies pertaining to these crystals are imperative. Our study evaluated the influence of distinct penalty functions within the Q.Clear algorithm on 18F-FDG scans, the β value is typically set within the range of 400–450 [18F-FDG. Thus, in our study, Q.Clear reconstruction employed 25 iterations and β values ranging from 100 to 500 in intervals of 100. OSEM + PSF (routine protocol), referred to as OSEM in this paper, was applied according to the manufacturer's suggestion [The images were acquired using our department's GE Discovery IQ PET-CT system . The scanner features five detector rings with 36 detector units per ring. Each detector is comprised of 6.3 × 6.3 × 30 mm BGO crystals. The scanner has a 70-cm trans-axial field of view (FOV) and a 26-cm axial FOV. It includes a 16-slice CT scanner. The PET images were reconstructed using Q.Clear and OSEM + PSF. According to previous studies, for oncology whole-body  400–450 , 26, 27.ggestion .18F-FDG solution to achieve lesion-to-background ratios (LBR) of 2:1, 4:1, and 8:1. The background was filled with activity concentrations of 5.1, 4.9, and 5 KBq/ml of 18F-FDG, respectively. A lung insert at the center of the phantom with an LBR of 8:1 was incorporated to simulate human lung tissue. This insert was filled with water and Styrofoam.A National Electrical Manufacturers Association (NEMA) image quality phantom with a total volume of 9780 ml and a height of 19.4 cm was utilized . This phAccording to NEMA phantom analysis standards, metrics such as maximum standard uptake value (zerland) , 31, 32.CR was defined as the following:where he image . BV is che image . The LE Contrast is determined as the mean voxel value in each sphere divided by the mean voxel value of the background (BG) ROIs, and SNR is the ratio of the mean voxel count of each sphere to the SD of BG ROIs.β values, the relative changes in each assessment parameter were examined at consistent intervals of β values. For this purpose, the relative difference between reconstruction a and reconstruction b was calculated as:To compare the effects of different 18F-FDG PET-CT scans of 15 patients (11 men and 4 women) diagnosed with rectal cancer. The patients, with a weight range of 45–100 kg, a median height of 160 cm, and a median age of 60 years, were intravenously administrated an activity concentration of 3.5–5 MBq of 18F-FDG per kilogram of body weight. All patients fasted for six hours before the injection, and the uptake time range was 60 ± 3.0 min. Images were captured with an acquisition time of 3 min per bed position (min/bp).We assessed the three-dimensional whole-body For the purpose of clinical image assessment, all lesions were identified by two nuclear medicine specialists and were subsequently enclosed with a volume of interest (VOI) to encompass them entirely. In the clinical data, we examined primary tumor in rectum and rectal cancer metastases spread throughout the lung and pelvic . These 45 lesions were assessed and categorized into two size groups: 27 small lesions (diameter ⩽ 10 mm) and 18 large lesions (diameter > 10 mm). Table t test in both our phantom and clinical studies. We conducted the t test individually for each evaluation parameter. This involved comparisons between two distinct reconstruction approaches, either comparing results derived from different β values or contrasting outcomes from a single β value with those from OSEM reconstruction. Statistical variances in image evaluation were assessed using the analysis of variances (ANOVA), followed by post hoc Bonferroni corrections to adjust comparisons among multiple reconstructions. P value < 0.05 was set as the significance level [We employed paired Student ce level . Statistβ value decreased. The β value escalated the RC. The percentage changes between OSEM and β values. Elevated noise in β values improved homogeneity for low LBRs, whereas lower β values were preferable for high LBRs compared to their low counterparts.Figure β values and lesion CR. Q.Clear consistently demonstrated higher CR and contrast compared to OSEM across all sphere dimensions and LBRs. For instance, at LBR 2:1, OSEM's CR values were 19.1%, 10.3%, and 6.1% lower than β values. At LBRs of 2:1, 4:1, and 8:1, OSEM had percentage disparities of − 8.1%, − 7.0%, and − 5.2%, compared to Figure β value. At LBR of 8:1, the LE values stood at 14.6 for p > 0.05) in SNR across different lesion sizes. At an LBR of 8:1  due to amplified noise. Conversely, elevated β values enhance image clarity and SNR by proficiently diminishing noise. Figure β values precipitate a reduction in both Figure β values and SNR, contrasting the trends observed in SBR and contrast. For small lesions, β values exceeding 100, and for larger lesions, β values surpassing 200 yielded a higher SNR compared to OSEM. The percentage difference in SNR between OSEM and p < 0.001) was observed in measured SBR and contrast between OSEM and Q.Clear. The percentage deviations in SBR and contrast between OSEM and There was a positive correlation between β value) with the prevalent OSEM + PSF algorithm (dubbed OSEM). Analyses were executed on NEMA phantoms filled with varying LBRs and on patients with rectal cancer administered with an 18F-FDG radiopharmaceutical.This investigation provided both quantitative and qualitative assessments of the Q.Clear reconstruction algorithm, contrasting a range of penalization factors . Q.Clear reconstructions exhibited superior ues Fig. . The BV n frames .β = 300), Q.Clear-constructed images exhibited quantitative superiority over OSEM. For instance, measurements in a 22 mm sphere at an LBR of 4:1 indicated decreases of 21.7% in ΔComparing OSEM and Q.Clear at matched noise levels . A prior study indicated that, in the case of cold lesions in the phantom, the CNR also rises with an increase in the β value, even if the LBR does not seem to influence it [The variation in BV resulting from reconstruction with different uence it .In Q.Clear, relative differences in p < 0.05, Table β values of 133, 267, 400, and 533 [In our phantom study with an LBR of 8:1, all Q.Clear reconstructions yielded significantly lower LE than OSEM. Thus, entclass1pt{minimaβ value escalated to 300. Nevertheless, minor alterations at high β values were reported. β value of > 500, plateaued.Both phantom and clinical data indicated that quantitative parameters shifted rapidly as the entclass1pt{minimaOur results demonstrate a greater relative difference in CR and BV when using Q.Clear with a BGO scanner compared to the results of previous studies with an LYSO scanner. At an LBR 4:1, the ectively . Our clipectively.β values could lead to reduced RC and, consequently, incorrect negative interpretations. Based on our phantom study, the optimal β value for small lesions ranges from 200 for LBR 2:1 to 300 for LBR 8:1, improving SUV while maintaining an acceptable noise level. For larger lesions, the optimal β value lies between 400 for LBR 2:1 and 500 for LBR 8:1 to enhance SNR. It is vital to recognize the variability in lesion ratios and the lack of preset lesion sizes within clinical studies. Detecting large lesions with high activity is relatively straightforward while pinpointing smaller lesions with low activity is more challenging. Therefore, if only one reconstruction is performed for diagnosis, a β value of 300 is recommended to achieve the most accurate interpretation of the images.Using a low penalizing parameter might increase noise, potentially leading to false positive enhancement when estimating lesion uptake or mistakenly identifying noise as lesions. Conversely, excessive smoothing in Q.Clear with higher β-factor for a broader patient population was not the focus of this investigation.Numerous factors strongly influence the optimization of the Q.Clear algorithm in PET scanning. One pivotal factor is the application or purpose of the PET scan. Different clinical or research needs might necessitate varying levels of image resolution, noise suppression, or contrast enhancement. Contrast is another important element. Lesions with higher contrast  may require distinct optimization strategies than those with lower contrast. Images with different noise and varying lesion sizes demand unique optimization. The patient count in our study was below the desired number, and the limited range of lesion sizes constrained our analysis. Larger studies are needed to confirm our findings. Assessing the effects of Body Mass Index (BMI), scan duration, various PET applications, and injected dose on optimizing the β value follows suit. For rectal cancer cases, we suggest using In conclusion, Q.Clear offers enhanced quantitative measurements while maintaining a noise level comparable to the OSEM algorithm. This results in superior image quality and lesion detection. The Q.Clear optimization depends on both lesion size and LBR. As lesion size and LBR decrease, the optimal


Chronic neuroinflammation is associated with many neurodegenerative diseases, such as Alzheimer’s. Microglia are the brain’s primary immune cells, and when activated, they release various proinflammatory cytokines. Several natural compounds with anti-inflammatory and antioxidant properties, such as epigallocatechin 3-gallate (EGCG), may provide a promising strategy for inflammation-related neurodegenerative diseases involving activated microglia cells. The objective of the current study was to examine the molecular targets underlying the anti-inflammatory effects of EGCG in activated microglia cells. BV-2 microglia cells were grown, stimulated, and treated with EGCG. Cytotoxicity and nitric oxide (NO) production were evaluated. Immunoassay, PCR array, and WES™ Technology were utilized to evaluate inflammatory, neuroprotective modulators as well as signaling pathways involved in the mechanistic action of neuroinflammation. Our findings showed that EGCG significantly inhibited proinflammatory mediator NO production in LPS-stimulated BV-2 microglia cells. In addition, ELISA analysis revealed that EGCG significantly decreases the release of proinflammatory cytokine IL-6 while it increases the release of TNF-α. PCR array analysis showed that EGCG downregulated MIF, CCL-2, and CSF2. It also upregulated IL-3, IL-11, and TNFS10. Furthermore, the analysis of inflammatory signaling pathways showed that EGCG significantly downregulated mRNA expression of mTOR, NF-κB2, STAT1, Akt3, CCL5, and SMAD3 while significantly upregulating the expression of mRNA of Ins2, Pld2, A20/TNFAIP3, and GAB1. Additionally, EGCG reduced the relative protein expression of NF-κB2, mTOR, and Akt3. These findings suggest that EGCG may be used for its anti-inflammatory effects to prevent neurodegenerative diseases. Neuroinflammation is a critical factor in the etiology of neurodegenerative diseases . The cenDue to microglial activation, cytokines and nitric oxide play a significant role in neuroinflammation. Cytokines serve as signaling molecules that mediate cellular growth, survival, and differentiation. They also modulate leukocyte traffic, the gathering of other inflammatory factors, and immune inspection . They seNeurodegenerative diseases such as Alzheimer’s disease (AD) are caused by immunosenescence, cellular bioenergetic dysregulation, deficient clearance mechanisms, and protein accumulation. Age-associated neurodegeneration alters these pathways’ typical function, leading to synaptic dysregulation, protein aggregation, blood–brain barrier disruption, chronic inflammation, and neuronal cell death. Nuclear Factor Kappa B (NF-κB) is a well-established transcription factor immunomodulator, aging controller, and mediator of inflammatory responses acting via proinflammatory cytokines, i.e., IL-6 and TNF-α ,15. The On the other hand, green tea (Camellia sinensis) is consumed all over the world ,19. GreeThis study aimed to identify EGCG’s anti-inflammatory and antioxidant activities in an established in vitro model system (BV-2) of microglial cells. Some of the significant findings from this study are that EGCG significantly reduced the protein and mRNA expression of NF-κB, PI3K-AKT, and mTOR and reduced the production of NO, IL-6, and CCL-2. Our results suggest that EGCG may be useful in delaying the onset of inflammation and oxidative stress–mediated neurodegenerative diseases involving excessively activated microglial cells.Escherichia coli O111: B4 γ-irradiated were purchased from Sigma Aldrich . Griess Reagent was purchased from Sigma Aldrich . The individual mouse Quantikine® ELISA kits for IL-6 and TNF-α were obtained from R&D Systems . Aurum total RNA mini kit, iScript™ Advanced cDNA Synthesis Kit,2-mercaptoethanol (14.2 M) 25 mL, ≥98% pure, SsoAdvanced™ Universal SYBR® Green Supermix, iScript™, Advanced cDNA Synthesis Kit, and mouse PrimePCR™ inflammation array were purchased from Bio-Rad . PrimePCR™ arrays utilized in this study were mTOR, NO, PI3K-Akt, and NF-B signaling pathways and were purchased from Bio-Rad . Pierce BCA Protein Assay kit was purchased from Bio-Rad . Protease and phosphatase inhibitors were obtained from G-Biosciences . In addition, the 12-230 Separation Module, 8 × 25 capillary cartridges, Anti-Rabbit Detection Module, HeLa Lysate Controls, and Erk1 Primary Antibody were purchased from ProteinSimple . Primary Antibodies NF-κB2 p100/p52, (Akt3 (ElZ3W), mTOR (7C10) Rabbit mAb), GAPDH, and Alpha-actinin, and secondary antibody Anti-rabbit IgG-HRP were purchased from Cell Signaling .BV-2 microglial cells were kindly provided by Elizabeta Blasi . Gibco D2/atmosphere; media was replaced every 2–3 days, and cells were sub-cultured. Plating media consisted of DMEM (minus phenol red), 2.5% FBS, and no penicillin/streptomycin for experiments. The EGCG stock was prepared by dissolving 9.2 mg in 0.400 mL of deionized water and 1.6 mL of Phenol Free DMEM Media to obtain 2 mL of 10 mM of EGCG. Different concentrations were prepared from a 10 mM stock solution of EGCG for further experiments. Activation of BV-2 cells was established using 1 µg/mL of LPS.Cells (BV-2) were grown in complete high-glucose media supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin. Culture conditions were maintained at 37 °C in 5% CO™, Thermo-Fisher Scientific, Waltham, MA, USA) indicator dye. Approximately 5 × 105 cells/mL (100 µL per well) were plated in 96-well plates overnight. The next day, cells were first stimulated with 1 µg/mL LPS for 1 h, then treated with different concentrations of EGCG (0–350 µM) and incubated at 37 °C for 24 h. Following the desired time points, 20 µL of AlamarBlue™ solution (0.5 mg/mL) was added and incubated for another 4–6 h. The fluorescent signal was monitored using a 485 nm excitation wavelength and a 590 nm emission wavelength. The fluorescent and colorimetric signal generated from the assay is proportional to the number of living cells in the sample.Cell viability was assessed using resazurin  were seeded overnight. The next day, plates were prepared following the manufacturer’s recommendations . An equal volume (50 μL) of each experimental sample (cell culture supernatant), standard, and Griess Reagent were combined, incubated for 10 min at room temperature, and protected from light. A standard curve for NO was generated from dilutions of sodium nitrite (NaNO2) (1–160 μM). Samples were analyzed at 550 nm on a UV microplate spectrophotometer .Nitric oxide (NO) production of BV-2 cells was evaluated using the Griess Reagent Assay. The Griess reagent was prepared by mixing an equal volume of 1.0% sulfanilamide in 0.5 N HCl and 0.1% N-(1-naphthyl)-ethylenediamine in deionized water. Briefly, BV-2 cells (5 × 105 cells/mL of BV-2 cells were seeded in 6-well plates (2 mL per well) overnight to attach. The next day, cells were treated with control (no treatment), 150 μM of EGCG only, 1 µg/mL of LPS only, and 150 μM of EGCG + 1 µg/mL of LPS, then incubated for 24 h. After 24 h exposure, the supernatant of each sample was collected into 5 mL tubes and centrifuged at 1000 rpm for 5 min at 4 °C to remove any particulate material. IL-6 and TNF-α ELISA kit reagents include the following: 96-well plate, the wash buffers, assay diluents, color reagents A & B, stop solution, conjugates, and standards. Briefly, seven serial diluted standards in pg/mL  and blank (no standard) were prepared. A total of 100 µL (in triplicate) of the standard, blank, and supernatant sample were added to each well in the plate, covered with an adhesive strip, and then incubated for 2 h at room temperature. Following this, the plate was washed; then, 100 µL of TNF-α or IL-6 conjugate was added to each well and incubated at room temperature for 2 h. The plate was washed again, and 100 µL of substrate solution was added to each well and incubated in the dark for 30 min. Finally, 100 µL of stop solution was added; then, the optical density of each well was read at 450 and 540 nm using the microplate reader . Optical errors were calculated by subtracting the absorbance of 540 nm from 450 nm. The standard curve was created by plotting the average of the triplicate optical density of each of the seven serial diluted standards. The concentration (pg/mL) of the samples was determined using the standard curve. This data obtained (concentrations of the sample [pg/mL]) were analyzed using GraphPad Prism 6 .This experiment used R & D Systems, mouse IL-6, and a TNF-α ELISA kit to measure IL-6 and TNF-α release into BV-2 cell supernatant quantitatively. A total of 5 × 105 cells/mL) in T-75 flasks (15 mL/flask) and treated for 24 h, as previously described. The cells were harvested after 24 h and then washed twice with PBS. Cell pellets were lysed with the 350 µL lysis solution and 350 µL of 70% ethanol and pipetted up and down. The homogenized lysate was put into an RNA binding column and centrifuged for 30 s. Then, 700 µL of high stringency wash solution was added to the RNA binding column and centrifuged for 30 s; 700 µL of low stringency wash solution was added to the RNA bind column after the column was replaced and centrifuged for 30 s and an additional 2 min to remove the residual wash solution . The RNA binding column was transferred to a 1.5 mL capped microcentrifuge tube, and 80 µL of the elution solution was added, allowing for the solution to saturate the membranes for one minute. The 1.5 mL microcentrifuge tube was centrifuged for 2 min to remove the total RNA. The eluted RNA was stored at −80 °C for later use. RNA purity and integrity were determined using Nanodrop . The cDNA strand was formulated from the RNA using Bio-Rad iScript™  advanced reverse transcriptase following the manufacturer’s instructions. A solution was created from 4 µL of the 5X iScript™ advanced reaction mix, 1 µL of reverse transcriptase, 5 µL of the RNA sample (250 ng/5 µL) for RT-PCR assay, 9 µL of nuclease-free water, and 1 µL of the RT control assay template, for a total of 20 µL. The reverse transcription thermal cycling program included two steps: 46 °C for 20 min and then 95 °C for 5 min.The Bio-Rad Aurum™ total RNA mini kit  manufacturer’s protocol was used for RNA extraction. Briefly, the BV-2 cells were seeded  and 75 °C for 5 s/step (melting curve) using the Bio-Rad™ CFX96 Real-Time System . Finally, the Bio-Rad® Mouse Inflammation, NF-κB, PI3k-Akt, mTOR, and NO signaling PrimePCR™ array data were analyzed via Bio-Rad CFX96 Manager software, version 3.1 , which calculates fold change/regulation using the ΔΔCT (threshold cycle) method using the 2−ΔΔCT formula. The p-values were calculated based on a student’s t-test of the replicate 2−ΔCT values for each gene in the control group and experimental groups. PrimePCR™ array results were normalized to GAPDH using the Bio-Rad CFX96 Manager software.The real-time PCR reaction was performed using the manufacturer’s protocol . A total of 10 µL of the cDNA of the sample (10 ng cDNA/reaction) plus 10 µL of Ssoadvanced™ BCA Protein Assay Kit. A series of concentration standards ranging from 0–2 mg/mL were prepared using bovine serum albumin (BSA). A total of 10 µL of each unknown protein sample and standard was used (sample to working reagent ratio 1:20). Then, 200 µL of the working reagent was added to each well of the 96-well microplates and mixed on a plate shaker for 30 s. The plate was covered and incubated at 37 °C for 30 min and left to cool at room temperature. Protein concentrations were quantified at 595 nm wavelength with the microplate reader Infinite M200 .Cells were treated, harvested, and centrifuged as discussed above. Then, the cell pellet was obtained and resuspended in 200 µL of lysis buffer (mixed with protease and phosphatase inhibitors) and incubated on ice for 30 min. Lysate was sonicated for 30 s using a Sonic Dimembrator  and centrifuged at 3000 RPM for 5 min. The supernatant (protein) was collected, and the protein concentration was determined using the PierceProteinSimple automated WES™ analysis was used for protein quantification . All reagents were provided by ProteinSimple, and the analysis was performed following the user’s manual. In brief, the first protocols were optimized for each antibody and protein loading. After optimization, the total protein concentration was 2 mg/mL for NF-κB and Akt3 and 1 mg/mL for mTOR. The protein extracts were mixed with a master mix to give a final concentration of 0.2 mg/mL total protein, 1× sample buffer, 1× fluorescent molecular weight markers, and 40 mM dithiothreitol. Samples were heated at 95 °C for 5 min. The primary antibody dilution factors used were: mTOR 1:150, Akt3 1:100, NF-κβ 1:100, GAPDH  1:125, and Alpha-Actinin  1:100. After samples were prepared and heated, they were loaded in the plate. Blocking solution (antibody diluent), appropriate primary antibodies, horseradish peroxidase-conjugated secondary antibodies, chemiluminescent substrate, separation and stacking matrices, and washing buffer were loaded into specified wells in a microplate. Then, the microplate was loaded into the device following the manufacturer’s instructions . Target proteins were confirmed via a primary antibody and immunosorbed using an HRP-conjugated secondary antibody and chemiluminescent substrate. Chemiluminescence was obtained via a charge-coupled device camera, and the digital image was analyzed and quantified using ProteinSimple Compass software. ProteinSimple WES™ data were normalized using GAPDH (NF-κB and Akt3), whereas α-actinin was used to standardize the mTOR data.t-test.Data evaluation was performed using GraphPad Prism (version 6.07). All data were expressed as mean ± standard error from 3 independent experiments, and the significance of the difference between the groups was calculated using a one-way ANOVA, followed by Tukey’s post hoc means comparison test or Student’s EGCG has been shown to have anti-inflammatory and antioxidative properties ,23. To aNitric Oxide (NO) is a valuable indicator of oxidative stress and inflammation . Colorim® mouse ELISA kits. The cytokine evaluation showed that concentrations of EGCG at 150 µM coupled with 1 µg/mL LPS significantly attenuated IL-6 compared to LPS (1 µg/mL)  . In FiguAs mentioned, cytokines and chemokines act as regulators of the innate neuroimmunological response in the microglia. The PrimePCR™ array was used to further validate the ELISA data and to investigate other proinflammatory cytokines affected by EGCG. Our results demonstrated that EGCG 150 µM coupled with 1 µg/mL LPS elevated the gene expression of Interleukin 3 (IL-3), Interleukin 11 (IL-11), and Granulocyte-macrophage colony-stimulating factor (GM-CSF or CSF2), as shown in Inflammatory signaling pathways have been shown to become dysregulated and maladjusted, leading to destabilized cellular metabolism, defective clearance mechanisms, and heightened microglial stress response due to neurodegenerative disorder ,31. PrimInflammatory signaling regulation at the protein level allows for the proper regulation of cellular metabolic and proliferative processes. Western blot validates the inflammatory signaling pathway action of inflammatory response and biomarkers. EGCG was shown to decrease the protein expression of NF-κB2 , Akt3 F, and mTOMicroglia are the immunomodulator cells of the central nervous system . The actNO has been used as a signal of inflammation and oxidative stress in vivo . NeuroinInflammatory cytokines and chemokines are significant indicators of neuroinflammation. The results of our ELISA experiments demonstrated that EGCG might decrease IL-6 while increasing TNF-α. IL-6 is an essential proinflammatory regulator that acts on JAK/STAT pathways in regulating inflammation. The reduction of IL-6 may be correlated to its regulation by nitric oxide in epithelial cells . It has The PrimePCR™ array evaluation of mTOR revealed that EGCG downregulated mTOR, which may act as an important mediator of various medicinal functions, i.e., anti-aging, autophagy regulation, neuroprotection, and glucose and bioenergetic mechanisms ,58. mTORThe link between PI3k-Akt and NF-κB was established with the upregulation of TLR4 and the downregulation of FOS (a transcription factor that can modulate apoptosis via AP-1). In addition, the PI3K-AKT pathway in our research outcomes displayed the downregulation of CDK1nb, which is associated with cell cycle arrest or cellular senescence. The inhibitory action of EGCG on the NF-κB pathway as well as its actions to downregulate TRAFs , allows for TLR to mediate TLR signaling , thus reducing inflammatory cytokine production (CCL5 and IL-1b). EGCG also reduced bcl3 expression, which is involved in directing NF-κB activity  and lipiRegarding the NO signaling pathway, our results show that EGCG quells overactive glutathione peroxidases (GPX1 and GPX4) or the overproduction of SOD by CYBA to reduce ROS. Nitric oxide has been shown to regulate ferroptosis, which may be a possible treatment for neurodegeneration ,68,69,70phox using an in vitro murine model system. Further investigation of AKT3 is its ability to modulate mitochondria and autophagy has been reported [The genes chosen for Protein Simple WES™ evaluation were indicative of EGCG effects on the signaling pathways themselves, i.e., NF-κB2, mTOR, and Akt3. The inhibition of NF-κB2 by EGCG may elicit further study on the effects of NF-κB in promoting inflammation and its role in mediating aging . AKT3 isreported ,76. Mostreported . Dubois reported  showed treported . EGCG domTOR is a multifaceted kinase that regulates autophagy, cellular senescence, aging, and neuroinflammation ,80,81. mNF-κB2 is the gene that encodes for the NF-κB family of proteins. NF-κB is an extensively studied inflammatory pathway. As previously mentioned, NF-κB regulates NO, which mediates oxidative stress and microglial activation via TLR signaling . Some leOur research showed that EGCG shows a profound mechanism of regulating PI3K-AKT, mTOR, and NO signaling in order to decrease neuroinflammation. EGCG’s anti-inflammatory and neuroprotective attributes were displayed by its ability to diminish the action of well-established inflammatory, stimulating cytokines, i.e., IL-6 and MIF. The neuroprotective capabilities were exhibited by the upregulation of IL-3 and IL-11. EGCG displayed some autophagic properties by acting on CTSB and PRKAB2. Although LPS was utilized to stimulate the BV-2 microglial cells, EGCG demonstrated the ability to suppress microglial initiation, which is a pivotal event in neurodegenerative disease.