Simulium guianense, the main vector of Onchocerciasis in South America, remain largely understudied. Here, we have characterized a salivary protease inhibitor (Guianensin) from the blackfly Simulium guianense.Salivary glands from blood-feeding arthropods secrete several molecules that inhibit mammalian hemostasis and facilitate blood feeding and pathogen transmission. The salivary functions from A combination of bioinformatic and biophysical analyses, recombinant protein production, in vitro and in vivo experiments were utilized to characterize the molecula mechanism of action of Guianensin. Kinetics of Guianensin interaction with proteases involved in vertebrate inflammation and coagulation were carried out by surface plasmon resonance and isothermal titration calorimetry. Plasma recalcification and coagulometry and tail bleeding assays were performed to understand the role of Guianensin in coagulation.S. guianense flies and belongs to the Kunitz domain of protease inhibitors. It targets various serine proteases involved in hemostasis and inflammation. Binding to these enzymes is highly specific to the catalytic site and is not detectable for their zymogens, the catalytic site-blocked human coagulation factor Xa (FXa), or thrombin. Accordingly, Guianensin significantly increased both PT (Prothrombin time) and aPTT  in human plasma and consequently increased blood clotting time ex vivo. Guianensin also inhibited prothrombinase activity on endothelial cells. We show that Guianensin acts as a potent anti-inflammatory molecule on FXa-induced paw edema formation in mice.Guianensin was identified in the sialotranscriptome of adult The information generated by this work highlights the biological functionality of Guianensin as an antithrombotic and anti-inflammatory protein that may play significant roles in blood feeding and pathogen transmission. Trypsin was obtained from Roche Molecular Biochemicals , and elastase was purchased from Elastin Products Company, Inc. . Kallikrein was obtained from Fitzgerald Industries International , Proteinase 3 was purchased from Merck-Millipore, and matriptase from R&D Systems . Human-purified fibrinogen and prothrombin-deficient plasma were from Diagnostica Stago . To evaluate the cleavage of the protease-activated receptor 2 (PAR-2) and for endothelial cell prothrombinase experiments, purified factor Va (FVa), prothrombin, factor X (FX), factor Xa (FXa), and endothelial culture media (MCDB 131) were purchased from Thermo Fischer Scientific . To measure the protease activity by fluorescence resonance energy transfer (FRET), substrates based on the mouse PAR-2 sequence (NSKGRSLIGR) were acquired from Anaspec .Purified human factors XIIa, Xa, and thrombin were purchased from Haematologic Technologies Inc. . Chromogenic substrates S-2222 (Methoxicarbonil-D-cyclohexylglicil-glicil-arginine-para-nitroanilide acetate) and S-2238  were obtained from Diapharma Group Inc. . Cathepsin G, human coagulation factor FXIa, urokinase plasminogen activator, and tissue plasminogen activator were obtained from Molecular Innovation . Plasmin, thrombin, α-chymotrypsin, chymase, control plasma, lipopolysaccharide , Simulium nigrimanum (ACZ28223), Zeugodacus cucurbitae (XP_011191995), Ixodes scapularis (XP_029848488), Pseudonaja textilis (Q90WA1), Vipera ammodytes ammodytes (AMH40733), and Homo sapiens (ABP02055).The amino acid sequence of Guianensin was obtained from the sialotranscriptome of uianense . SignalPuianense . The seq2.32, 0.01 M MgSO4, and 0.02 M Glucose) according to the manufacturer’s instructions (Invitrogen Life Technologies). The cells were propagated on agar plates supplemented with ampicillin (10 µg/mL) and chloramphenicol (50 µg/mL) at 37°C. Individual colonies were expanded in 1L of Escherichia coli culture following the same protocol and antibiotic concentrations described above until they reached the O.D of 0.8 at 37°C and shaking at 2,500 RPM. Bacterial growth was induced with 0.1 mM final concentration of isopropyl-ß-d-thiogalactopyranoside (IPTG) overnight at 20°C and shaking at 100 RPM. Bacterial cells were harvested by centrifugation at 6,000 RPM for 20 min and stored at -80°C until protein extraction and purification.The mature peptide of Guianensin was chemically synthetized containing two restriction enzyme sites (NdeI 3’end and XhoI 5’-end) and a 6x-His tag before the stop codon . The synthetic gene coding for mature Guianensin was optimized for bacterial expression and subcloned into pET17b . Chemically competent BL21(DE3) pLysS cells were transformed with the pET17b-Guianensin plasmid and cultured in 250 µL of SOC medium ). After sonication, the soluble fraction was separated from the cell debris by centrifugation at 12,000 RPM for 30 min. The supernatant was supplemented with 500mM NaCl and 5 mM Imidazole and loaded onto an affinity column , followed by size-exclusion chromatography . The identity of purified Guianensin was confirmed by Edman degradation. Purified protein was further processed to remove any LPS contaminant and verified using Endosafe nexgen-PTS  as described in a previous report . The con2.5(CD) analyses, 0.1 mg/mL of purified Guianensin in 20 mM Tris-HCL 150 mM and NaCl pH 8.0 was used. Continuous measurements with a pitch of 0.2 nm were recorded from 200–250 nm wavelengths with a bandwidth of 1 nm. Mean residue ellipticity was calculated with the following equation:  × θλ))/(10 × pathlength in cm × protein concentration in g/mL). All readings were normalized by subtracting with blank (buffer) mean residue ellipticity. Data were analyzed using CAPITO  at 10 s intervals for over 60 min. Recalcification was determined as the time for each sample to reach 0.025 absorption units at 650 nm. aPTT and PT were measured on a Start 4 Stago coagulometer  following the manufacturer’s instructions. Lyophilized citrated normal human control plasma  was reconstituted with distilled water, and 50 µL was transferred to an individual cuvette containing 5 µL of phosphate-buffered saline (PBS) or different concentrations of Guianensin . Each reaction was incubated for 2 min at 37°C. For aPTT, the mixture was supplemented with 50 µL of prewarmed aPTT reagent followed by 3 min of incubation. Coagulation was initiated by the addition of 50 µL of CaCl2 . For the PT assay, 100 µL of Neoplastine CI Plus reagent (Diagnostica Stago) was added to the sample to initiate the clot formation. All experiments were carried out in duplicate.The recalcification time of human plasma was carried out as described previously . Briefly2.72. Calcium chloride was not used for thrombin preparation. Then, samples were incubated for 15 min at 37°C followed by the addition of 5 µL of chromogenic substrates (S-2222 for FXa or S-2238 for thrombin) at final concentrations of 5 mM, with a final reaction volume of 105 µL. Absorbance at 405 nm was measured on a VersaMax microplate reader  at 37°C, with reads every 10 sec for 1h.The role of Guianensin on two common points (Factor Xa activation and thrombin formation) of the coagulation pathway was investigated. In a 96-well plate, 25 µL of TBS  or Guianensin in TBS at 83 µg/mL or 57 µg/mL was added to each well, in triplicate, for FXa or thrombin assay, respectively. Seventy-five microliters of FXa (0.5 nM) or thrombin (0.1 nM) solution were added to the wells. FXa was prepared in TBS supplemented with 0.5% BSA and 5 mM CaCl2.82, 0.3% [w/v] BSA, and pH 7.5). Guianensin (125 pM–0 pM) was incubated with FX  for 20 min at RT. Cascade activation was followed by the addition of human factor Va (1 nM) and phosphatidylcholine and phosphatidylserine (PC/PS) at 10 µM with an incubation time of 5 min. Prothrombinase complex was triggered by the addition of human prothrombin (1.4 nM), and aliquots of 25 µL was collected every other minute and transferred to a new plate containing stop solution . Twenty-five microliters of S2238 (312.5 µM) were added to each collected aliquot. A kinetic evaluation of the effect of Guianensin in prothrombin assembling was assessed by measurement of absorbance at 405 nm at 37°C during 15 min with 11-sec intervals between reads using a VersaMax microplate reader .Factor Xa inhibition properties of Guianensin were evaluated regarding its role to interfere in the activation of the prothrombin complex by human FXa. The assay was performed as described previously . Briefly2.950 (nM) and the Ki (nM) of each interaction were calculated as described elsewhere (Guianensin was screened against a broad panel of various serine proteases involved in blood coagulation and inflammation pathways. The assay was performed as described previously  using a lsewhere .2.10a) and dissociation (Kd) rate constants and affinity constant (KD) were obtained using a global fitting and 1:1 interaction model using the Biacore T200 Evaluation software.Affinity experiments were carried out by surface plasmon resonance (SPR) using an SPR T200 instrument . Materials  were obtained from GE Healthcare. Guianensin was immobilized at 1250 RU on a CM5 sensor chip by the amine coupling method followed by ethanolamine blocking of empty sites. An additional flow cell on the same sensor chip was immobilized with a running buffer only and served as a non-specific binding parameter during the analysis. The sensor was regenerated with 10 mM glycine-HCl pH 2.0 after each cycle. A blank sensogram, obtained from running buffer only as an analyte, was used to subtract the bulk refractive index background against running curves. Biacore T200 Evaluation Software package was used for the analysis. The analytes investigated included human FXIa; human FXa; human FIXa; Plasmin; Kallikrein; zymogens (Protein Z); Gla-domainless factor Xa (hGla (–)), which is a truncated form of factor Xa, human factor Xa, bovine factor Xa, mouse factor Xa, human ß-factor Xa (hß), and the human factor Xa that had the catalytic site blocked (hDEGR). A kinetic assay was performed for each confirmed molecular partner of Guianensin  with a 500 nM starting concentration and six serial dilutions. Binding running parameters were set at a contact time of 180 sec, a flow rate of 30 µl/min, and 600 sec for dissociation. The individual association . Samples were prepared in TBS buffer (20 mM Tris pH 8.0 and 0.15M NaCl) and degassed using a MicroCal ThermoVac  prior to loading. Titration experiments were performed by successive injections of FXa at 30 μM into the sample cell containing 3 μM Guianensin. Assays were performed at 30°C, and measured heats were converted to enthalpies and analyzed by fitting to a single-site binding model on the Microcal Origin software package version 7 (OriginLab).2.12Ten micromolar of Guianensin or FXa alone, or at an equal 1:1 molar ratio, were incubated for 16 h at 37°C in TBS-Ca in a 25 µL total reaction volume. Following the incubation period, 10 µL of LDS buffer (Invitrogen) was added to each sample in the presence of a 4 µL reducing agent (Invitrogen). Samples were loaded onto a 4%–12% SDS-PAGE gel under reducing and non-reducing conditions and blotted to a nitrocellulose membrane. Bands were stained with Coomassie blue without acetic acid. Fragments from the Guianensin cleavage were excised and submitted for N-terminal sequencing for identification.2.13g for 5 min in a bench-top centrifuge. Blood samples were diluted 1:5 in water and added to microtiter flat-bottom 96-well plates  in technical triplicates. The absorbance was measured at 540 nm in a VersaMax microplate reader (Molecular Devices).All procedures were performed in accordance with the animal study protocol approved by the NIAID Animal Care and Use Committee (ASP#: LMVR3). Sixteen to twenty-week-old female mice (C57BL/6) were anesthetized using a mixture of ketamine and xylazine (90 mg/kg and 10 mg/kg IP) and placed on a warming pad at 37°C. The tail was measured and marked with ink at 1 cm from the tip and transected with a scalpel to lacerate the tail vein. The tail was hung over the edge of the warming pad and immersed in warmed tubes at 37°C containing 100 µl of either water as a control or Guianensin at two different concentrations (0.3 µM and 1 µM), ensuring that the tail tip did not touch the walls or the bottom of the tube. Shed blood was collected for 15 min. The blood volume and concentration of hemoglobin were used to estimate the quantity of shed blood. To measure the hemoglobin content, 900 µl of distilled water was added to each tube to lyse red blood cells and release hemoglobin. Tubes were vortexed, incubated for 10 min at room temperature, and spun down at 12,000 x 2.14at p < 0.05 .In this study, we utilized 8-week-old female C3H/HeJ mice obtained from Charles River Laboratories and housed in the NIAID Animal Care Facility. Posterior footpads were injected with PBS, Guianensin (10 µg), FXa alone (10 µg), or FXa incubated with Guianensin (10 µg + 10 µg) for 15 min. Intradermal/subcutaneous (s.c.) injections were performed by injecting 50 µl of sample into the footpads using BD Microfine IV needles. Before each injection and at 15, 30, 45, and 60 min, a caliper  was used to measure the thickness of the posterior footpad. In these experiments, statistical significance was determined by two-way ANOVA, using the FXa alone group as the positive reference of inflammation. Statistical differences were set 2.152 environment following standard cell culture procedures. HMEC-1 cells (1 x 103) were seeded in 96-well plates until they reached a confluent monolayer. The endothelial cells were then activated for 6 and 24 h with 1 μg/mL LPS (O111:B4) in the absence of fetal bovine serum. After the incubation time, culture media was removed, and human plasma (50 μL) was added in the presence or absence of different concentrations of Guianensin  in a final volume of 150 μL. The coagulation process was triggered by 10 mM CaCl2, and clot formation kinetics was followed directly on endothelial cell monolayers at 650 nm for 40 min using a Cytation 5 microplate reader . Coagulation time (measured in seconds) was defined as the time taken to reach the 0.05 absorbance value .Human Dermal Microvascular Endothelial Cells (HMEC-1) from ATCC  were cultured in MCDB 131 medium supplemented with epidermal growth factor (10 ng/mL), hydrocortisone (1 μg/mL), glutamine (10 mM), and fetal bovine serum (10%). The cells were maintained without antibiotics in a 37°C/5% CO2.162. The kinetics of FXa formation was measured directly on endothelial cell monolayers at 405 nm in the presence of 2 mM S2222 substrate for 40 min using a Cytation 5 microplate reader . In another set of experiments, thrombin generation was also investigated on HMEC-1 by measuring prothrombinase complex activity. In this case, prothrombinase components  were assembled on HMEC-1 confluent monolayers and incubated with Guianensin. Thrombin formation was detected after adding human-purified fibrinogen  in the presence of 10 mM CaCl2. Similarly, the kinetics of fibrinocoagulation was followed at 650 nm in the Cytation 5 microplate reader .FXa generation was measured on HMEC-1 surfaces through tenase complex activation using human plasma deficient in prothrombin. For this purpose, confluent HMEC-1 cells were incubated with Guianensin  in the presence of prothrombin-deficient plasma (50 µL). Then, the tenase complex was activated by adding thromboplastin (10 µL) in the presence of 10 mM CaCl2.172O, and 1% FA). Samples were introduced using a syringe pump  with a flow rate of 5 μL/min. Xtract algorithm of the Freestyle  was used to deconvolute the raw data. Deconvolution parameters were set as output mass, MH+; charge range, 1–5; minimum number detected charge, 2.FXa cleavage of PAR-2 peptide in the presence of Guianensin was performed as described previously . FXa  software.The data are presented as means ± SE, and significant differences were analyzed by one-way ANOVA followed by an unpaired 33.1S. guianense revealed the presence of a typical single Kunitz protein deducted from three expressed sequence tags (ESTs). The cDNA of Guianensin (GeneBank: JI626169) has an open reading frame of 321 base pairs (bp) that encodes for a protein of 106 amino acids (AA). It has a predicted signal peptide  indicates that one molecule of Guianensin binds to one molecule of FXa  experiments were performed. Typical kinetic sensograms revealed that Guianensin exhibits high affinity binding to FXa Table 1. species .3.4S. guianense blood-feeding behavior. Guianensin significantly reduced the activity of cathepsin G, kallikrein, trypsin, α-chymotrypsin, β-tryptase, plasmin, elastase, proteinase 3, and the coagulation factor XIa (50) and inhibition constant (Ki) values for each serine protease were calculated as described previously (Ki) values calculated for each Guianensin-serine protease were in the low nM range, suggesting a strong inhibitory activity against these enzymes .Planned the experiments: PV-L, AC, LT, and EC; performed the experiments: PV-L, AC, IM-M, MK, GS, AW, LT, and EC; analyzed data: PV-L, AC, IM-M, AP, MK, LT, and EC; wrote the paper: PV-L, AP, and EC. All authors contributed to the article and approved the submitted version.


The role of HbA1c in women with gestational diabetes mellitus (GDM) is still unclear, particularly in the Asian population.To investigate the association between HbA1c levels and adverse outcomes considering maternal age, pre-pregnancy body mass index (BMI), and gestational weight gain (GWG) in women with GDM.A retrospective study included 2048 women with GDM and singleton live births. Using logistic regression, the associations between HbA1c and adverse pregnancy outcomes were assessed.Compared to women with HbA1c ≤ 5.0%, HbA1c was significantly associated with macrosomia , pregnancy-induced hypertension , preterm birth , and primary Cesarean section  in GDM women with HbA1c ≥5.5% while significantly associated with PIH  in women with HbA1c 5.1-5.4%. The associations between HbA1c and adverse outcomes varied with maternal age, pre-pregnancy BMI, and GWG. In women aged ≤29 years, there’s significant association between HbA1c and primary C-section when HbA1c was 5.1-5.4% and ≥5.5%. In women aged 29-34 years and HbA1c ≥5.5%, HbA1c was significantly associated with macrosomia. In women aged ≥35 years, there’s significant association between HbA1c and preterm birth when HbA1c was 5.1-5.4% and macrosomia and PIH when HbA1c ≥5.5%. In pre-pregnant normal-weight women, HbA1c was significantly associated with macrosomia, preterm birth, primary C-section, and PIH when HbA1c ≥5.5% while HbA1c was significantly associated with PIH when HbA1c was 5.1-5.4% . In pre-pregnant underweight women with HbA1c 5.1-5.4%, HbA1c was significantly associated with primary C-section. HbA1c was significantly associated with macrosomia among women with inadequate GWG or excess GWG and HbA1c≥5.5%. In women with adequate GWG, there’s significant association between HbA1c and PIH when HbA1c was 5.1-5.4% and ≥5.5% .Conclusively, HbA1c at the time of diagnosis is significantly associated with macrosomia, preterm birth, PIH, and primary C-section in Chinese women with GDM. The incth percentile , and gestational weight gain (GWG) among GDM women, which might provide evidence for the prevention of adverse outcomes in GDM pregnant women.22.1A retrospective study was conducted among women with gestational diabetes who received regular prenatal care and delivered at the Women’s Hospital, School of Medicine, Zhejiang University from 1-July-2017 to 30-June-2018. Women who were diagnosed with GDM by OGTT in the second trimester of pregnancy, delivered a live singleton more than 28 gestational weeks, and had complete medical records were included. Women who had a prior history of diabetes mellitus, chronic diseases , autoimmune diseases , or tumors were excluded. Finally, 2048 GDM women were included in this study.l hypertension, preeclampsia, eclampsia) was obtained.Relevant information about pregnant women, including age, height, weight before pregnancy (within one month before pregnancy), weight gain during pregnancy, gravidity, parity, OGTT value , HbA1c, mode of delivery, gestational week of delivery, neonatal birth weight, pregnancy complications such as macrosomia, pregnancy-induced hypertension  automatic analyzer. HbA1c was measured by high-performance liquid chromatography (HPLC) on an automated glycosylated hemoglobin analyzer (HLC-723G8), which has been certified by the National Glycohemoglobin Standardization Program (NGSP) to conform to the results of the Diabetes Complications and Control Trial and standardized according to International Federation of Clinical Chemistry (IFCC) reference system.2.2.22), normal weight (18.5 kg/m2-23.9 kg/m2), overweight (24.0 kg/m2-27.9 kg/m2), and obese (≥28.0 kg/m2) groups according to Chinese criteria. ];2013 Available online: http://www.nhc.gov.cn/ewebeditor/uploadfile/2013/08/20130808135715967).BMI was calculated as pre-pregnancy weight in kilograms(kg) divided by the square of height in meters(m). Pre-pregnancy BMI was categorized into underweight (<18.5 kg/m2.2.3GWG was the difference between pre-delivery and pre-pregnancy weight. According to the standard definition of the Institute of Medicine (IOM) guidelines in 2009 , appropr2.2.4Neonates were defined as LGA if their birth weight was >90th percentile based on national population references for age and sex. Neonates with gestational age ≥ 28 weeks and < 37 weeks were considered as preterm neonates. Neonates with birth weight ≥4000g were defined as macrosomia. PIH was diagnosed in women with no previous history of hypertension with systolic blood pressure (SBP) ≥140 mmHg and diastolic blood pressure (DBP) ≥90 mmHg on two occasions at least 4 hours apart after 20 gestational weeks with or without proteinuria .2.3p-values less than 0.05 were considered significant. All statistical analyses were done with SPSS 26.0 software.Maternal and neonatal demographic and clinical features were reported as frequency (%) or means ( ± SD). Categorical variables, including maternal age groups, parity, gravidity, pre-pregnancy BMI group, GWG groups, and difference in the incidence of adverse pregnancy outcomes among HbA1c groups, were evaluated by chi-squared test. Continuous data, including birthweight, FPG, 1h-PG, 2h-PG, and maternal age, were evaluated using one-way ANOVA. HbA1c level was divided into three different categories by quartiles, which included ≤25th , 25th-75th  and ≥75th . Logistic regression was used to explore the association between HbA1c level and adverse outcomes in different maternal age groups, pre-pregnancy BMI groups, and GWG groups. Two-sided 33.1Our study enrolled 2048 women with GDM of live singleton births without missing data Figure 13.2In GDM women with HbA1c ≥5.5%, HbA1c was significantly associated with preterm birth , macrosomia , and primary C-section  compared to their counterparts with HbA1c ≤5.0%. Interestingly, both GDM women with HbA1c 5.1%-5.4% and HbA1c ≥5.5% had significantly increased risk of PIH , respectively compared to their counterparts with HbA1c ≤5.0% Table 2.3.3There w ere significantly positive associations between HbA1c level and primary C-section in women aged ≤29 years with HbA1c 5.1 - 5.4%  or HbA1c ≥5.5%  compared to their counterparts with HbA1c ≤5.0%. Interestingly, young women aged ≤29 years showed an increased risk of PIH when their HbA1c was≥5.5% . Additionally, women aged ≥35 years with HbA1c ≥5.5% also showed an increased risk of PIH  compared to women ≥35 years with HbA1c ≤5.0%. HbA1c ≥5.5% was significantly associated with macrosomia among women aged 30 -34 years old  and those aged ≥35 years  compared to HbA1c ≤5.0% Table 3.3.4Pre-pregnant normal-weight women with HbA1c ≥5.5% had significantly increased risk of preterm birth , macrosomia , PIH  and primary C-section  compared to pre-pregnant normal weight women with HbA1c ≤5.0%. Interestingly, pre-pregnant underweight women with HbA1c 5.1 - 5.4% at the time of GDM diagnosis were significantly associated with a higher risk of primary C-section compared to their counterparts with HbA1c ≤5.0%  compared to their counterparts with HbA1c ≤5.0%. On the other hand, women with inadequate GWG or excess GWG with HbA1c ≥5.5% also showed an increased risk of macrosomia compared to women with inadequate GWG or excess GWG who had HbA1c ≤5.0%  Table 5.4This retrospective study demonstrated a strong relationship between HbA1c at the time of GDM diagnosis (24–28 weeks) and adverse pregnancy outcomes  in Chinese women with GDM. Chinese women below recommended HbA1c (6.0%) by ADA might be at high risk of adverse outcomes. In our study, women with HbA1c ≥5.5% had a higher rate of adverse outcomes compared to women with HbA1c 5.1%-5.4% and ≤5.0%. Compared to HbA1c ≤5.0%, HbA1c ≥ 5.5% was significantly associated with an increased risk of macrosomia, preterm birth, PIH, and primary C-section. Our results support the existing evidence that HbA1c might be a biomarker for predicting adverse pregnancy outcomes in GDM women; however, we innovatively demostrated that maternal age, pre-pregnancy BMI, and GWG should be considered when determining the relationship between HbA1c and adverse outcomes. Therefore, our findings may help initiate focused individual prenatal care, health education, and strict counselling to prevent adverse outcomes in high-risk GDM women.HbA1c during mid-pregnancy have been reported to have the risk of adverse outcomes; however, findings are still controversial. This is due to the measurement of HbA1c in different gestational age, different population involved in the study, and different GDM diagnostic criteria. Given this background, there is still lack of optimum HbA1c for identifying adverse outcomes for GDM women. Surprisingly, HbA1c <5.0% (31mmol/mol) in Asian Indian women with GDM was associated with an increased risk of adverse outcomes . A studyStudies have indicated a strong relationship between HbA1c lower than recommended cutoff <6.0%(42mmol/mol) and macrosomia in Asian women with GDM, similar to our findings , 21, 25.Preterm birth is the leading cause of neonatal mortality and morbidity . ContrarAsian women have lower HbA1c levels compared to other women; thus, the ADA HbA1c cutoff of <6.0%(42mmol/mol) used based on studies that involved only Caucasian women might be higher for Chinese GDM women. An increase in HbA1c is related to the occurrence of microvascular disease, which may play a certain role in the pathogenesis of PIH . Moreove2 and HbA1c ≤5.5% (37mmol/mol) had a lower risk of C-section (2 and HbA1c ≥5.5%(37mmol/mol) were not evaluated in Antoniou et al.’s study. Our findings are in accordance with the HAPO study that showed HbA1c ≥5.8%  was significantly associated with an increased risk of primary C- section compared to lower HbA1c levels in pregnant women with hyperglycemia  is well established, there is still a lack of specific guidelines on HbA1c to prevent adverse outcomes in GDM. Our findings indicated that even though the recommended HbA1c cutoff for pregnant women with diabetes is <6.0%(42mmol/mol), it is still crucial to consider HbA1c cutoffs specific for women with GDM in consideration of race. Disregarding relatively higher HbA1c within the normal range in Chinese women with GDM can lead to severe adverse pregnancy outcomes ; thus, eTo the best of our knowledge, this study is the first to explore the association between HbA1c levels and adverse outcomes considering maternal age, pre-pregnancy BMI, and GWG. Our findings may help healthcare providers to manage GDM pregnant women personally and reduce the risk of adverse outcomes using HbA1c level, pre-pregnancy weight, maternal age, and GWG.There are several limitations to our study. Firstly, we included a relatively small-size sample. Secondly, there was no further exploration of demographic characteristics, nutrition, and lifestyle, which may influence the results of our study despite the adjustment of confounders. Finally, this was a single-center and retrospective study; further multi-center and future research is required to investigate the utility of HbA1c in predicting adverse outcomes in different ethnicities and gestational age in consideration of pre-pregnant BMI, maternal age, and GWG.Conclusively, HbA1c is significantly associated with macrosomia, preterm birth, PIH, and primary C-section in GDM women, particularly in women with HbA1c≥5.5%. Our findings may help healthcare providers identify women at high risk of adverse outcomes and manage pregnant women with GDM through counselling and health education by their HbA1c, thereby reducing the incidence of adverse outcomes in GDM. Nonetheless, Chinese women with HbA1c below the recommended HbA1c cut-off are also at high risk of adverse outcomes, which should not be disregarded. Thus, further advanced studies are needed to determine optimal HbA1c cut-offs for predicting adverse outcomes in consideration of Chinese population characteristics. Most importantly, maternal age, pre-pregnancy BMI, and GWG should be considered while evaluating the association between HbA1c and adverse outcomes.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was approved by the Human Ethics committee at Women’s Hospital, School of Medicine, Zhejiang University (IRB-20210269-R). The patients/participants provided their written informed consent to participate in this study.Conception and design: ZL, DC, and LQ; Analysis and interpretation of the data: All authors; Drafting of the paper: MM and LZ; Paper revision and editing: ZL; Revising paper critically for intellectual content: All authors; Data collection: QW and LZ; Final approval of the version to be published: All authors. All authors agreed to the final content of the manuscript for submission and accountability for all aspects of this work.


Sargentodoxa cuneata (EAdSc) has potential anti-inflammatory activity, its effects on UC remain unknown. In this study, the constituent compounds discussed in the literature and identified by gas chromatography and mass spectrometry (GC–MS) were collected, and the blood-soluble components of EAdSc were identified by liquid chromatography–mass spectrometry. The network pharmacology analysis and molecular docking analysis were performed to explore the potential underlying mechanism and active ingredients of EAdSc against UC. Furthermore, mice with dextran sulfate sodium (DSS)-induced UC were used to study the therapeutic effects and validate the mechanism of EAdSc against UC. A total of 53 compounds from EAdSc were identified in the literature and by GC–MS, and 22 blood-soluble EAdSc components were recognized. Network pharmacology analysis revealed that multiple inflammatory signaling pathways are involved in EAdSc’s anti-UC activity. Furthermore, molecular docking analysis showed that the eleutheroside A, liriodendrin, epicatechin, 2-methoxy-4-vinylphenol, catechin, androsin, coumaroyltyramine, and catechol may be active against UC through the TLR4/NF-κB/NLRP3 pathway. EAdSc reduced the disease activity, macroscopic colon damage, and histological damage indices, as well as inhibiting DSS-induced spleen enlargement and colon shortening. In addition, EAdSc decreased the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-17, as well as the expression of TLR4, NF-κB p65, NLRP3, and Caspase-1 mRNA in colon tissues. These results provide insights into the anti-UC effects and underlying mechanisms of EAdSc and help elucidate the active ingredients of EAdSc in the treatment of UC.Ulcerative colitis (UC) is an intractable disease prevalent worldwide. While ethyl acetate extract from decoction of  Ulcerative colitis (UC) is a chronic intestinal immune inflammatory disease, the course of which is protracted. As lifestyles continue to change, the worldwide prevalence of UC has increased annually . CurrentSargentodoxa cuneata grows in sunny forests and is mainly distributed in the subtropical zone of China  and [No. 20220003].Gas chromatography–mass spectrometry (GC–MS) was performed on an Agilent HP6890/5975C gas chromatograph with an Agilent HP-5MS mass spectrometer (60 m × 0.25 mm × 0.25 μm). An elastic quartz capillary column was used for chromatographic separation. The programmed oven temperatures were as follows: initial temperature at 40 °C for 2 min, increased to 215 °C at a heating rate of 3.5 °C/min, and then increased to 310 °C at a heating rate of 8 °C/min. The total analysis time was 74 min. The gasification chamber temperature was 230 °C, and the carrier gas was 1.0 mL/min helium. The temperature of the ion source was set at 230 °C, and the mass scanning range was set between 29 and 500 amu in the full scan mode. The injection was performed in the split mode with a split ratio of 10:1. Compounds were identified by comparing the obtained mass spectra of the analytes with those of authentic standards from the NIST 17 library and Wiley 275 standard mass spectra. The relative content of each chemical compound was calculated using the peak area normalization method.Ten rats were intragastrically administered EAdSc (200 mg/kg) for 3 days. At 15 min, 30 min, 1 h, 2 h, and 4 h after the last administration, two rats were anesthetized and sacrificed at each time point, and blood was collected through their femoral arteries. The collected blood was anticoagulated using sodium heparin. The samples were centrifuged at 10,000 rpm for 10 min at 4 °C, and the resulting supernatant was considered the drug-containing plasma. The drug-containing sera obtained at each of the five time points were mixed, and 5 mL of the mixed sera was added to 50 mL of extract liquor (methanol), vortexed for 2 min, and centrifuged at 13,000 rpm for 10 min. The resulting supernatant was evaporated until dry, dissolved into 500 μL methanol, and centrifuged once again at 13,000 rpm for 10 min again. The supernatant was used for LC–MS analysis. Additionally, a control group was established. Except for the administration of EAdSc, all procedures were the same as those for the administration group.TM Triple TOF5600+ mass spectrometer were used for analysis. Elution was performed in a Waters BEH C18  with a column temperature of 40 °C and a constant flow rate of 0.3 mL/min. The injection volume was 10 µL for each determination. The elution program was as follows: 0–2 min, 5–5% B; 2–10 min, 5–95% B; 10–13 min, 95–95% B; and 13–15 min, 95–5% B . Spectra were acquired in both positive and negative ionization modes, with the capillary voltage set at −4400 V and +5500 V. The collected data were further processed using MS-DIAL 4.60 software and searched qualitatively through MassBank, Respect, and the GNPS library.The blood-soluble components of the EAdSc were identified. Briefly, a SHIMADZU Nexera UPLC LC-3A system  and an AB SCIEXhttps://www.ncbi.nlm.nih.gov/pccompound/, accessed on 14 September 2022) and Swiss Target Prediction platforms  were used to identify the potential targets of the EAdSc components. Gene data were obtained from the GSE206285 gene chip test results in the GEO database , and the absolute values of the gene difference between healthy subjects and UC patients (|log FC|) ≥ 1, and p < 1 × 10−15 were used as screening criteria to analyze the gene expression information and obtain the differential genes of UC. Using EAdSc components, EAdSc component targets, and UC targets combined with the STRING platform , the “component–target–disease” network of EAdSc components was constructed by Cytoscape 3.9.1. The maximal clique centrality (MCC), maximum neighborhood component (MNC), Closeness Centrality, Betweenness Centrality, and Degree algorithms in the plug-in CytoHubba were used to screen core targets, and the targets that simultaneously satisfied greater than the median of the above five parameters were considered core targets.The NCBI PubChem Compound  and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on core targets of EAdSc components and UC. Using the R language, the data package “org.Hs.eg.db” was used to encode the gene. Then, the data packages “colorspace” and “stringi,” as well as the “DOSE,” “clusterProfiler,” and “pathview” data packages of the Bioconductor platform and the ClueGo plug-in in the Cytoscape 3.9.1 software were used to perform GO biological process enrichment analysis and KEGG pathway enrichment analysis.https://www.rcsb.org/, accessed on 5 August 2023). The core protein PyMol software 2.6 was used to virtually perform dehydration and hydrogenation of the proteins, and the format of the core compounds and target proteins was converted to *pdbqt format using AutoDock software 4.2. Docking analysis was performed using AutoDock Vina software. The grid boxes for TLR4 , NF-κB p65 , NLRP3 , ASC , and Caspase-1  Å were applied. The docking results were visualized using Discovery Studio Visualizer software 2016. The protein–ligand complex with the lowest binding energy  was determined to be the most suitable structure.Molecular docking technology was used to identify the components of EAdSc effective against UC based on the TLR4/NF-κB/NLRP3 pathway. The core non-blood-soluble EAdSc compounds (*mol2 format) were downloaded from the Traditional Chinese Medicines Systems Pharmacology (TCMSP) database, while the 3D structures (*PDB format) of the various target proteins, TLR4 (PDB:3FXI), NF-κB p65 (PDB:7O6J), NLRP3 (PDB:7ALV), ASC (PDB:5H8O) and Caspase-1 (PDB:6F6R) were obtained from the Protein Data Bank (PDB) database (Sargentodoxa cuneata. UC was induced in mice by administration of 3.0% (w/v) DSS in drinking water for 7 days. Mice were then given normal drinking water for another 3 days before sacrifice. From the first day of giving DSS in drinking water, the control and UC groups were given free water, the mesalazine group was administered 0.52 g/kg mesalazine by gavage, and the EAdSc groups were dosed by intragastric gavage with corresponding oral doses of EAdSc daily for 10 days. On Day 10, the mice were sacrificed by cervical dislocation, and the lengths of the colon tissue from the proximal end of the rectum to the ileocecal junction were measured. Simultaneously, the spleen was weighed, and the spleen index was calculated using the following formula: Spleen index (100%) = spleen weight/mouse body weight × 100. Colon tissues were immobilized in 4% paraformaldehyde solution or stored at −80 °C for further analyses.After acclimatization for 7 days, the mice were randomly assigned to one of six groups (n = 8 per group): control group, UC group, mesalazine (0.52 g/kg) group, and three EAdSc groups of varying concentrations . The mesalazine and EAdSc doses were determined based on the clinical doses and yield of the extract. The 14.63 mg/kg dose of EAdSc was equivalent to a human clinical dose of 0.25 g/kg of Body weight, stool consistency, and occult blood levels were recorded daily during UC induction. The DAI was calculated as described in a previous study using the following formula: DAI = /3 . The CMDColon tissues were fixed in 4% paraformaldehyde, and the hematoxylin and eosin (H&E) stain was performed, and histopathological changes were observed and evaluated in a blinded fashion, as previously described . The hisThe supernatants of the subsequently homogenized colon tissues were obtained to detect production of the pro-inflammatory factors IL-1β, TNF-α, IL-6, and IL-17 using corresponding ELISA kits  according to the manufacturer’s instructions.−ΔΔCt.For RT-PCR analysis, total cellular RNA was isolated from the colon tissue using RNA TRIzol Reagent , and cDNA was synthesized from the resulting total RNA using the PrimeScript RT Reagent Kit . The amplification reactions were conducted in 96-well reaction plates with a 20 μL reaction volume . In this study, the following primer sequences were used: NLRP3 F: 5′-CCAGATGGAGAAGGCAGATCACTTG-3′, R: 5′-TCGCAGCAAAGATCCACACAGC-3′; p65 F: 5′-CCAAGACACACCCCACCATCAAG-3′, R: 5′-AGGTCAGCCTCATAGTAGCCATCC-3′; Caspase-1 F: 5′-AGAGGATTTCTTAACGGATGCA-3′, R: 5′-TCACAAGACCAGGCATATTCTT-3′; TLR4 F: 5′-GCCATCATTATGAGTGCCAATT-3′, R: 5′-AGGGATAAGAACGCTGAGAATT-3′; and ACTB F: 5′-CATCACTATTGGCAACGAGCGGTTCC-3′, R: 5′-ACGCAGCTCAGTAACAGTCCGCCTA-3′. The fold change in the level of the target mRNA between the control and treated animal groups was corrected to the level of ACTB. Finally, the change in gene expression was calculated by 2p < 0.05 was considered statistically significant.Data are presented as mean ± standard error of the mean (SEM). Using SPSS software , the means were assessed using one-way analysis of variance (ANOVA), and the Fisher’s protected least significant difference (LSD) post hoc test was used to determine the significance for multiple comparisons. In this work, 29 compounds were identified in EAdSc using GC–MS, and 22 blood-soluble EAdSc components were recognized. Network pharmacology analysis revealed that multiple inflammatory signaling pathways are involved in EAdSc anti-UC activity. Furthermore, molecular docking analysis found that eleutheroside A, liriodendrin, epicatechin, 2-methoxy-4-vinylphenol, catechin, androsin, coumarouyltyramine, and catechol may be the active components of EAdSc in the treatment of UC through the TLR4/NF-κB/NLRP3 signaling pathway. In addition, we first demonstrated that EAdSc significantly improved the DSS-induced clinical symptoms and intestinal inflammatory injury of mice with UC. EAdSc could regulate the TLR4/NF-κB/NLRP3 signaling pathway and decreasing the pro-inflammatory cytokine levels in colon tissues. This study provides a basis for the use of EAdSc in the treatment of UC and provides support for determining the mechanisms of action of various active constituents.


The interaction between monocytes and endothelial cells in inflammation is central to chemoattraction, adhesion, and transendothelial migration. Key players, such as selectins and their ligands, integrins, and other adhesion molecules, and their functions in these processes are well studied. Toll-like receptor 2 (TLR2), expressed in monocytes, is critical for sensing invading pathogens and initiating a rapid and effective immune response. However, the extended role of TLR2 in monocyte adhesion and migration has only been partially elucidated. To address this question, we performed several functional cell-based assays using monocyte-like wild type (WT), TLR2 knock-out (KO), and TLR2 knock-in (KI) THP-1 cells. We found that TLR2 promotes the faster and stronger adhesion of monocytes to the endothelium and a more intense endothelial barrier disruption after endothelial activation. In addition, we performed quantitative mass spectrometry, STRING protein analysis, and RT-qPCR, which not only revealed the association of TLR2 with specific integrins but also uncovered novel proteins affected by TLR2. In conclusion, we show that unstimulated TLR2 influences cell adhesion, endothelial barrier disruption, migration, and actin polymerization. Monocytes, which comprise approximately 10% of total human leukocytes, play a fundamental role in protective immunity and are involved in both the initiation and resolution of inflammation . Early iTLRs, which are among the best-known pattern recognition receptors, are key elements in fighting invading pathogens ,15. TheyRecent evidence suggests that TLRs, especially TLR2 and TLR4, are involved in integrin-dependent leukocyte adhesion and transmigration ,20. The All previous findings have indicated that TLR2, as an activated receptor, promotes the process of homing. Our study aims to show that there is a significant difference in cell adhesion and migration induced by TLR2, even in its inactive state, affecting the gene expression of specific proteins essential for these processes. Therefore, we stably transfected THP-1 TLR2 knock-out (KO) cells with human full-length TLR2 and compared THP-1 wild-type cells (WT), KO cells, and the generated TLR2 knock-in (KI) cells using various cell-based assays. We also performed proteomics and gene expression analysis with a focus on adhesion- and migration-associated proteins and examined the actin polymerization. We found that TLR2 per se, without prior activation, plays a significant role in the homing process, including chemoattraction, cell adhesion, and cell migration.®, Manassas, VD, USA), the THP-1-Dual™ KO-TLR2 cell line (KO) , and the THP-1-Dual™ KI-TLR2 cell line (KI) (see Escherichia coli purified by gel-filtration chromatography (Sigma-Aldrich) at a final concentration of 100 ng/mL. After activation, blood was centrifuged at 2500× g for 15 min to isolate the plasma. Plasma was further diluted 10-fold in fresh medium (M199 or huMEC) to obtain conditioned medium.The human acute monocytic leukemia-derived THP-1 cell line (WT) (ATCC(KI) see  were maiEscherichia coli strain GCI-5α (GeneCopoeiaTM) and verified by sequencing. DNA sequencing was carried out by Microsynth Austria GmbH with the following primers: primer sequence 1: GTTTCGTTTTCTGTTCTGC; sequence 2: CGGAGGCTGCATATTCCAAG; sequence 3: TGTGTCTTCATAAGCGGGACT.To generate a TLR2 knock-in cell line (KI), full-length human TLR2 was cloned into the lentiviral vector pEZ-Lv195 with a plasmid size of 9784 base pairs and a puromycin selection marker (GeneCopoeiaTM). The construct was then transformed into the 6 THP-1 KO cells were infected with 1 mL of virus suspension that was diluted in complete culture medium with 8 µg/mL polybrene. After 24 h, the medium was replaced with complete cell culture medium without polybrene, and 48 h later, selection was started with 1 µg/mL puromycin dihydrochloride (Thermo Fisher Scientific).For lentiviral transduction, 293Ta lentiviral packaging cells and the Lenti-Pac™ HIV Expression Packaging Kit (both GeneCopoeiaTM) were used according to the manufacturer’s instructions. Supernatant containing viral particles was used for infection or stored at −80 °C. For transfection, 1 × 106 cells were harvested for each cell line . Cells were then washed with PBS and lysed in 100 µL ice-cold lysis buffer consisting of 500 mM NaCl, 50 mM Tris-HCl, pH 7.4, 0.1% SDS, 1% NP-40 and 0.05% NaN3, 1U DNase I, 1 U protease, and phosphatase inhibitor cocktail . Cell lysates were frozen at −80 °C for 2 h and centrifuged at 12,000 rpm for 30 min at 4 °C, and the supernatants were resuspended in 4X Laemmli sample buffer  containing 10% ß-mercaptoethanol (Merck). Proteins were separated on 7.5% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad) using PageRuler™ Plus Prestained Protein Ladder (Thermo Fisher Scientific) as a molecular weight marker and run at 100 V. The gels were then transferred onto 0.2 µm nitrocellulose membranes (Bio-Rad) using a Trans-Blot® Turbo Transfer System (Bio-Rad). To saturate non-specific binding sites, immunoblots were blocked with 5% nonfat dry milk  in PBS supplemented with 0.1% Tween 20 (PBS-T) overnight at 4 °C. Primary anti-TLR2 antibody  and vinculin  were diluted in 5% nonfat dry milk in PBS-T, and the membranes were incubated for 2 h at room temperature. After thorough washing with PBS-T, the blots were incubated for 1 h with secondary HRP-conjugated anti-rabbit antibody (Thermo Fisher Scientific). Membranes were washed with PBS-T and developed with the Clarity Western ECL Substrate (Bio-Rad) according to the manufacturer’s instructions. Protein visualization was performed using ChemiDoc MP Imaging Systems with Image LabTM (Bio-Rad).For the preparation of whole cell lysates, a total number of 2 × 106 cells/mL in a 96-well plate, and, after reaching 90% confluence, they were stimulated with conditioned media from LPS-stimulated whole blood for 4 h. An amount of 0.5 × 106 cells/mL of THP-1 KO, WT, or KI cells was labeled with Hoechst 33342  and added to the LECs for 15 min under shaking conditions. After thorough washing, fluorescence was measured using the Mini Max 300 imaging cytometer .LECs were seeded at a density of 0.25 × 106 cells/mL and cultured as previously described [2), and slides were then connected to fluidic units and cultured for 1 h at 2 dyn/cm2. The flow rate was further increased to 5 dyn/cm2. After reaching 100% confluence, the cells were activated with conditioned medium for 4 h at a shear stress of 5 dyn/cm2. After endothelial activation, the reservoirs were spiked with either THP-1 WT, KO, or KI cells to reach a final concentration of 0.5 × 106 cells/mL in the reservoirs. Monocyte adhesion was assessed using the CKX-41 inverted cell culture microscope and cellSens software , with images taken every 15 s for 15 min.To measure adhesion under flow, HUVECs were seeded onto fibronectin-coated channel slides  at a density of 1 × 10escribed . Briefly® 24-well permeable plates with 5 µm pore polycarbonate membranes  were used for the migration assay. First, LECs were seeded into T25 cell culture flasks , and, after reaching 90% confluence, the cells were washed with PBS. The cells were incubated in huMEC media without FCS and treated with or without LPS (100 ng/mL) for 4 h before the media was applied to the lower compartment of the transwell chambers. THP-1 cell lines (KO and WT) with a density of 1 × 106 cells/mL were stained with Hoechst 33342 (2 µg/mL), added to the upper chamber of the transwell plate, and incubated for 2 h to allow the cells to migrate through the filter. After aspiration of the remaining cells, the cells adhering to the filter were gently removed with a cotton swab, and the migrated cells (bottom of the filter) were visualized with an inverted microscope  using a 40× objective and analyzed with the Leica Application Suite Version X 3.8.0 software.To evaluate and compare the migration capacity of THP-1 WT and KO cells, Transwell2. Wells were then inoculated with LECs at a seeding density of 30,000 cells per well in 100 µL huMEC complete media at 37 °C and 5% CO2. The run was performed under a single frequency of 4000 Hz and continuously monitored every 60 s. Impedance (Z) was determined by Ohm’s law: Z = V/I. After 20 h, when an endothelial monolayer was reached, KO and WT THP-1 cells with and without stimulants were added at a density of 100,000 cells per well in 50 µL huMEC complete media. For stimulation, 100 ng/mL LPS (Thermo Fisher Scientific) or 300 ng/mL of either TLR2-specific ligand Pam2CSK4 (Pam2) or Pam3CSK4 (Pam3) (both InvivoGen) was used. Endothelial monolayer disruption was observed for 10 h.Endothelial cell monolayer disruption (transmigration) by monocytes +/− LPS and TLR2 ligand stimulation was measured using the 9600Z Electrical Cell-Substrate Impedance Sensing (ECIS) system . A 96-well plate containing 20 gold film electrodes per well (ibidi) was coated with 1 mg/mL neutralized rat tail collagen type I (Thermo Fisher Scientific) for 10 min at room temperature and 2 µg/mL bovine plasma fibronectin (Thermo Fisher Scientific) for 45 min at 37 °C and 5% CO6 cells per well in a 12-well plate and incubated for 3 h at 37 °C and 5% CO2. The cells were then collected and washed with PBS twice.For quantitative mass spectrometry, THP-1 KO, WT, and KI cells were seeded at a density of 1 × 10g for 10 min, the supernatants were transferred to new tubes to remove most of the DNA and stored at −20 °C until further use.For sample preparation and digestion, the cell pellets were dissolved in 40 µL lysis buffer containing 8 M urea and 50 mM NH₄HCO₃ (both Sigma-Aldrich) and sonicated for 10 min. After centrifugation at 14,000× After protein concentration was determined using the BCA assay (Sigma-Aldrich), 20 µg of protein per sample was used for digestion. Samples were reduced and alkylated with TCEP and IAA and sequentially digested with Lys-C  for 1 h and trypsin  for 16 h. Peptides were purified using Sep-Pak tC18 1 cc Vac cartridges , dried, and stored at −20 °C until analysis.Samples were analyzed using an Ultimate 3000 RSLCnano system coupled with an Orbitrap Eclipse Tribrid mass spectrometer (both Thermo Fisher Scientific).2O, 2% ACN, and 0.1% FA). A total of 2 µL was injected into a PepMap 100 (C18 0.3 × 5 mm) TRAP column and analyzed using a PepMap RSLC EASY-spray column . Separation was performed at 300 nL·min−1 with a flow gradient of 2–35% mobile phase B  within 60 min, resulting in a total method time of 80 min. The mass spectrometer was operated in DIA mode with the FAIMS Pro system in positive ionization mode at CV-45. MS1 scans were acquired in the scan range of 350–1400 m·z−1 with a resolution of 120,000 @200 m·z−1. For DIA scans, the precursor mass range was set to 400–1000 m·z−1 with a 14 m·z−1 isolation window and 1 m·z−1 window overlap for a total of 43 independent scans. HCD fragmentation was performed at 30% NCE, and fragments were analyzed in the Orbitrap at a resolution of 30,000 @200 m·z−1.Dried samples were suspended in 40 µL of mobile phase A  and isolation windows of 4 m·z−1 with 2 m·z−1 window overlap.To deepen the analysis, a pool of all samples was created and used for gas phase fractionation (GPF) . Sample DIA-NN (version 18.1.1)  was usedFurthermore, upregulated genes in THP-1 WT and/or KI vs. KO cells associated with cell adhesion + TLR2 were analyzed for interactions using the © STRING CONSORTIUM 2023 (version 11.5). This generated protein–protein network shows interactions based on data from experiments, genomic context predictions, automated text mining, co-expressions, and curated databases .The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner ® Gene Expression Master Mix (Thermo Fisher Scientific) were used for quantification. Amplification parameters were 10 min at 95 °C for initial denaturation, followed by 40 cycles of 95 °C for 20 s and 60 °C for 1 min run on the Quant Studio 7 Flex (Applied Biosystems). The fold change in the mRNA expression was determined using the ΔΔCt method [Total RNA was isolated using the RNeasy kit , and 1 µg RNA was reverse transcribed using the Hight Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific), both according to the manufacturer’s instructions. Pre-designed TaqMan gene expression assays with FAM-labeled primers/probes  and TaqMant method .5 cells/mL) were added to each well, and cells were allowed to attach overnight at 37 °C and 5% CO2. The cells were washed with PBS and fixed for 10 min with a 4% paraformaldehyde solution at room temperature. The acidity of the paraformaldehyde was neutralized with a 50 mM ammonium chloride solution (Sigma-Aldrich) for 10 min at room temperature. Cells were washed with PBS and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for 10 min at 4 °C and blocked with 5% goat serum (Invitrogen) for 30 min at room temperature. Cells were washed with PBS, and F-actin was stained with phalloidin conjugated to AlexaFluor 635 (Invitrogen) diluted 1:200 in 1% bovine serum albumin solution (Sigma-Aldrich) for 60 min at room temperature in the dark. Cells were counterstained with DAPI (Sigma-Aldrich) and mounted with Fluoromount aqueous mounting medium (Sigma-Aldrich) onto high-precision microscope coverslips . Images of the stained cells were acquired with a confocal laser scanning microscope  using a 63X glycerol objective . The 3D images of the beads were obtained by z-stack imaging . Image analysis was performed using the LAS X software (Leica).F-actin expression was visualized by confocal microscopy. Briefly, 8-chamber glass slides (ibidi) were precoated with 11 µg/mL fibronectin (Thermo Fisher Scientific). THP-1 WT, KO, and KI suspensions (4 × 106 cells/mL), washed, fixed, and permeabilized as described above , and the proteomics data were evaluated using Perseus (version 2.0.6.0). Data are expressed as mean ± standard deviation and significance was accepted at To investigate whether TLR2 plays an important part in rolling, adhesion, and cell migration/transmigration during inflammatory events, we used a monocytic THP-1 WT, a KO, and a stable KI cell line. As indicated in To investigate whether TLR2 also induces endothelial barrier disruption, and the transendothelial migration of THP-1 WT and KO cells, endothelial cells were plated on a 96-well plate with gold film electrodes (ECIS), precoated with collagen type I and fibronectin, and allowed to form a monolayer. Cells were left untreated or were treated with LPS, which is a potent stimulator of TLR4, Pam2, and Pam3, which are TLR2 ligands, and THP-1 KO and WT cells were added directly to the endothelial cells. Endothelial monolayer resistance was then measured by real-time impedance quantification, demonstrating changes in endothelial barrier disruption. As shown in To gain more insight into the protein expression affected by TLR2, we performed label-free quantification mass spectrometry. For this purpose, THP-1 WT, KO, and KI cells were harvested, and a data-independent acquisition (DIA) method was applied to compare THP-1 WT and KI vs. KO cells. In general, approximately 7200 proteins were identified in our samples, of which 858 proteins were significantly regulated when comparing WT vs. KO cells, and 951 proteins were significantly regulated when comparing KI vs. KO cells . Furthermore, 236 common proteins were found to be significantly upregulated in WT and KI vs. KO cells. . RelevanTo obtain a comprehensive and objective connectivity network of TLR2, including both direct and indirect interactions, a STRING network analysis was performed. For this purpose, the highly expressed proteins in THP-1 WT and/or KI vs. KO cells related to cell adhesion and migration were used. This network determines interactions based on data from experiments, genomic context predictions, automated text mining, co-expressions, and curated databases. As shown in To deepen our understanding of the influence of TLR2 on gene expression, we examined the mRNA expression levels of certain molecules that were upregulated in THP-1 WT and/or KI cells in the proteomics results shown above. The expression of the integrins ITGAL, ITGAX, and ITGB1 reflected a similar pattern to that seen in the proteomics data. However, the increase between THP-1 WT and/or KI cells and KO cells was less pronounced, often not significant, or not present at all a–c. A coActin reorganization and polymerization are critical for leukocyte adhesion  and tranThe importance of TLR2, not only as a pattern recognition receptor but also in cell adhesion and migration with a focus on TLR2 ligand activation, has been clearly demonstrated ,20,26. TTherefore, we next performed quantitative mass spectrometry to investigate the protein expression patterns affected by TLR2 in THP-1 cell lines. Compared to in KO cells, we found that ITGAL, ITGAX, ITGB1, and ITGB2 were significantly upregulated in WT cells. In KI cells, in which TLR2 was expressed at much lower levels than in WT cells, all of the above integrins showed increased expression, but only ITGAX was significantly upregulated. Both ITGAX and ITGAL belong to the β2 integrins and are upregulated in monocytes during inflammation, resulting in increased cell adhesion to the endothelium . An in vDirect interactions between TLR2 and all integrins, except for ITGB1, were found in the STRING analysis. We observed the same pattern in the mRNA expression levels of the integrins ITGAL, ITGAX, and ITGB1 as in the proteomics data. However, the upregulation between THP-1 WT and/or KI cells and KO cells was less pronounced, often not significant, or absent, and a completely different expression was observed for ITGB2. In contrast to the proteomics data, the mRNA level was significantly decreased in cells expressing TLR2. This led us to hypothesize that the heterogeneity may be due to post-translational modifications and/or alternative splicing, which is not an uncommon event to observe ,43. MoreIn addition to integrins, the proteomic analysis and the subsequent protein network analysis highlighted several significant proteins involved in cell adhesion and migration, including CD44, LGALS3, LGALS3BP, VTN, and MYO1G. It has been shown that monocyte rolling under inflammatory conditions is strongly dependent on CD44 expressed in monocytes ,61. CD44p-value 0.06). RRAS was first recognized because an overexpressed mutant of RRAS made cells highly integrin-dependent and adherent [The coordinated polymerization of F-actin plays a fundamental role in proper whole cell migration . Wang etadherent . Furtheradherent ,81. A readherent . The ideOverall, by comparing these three monocytic cell lines, we were able to demonstrate that TLR2 is a key mediator of cell adhesion and cell migration to the endothelium, even without prior stimulation. We discovered altered mRNA and protein expression patterns in several adhesion molecules, such as integrins, including ITGB1 and ITGAL, CD44, and LGALS3, that are dependent on TLR2 expression. In addition, novel TLR2-dependent proteins, such as P4HB and MYO1G, which are essential for the homing process, were uncovered, and new connections between TLR2 and actin polymerization were identified. Our results suggest that TLR2 in monocytes may be an important contributor not only in the initiation of inflammation but also in adhesion and cell migration by influencing the protein expression of integrins, adhesion molecules, and actin regulatory proteins.


Escherichia coli strain that was isolated from municipal biosolids collected from a Canadian wastewater treatment plant. This strain contains multiple metal, antimicrobial, and heat resistance genes, as determined by genome sequencing, and could be a useful bacterial model for future studies.This announcement reports the complete genome sequence of a non-Shiga toxin-producing  Escherichia coli is a Gram-negative bacterium, either harmless or pathogenic, that is commonly found in the intestines of humans and animals and in environmental habitats, including agricultural soils and biosolids (–E. coli strain (HH107) that was isolated from a posttreated (biosolids) sample collected from a Canadian wastewater treatment plant in 2010. The strain was isolated by enrichment in lauryl sulfate tryptose broth at 35°C for 24 h, followed by E. coli broth at 45°C for 24 h, and isolation using Levine's eosin methylene blue agar at 35°C for 24 h (E. coli using API 20 E (bioMérieux Canada Inc.) and confirmed by genome sequencing.iosolids –3. This N50 of 17,401 bp) were obtained, followed by base calling using Guppy v6.1.2, trimming using Porechop v0.2.4, and filtering using NanoFilt v2.8.0 (dnaA as the starting point were determined by Unicycler v0.5.0. The coverage depth (262×) was assessed using SAMtools v1.13 , with subsequent treatment using the Short Read Eliminator XS kit (PacBio). Illumina sequencing was conducted by library preparation using a DNA preparation kit  and sequencing on a MiSeq platform (Illumina) using the MiSeq reagent kit v3 to generate a total of 2,684,745 paired-end (300-bp) reads, which were filtered and trimmed using Fastp v0.23.2  under SRA accession numbers SRR23100672 and SRR23100675, respectively.The chromosome and plasmid sequences of strain HH107 have been deposited in GenBank under accession numbers


Rottlerin, a type of natural extract, can inhibit the activity of various viruses. Feline coronavirus (FCoV) causes a devastating disease in cats. In this study, we investigated whether rottlerin has inhibitory effects on FCoV. We demonstrated that rottlerin inhibits FCoV replication and FCoV-induced PKCδ phosphorylation. We showed that rottlerin affects the early (endocytosis) and late (syncytium formation) stages of FCoV replication. We also observed that the liposome encapsulation technique overcomes the drawbacks of rottlerin and enhances its efficacy. Therefore, we suggest that rottlerin-liposome is worth further investigation as a potential treatment for FCoV.Mallotus philippensis with antiviral properties. Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV) that is characterized by systemic granulomatous inflammation and high mortality. We investigated the antiviral effect of liposome-loaded R, i.e., rottlerin-liposome (RL), against FCoV. We demonstrated that RL inhibited FCoV replication in a dose-dependent manner, not only in the early endocytosis stage but also in the late stage of replication. RL resolved the low solubility issue of rottlerin and improved its inhibition efficacy at the cellular level. Based on these findings, we suggest that RL is worth further investigation as a potential treatment for FCoV.Rottlerin (R) is a natural extract from  Mallotus philippensis, commonly known as the Kamala tree. A traditional Indian medical journal, Ayurveda, has referred to the powder as Kampillaka and recommended its use as a remedy for topical and oral medicine in the treatment of various diseases since the 8th century [Kampillaka has been known to alleviate skin disease and injuries and was especially used to treat carbuncles in combination with cow’s ghee to form an ointment [Kampillaka has been used to treat ascites, abdominal tumors, parasite infections, blood disorders, contraception, and leprosy [Kampillaka for children is 500 mg to 750 mg and for adults is 8 g [Rottlerin (R) is a natural polyphenol ketone compound derived from the fruit powder of  century ,2,3,4. Ws is 8 g . Intrigus is 8 g ,11,12,13Mycobacterium tuberculosis infection, and fibrosis [R has been widely used as a protein kinase C δ (PKCδ) phosphorylation inhibitor ,12,14. Rfibrosis ,18,19. ADrug delivery systems (DDSs) have been widely used in various fields to increase the efficacy of pharmaceutical substances ,21,22. ANidovirales; subfamily: Coronavirinae; family: Coronaviridae) is a known causative agent of a devastating disease, feline infectious peritonitis (FIP), which is characterized by systemic granulomatous inflammation [Feline coronavirus /mL) in a PKCδ-dependent manner. In animal studies, intranasal administration of RL significantly reduced viremia levels, interstitial pneumonia, and clinical scores of PRRSV-infected piglets compared to the control group. Previously, our group demonstrated that rottlerin-liposome (RL), i.e., liposome-encapsulated rottlerin, successfully inhibited porcine reproductive and respiratory syndrome virus (PRRSV) infection in MARC-145 cells and piglets . In detaNidovirales, along with PRRSV, may also be inhibited by RL. In this study, we confirmed that RL can inhibit the replication of FcoV. We can observe a slight reduction in PKCδ phosphorylation by RL treatment during the early stages of FCoV infection. RL affects at least two stages of FCoV replication, one during the early endocytosis of FCoV and the other during the late stage, which is possibly related to the syncytia formation stage. Our study provides preliminary evidence of the inhibitory effect of RL on FCoV and its possible mechanism and highlights the potential advantages of applying liposomes to antiviral drugs.Thus, we hypothesized that FCoV, which belongs to the order In this study, we used the WSU 79-1146 (VR-990) virus as a model for FCoV ,47,48. Av/v). Then, R was added at 1/20 of the weight of the mixture. The mixture was dissolved by stirring at room temperature for 1 h, the flask was mounted onto a rotary evaporator , and the device was operated under conditions of 54 °C, 50 rpm, and 300 mbar to form a thin layer of film on the surface of the flask. Distilled water (DW) was added to hydrate the layer at 65 °C and 60 rpm. After the film was fully hydrated, the recovered product was dialyzed against DW overnight at room temperature.R  was dissolved in DMSO at a concentration of 10 mg/mL. RL was prepared using the thin-film hydration method with reference to previous studies ,24. Hydrv/v), and the absorbance was measured at a wavelength of 405 nm using a spectrophotometer  [To verify the quality of the RL, we measured its shape, size, and concentration. The shape of the particles was evaluated by utilizing a transmission electron microscope (TEM), while the size distribution was determined using a dynamic light scattering (DLS) system . To determine the amount of R encapsulated in liposome, triton-x100 was mixed with the prepared RL at a ratio of 19:1 to dissolve the lipid (zerland) . The abs5 cells/mL in 96-well plates. Various concentrations of R and RL were prepared by diluting them with a cell culture medium, and 100 µL of each concentration was added to the wells. DMSO was added as a control. The plates were then incubated at 37 °C in 5% CO2 for 48 h. Following this, 10 µL of EZ-Cytox  solution  was added to each well and further incubated for 4 h [Twenty-four hours prior to the test, CRFK cells were seeded at a concentration of 2 × 10 for 4 h . At the 5 cells per well in 24-well plates. The following day, we tested the anti-FCoV activity of various concentrations of R or RL, which was determined based on the cell viability assay results. The CRFK cells were seeded at a concentration of 10The cells were pre-treated with various concentrations of R, RL, or DMSO (as vehicle controls) for 2 h before infection with FCoV at a multiplicity of infection (MOI) of 0.008 with the same drug concentrations. The cytopathic effect (CPE) induced by the virus was monitored for up to 72 h. Following this, the supernatant of the cell lysates was collected and subjected to titration or RT-qPCR.® 96 Real-Time PCR System . The obtained quantification cycle (Cq) value was converted into a log (TCID50/mL) equivalent using a standard curve.Viral RNA was isolated using a Viral Gene-spin™ Viral DNA/RNA Extraction Kit . To quantify viral RNA, we used the primers and probe set, targeting the 3’UTR region of FCoV from the previous study by modifying the reporter fluorescence to FAM . The rea5 cells/mL) were prepared in a 24-well plate and incubated for 24 h. After removing the medium, a medium containing 1 MOI of FCoV and 2.5 μM of RL was added. After 15, 30, 45, and 60 min, the cell lysates were collected using M-Per® Mammalian Protein Extraction Reagent , and all cell debris was removed by centrifugation at 13,000 rpm for 10 min at 4 °C. The CRFK cells (2 × 10For sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), samples were processed with a 4× LDS sample buffer  according to the manufacturer’s guidelines. An amount of 10 μL of the prepared sample was loaded onto Bolt™ 4 to 12%, Bis-Tris gel, and electrophoresed at 200 volts for 25 min. Then, the bands were transferred to a nitrocellulose membrane under the condition of 20 volts for 1 h, as suggested by the manufacturer. The transferred membrane was washed 3 times with the washing buffer (PBS containing 0.05% Tween20) and stained with the primary antibody diluted with a blocking buffer (PBS containing 0.05% Tween20 and 5% BSA). After overnight incubation at 4°C, the membrane was washed 3 times and incubated with the secondary antibody diluted with a blocking buffer. After incubation for 2 h, the membrane was washed 3 times with PBS and developed using the Clarity Western ECL Substrate . Images were taken using the FUSION SOLO system . For the β-actin signal, the previously stained membrane was stripped with the Restore™ Stripping Buffer  for 15 min and then stained with Anti β-actin antibody, HRP  for 2 h and analyzed. The captured images were quantified using ImageJ . Values 5 cells/mL CRFK cells were prepared on a 24-well plate. At a predetermined point, the medium in each well was removed and 2.5 μM of the RL was added. At +0 h, a medium containing 0.008 MOI of FCoV and 2.5 μM of RL was added, incubated for an hour, and removed. The cells were further incubated for up to 72 h with the media containing 2.5 μM of the RL and the cell lysates were collected. The samples were subjected to titration or qRT-PCR. Based on the virus inoculation time point (+0 h), 7 groups (A to G) of the inoculation times of the RL were created. Twenty-four hours before the inoculation, the 2 × 105 cells/mL in a 24-well plate and cultured for 24 h. After removing the medium, a medium containing 0.008 MOI of FCoV and 2.5 μM of R or RL was added. After 1 h, the medium was removed, and the cells were washed with PBS. After that, a medium containing 2.5 μM of R or RL was added and cultured for 24 h. After treatment, the cells were washed once with PBS and then fixed with 4% paraformaldehyde for 15 min, followed by permeabilizing for 15 min using 0.05% triton-X100. After washing 3 times with PBS, the primary antibody diluted with a blocking buffer was added to the cells. After overnight incubation at 4 °C, the cells were washed 3 times with a washing buffer, and the diluted secondary antibody was added. After incubation for 3 h, the cells were washed 3 times and mounted. The cells were observed using EVOS® FL Color . The captured images were quantified using ImageJ [The CRFK cells were prepared at 2 × 10g ImageJ .5 cells/mL and incubated for 24 h. The cells were pre-treated with the RL at a concentration of 2.5 μM and incubated for 1 h. After discarding the medium, the cells were inoculated with a mixture of 1 MOI of FCoV and 2.5 μM of RL. After 1 h of incubation for viral binding and internalization, the cells were washed and replenished with a medium containing 2.5 μM of RL. In the mock test group, a fresh medium containing DMSO was used. After 2, 4, and 6 h, each well was treated with PBS either containing or not containing protein kinase  for 45 min at 4 °C, then fixed, stained, and observed under an LSM-900 . Microscopic images were processed using ImageJ software and fluorescence signals were quantified [The CRFK cells were seeded on a chamber slide at a concentration of 1 × 10antified . The flu50) and 50% effective concentrations (EC50) were calculated using nonlinear regression analysis. Depending on the variability of the data, either one-way ANOVA and Dunnett’s multiple comparison or two-way ANOVA and Tukey’s multiple comparison were applied. Statistical significance is indicated by the symbols (*), (**), (***), and (****), representing p < 0.05, p < 0.005, p < 0.0005, and p < 0.0001, respectively.All statistical analyses were conducted using GraphPad Prism version 8 . The 50% cytotoxicity concentrations (CC50) of RL was 3.53 µM, which was similar to R (3.90 µM)  in the presence of RL or R. After 72 h post-infection, viral RNA synthesis, N protein expression, and virus titer were measured. As shown in 50) of RL and R were 1.40 µM and 1.60 µM, respectively (R2 > 0.95). Interestingly, RL showed a significantly enhanced antiviral effect (p < 0.05) compared to R in terms of viral RNA, protein, and titration, suggesting that loading R onto a liposome could increase its potency  and RL (2.5 μM), and the cell lysates were collected at 15, 30, 45, and 60 min after infection for Western blotting to measure total PKCδ, phosphorylated PKCδ (PKCδ-S645), and β-actin. p < 0.0001, not indicated), suggesting that FCoV infection induced the phosphorylation of PCKδ (p < 0.05) (The levels of phosphorylated PCKδ in the FCoV-infected groups (FCoV+) were significantly increased compared to those of the negative control group (FCoV−/RL−) at all time points  B. There p< 0.0001 or p < 0.0005). RL pre-treatment at −2 (group A) showed the highest reduction in viral RNA (4.7 ± 0.5 log (TCID50/mL) equivalent) and titer (5.2 ± 0.1 log (TCID50/mL)) among all groups, suggesting that RL affects an early stage of FCoV infection. RL treatment at +0, +1, +3, +5, and +9  resulted in the intermediate inhibition of viral replication. Interestingly, RL treatment at +24 h (group G) significantly decreased the viral titer but only slightly decreased the viral RNA. These intermediate inhibitions indicate that RL also affects the middle and late stages of FCoV infection, which may be associated with disrupting viral protein translation, capsid assembly, virus release, or virus spread. To investigate which stage of FCoV replication was inhibited by RL, a time of drug addition assay was conducted. The experimental design is presented in 7 ± 4.06) and R (1.8 × 107 ± 2.8 × 106) showed significantly lower fluorescence intensity than the mock (2.2 × 107 ± 3.9 × 106) or liposome (2.3 × 107 ± 2.5 × 106) groups  groups A. Surpri) groups B. These Since pre-treatment of RL showed the most significant reduction in FCoV replication, we hypothesized that RL treatment could disturb the entry process of FCoV. To test this hypothesis, we observed the early localization of FCoV in CRFK cells after treatment of RL or mock (DMSO) with or without proteinase K (proK). The proK treatment was capable of removing outside viruses from the plasma membrane, thereby enabling us to observe only endocytosed virus particles . Two houIn this study, we have presented preliminary evidence showing that rottlerin liposome (RL) exhibits inhibitory activity against FCoV in CRFK cells. Rottlerin (R) has shown antiviral activity against many viruses, especially enveloped viruses, including chikungunya virus, dengue virus, human immunodeficiency virus-1 (HIV-1), human T-cell leukemia virus-1 (HTLV-1), la crosse virus, porcine reproductive and respiratory syndrome virus (PRRSV), rabies virus, rift valley fever virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and zika virus ,53,54,55+, K+-ATPase, resulted in a similar phenotype of R showing inhibition of the entry of mouse hepatitis virus [We showed that RL inhibited the replication of FCoV by interrupting its endocytosis . This efis virus . In zikais virus . Therefo50/mL) was significantly more inhibited than viral RNA  at +24 h  or PKCδ-independent pathways that reduce ATP levels (such as in rabies and zika viruses), suggesting that R may inhibit multiple stages of viral replication ,12,16. TWe demonstrated the advantages of liposomes in enhancing the potency of R . Liposom50) values of R and RL measured in CRFK cells were 3.90 and 3.53 μM, respectively, which are lower than those of other known small-molecule inhibitors  efficiently inhibits FCoV replication in cell cultures by reducing FCoV endocytosis. We have also observed that RL reduces both fluorescent signals for FCoV N protein and the formation of syncytia in FCoV-infected cells. Additionally, we have found that encapsulating rottlerin in liposomes enhances its inhibitory effect compared to using rottlerin alone. These results suggest that RL has the potential to be further investigated for its antiviral activity.


Meanwhile, manoalide caused reactive oxygen species (ROS) overproduction and disrupted antioxidant proteins, activating the apoptotic proteins caspase-9/-3 and PARP (Poly (ADP-ribose) polymerase). Excessive levels of ROS in the mitochondria affected oxidative phosphorylation, ATP generation, and membrane potential (ΔΨm). Additionally, manoalide down-regulated mitochondrial fusion protein and up-regulated mitochondrial fission protein, resulting in mitochondrial fragmentation and impaired function. On the contrary, a pre-treatment with n-acetyl-l-cysteine ameliorated manoalide-induced apoptosis, ROS, and antioxidant proteins in OS cells. Overall, our findings show that manoalide induces oxidative stress, mitochondrial dysfunction, and apoptosis, causing the cell death of OS cells, showing potential as an innovative alternative treatment in human OS.Osteosarcoma (OS) is the most common primary malignant bone tumor that produces immature osteoid. Metastatic OS has a poor prognosis with a death rate of >70%. Manoalide is a natural sesterterpenoid isolated from marine sponges. It is a phospholipase A2 inhibitor with anti-inflammatory, analgesic, and anti-cancer properties. This study aimed to investigate the mechanism and effect of manoalide on OS cells. Our experiments showed that manoalide induced cytotoxicity in 143B and MG63 cells (human osteosarcoma). Treatment with manoalide at concentrations of 10, 20, and 40 µM for 24 and 48 h reduced MG63 cell viability to 45.13–4.40% ( Luffariella variabilis  × 100%. The data were expressed as the mean ± SEM.Cell proliferation (viability) was assessed using an MTT assay following treatment with different concentrations of manoalide for 24 and 48 h. MTT is a yellow substance and will interact with succinate dehydrogenase (complex II) in the electron transport chain in living cells to generate a purple substance. The cells can be lysed with DMSO to release the purple substance, and the number of living cells can be directly estimated by detecting the absorbance at 570 nm. The cells were plated in triplicate at a density of 5 × 105 cells/mL) in 1× binding buffer. The samples were treated according to the manufacturer’s instructions for the FITC Annexin V Apoptosis Detection Kit . Cells were first resuspended in 100 µL 1× binding buffer (6 × 104 cells), and then 3 µL Annexin V-FITC and 3 µL PI-PE were added to each sample for fluorescent labeling. The samples were gently vortexed and placed at room temperature for 15 min in the dark. At the end of the incubation, 400 µL of 1× binding buffer was added to each sample, and the samples were analyzed using a CytoFLEX LX flow cytometer  with CytExpert analysis software version 2.0. We used four-quadrant flow cytometry software to detect live cells (bottom left), early apoptotic cells (bottom right), late apoptotic cells (top right), and necrotic cells (top left). At least 20,000 cells were analyzed per sample.MG63 cells were treated with manoalide at the indicated concentrations of 0–10 µM for 24 h; then, the culture medium was removed, washed in PBS, trypsinized, and centrifuged, and cells were resuspended (6 × 10TM Red superoxide (O2•−) indicators are novel fluorogenic dyes specifically targeted to mitochondria in live cells. The oxidation of the MitoSOXTM Red reagent by mitochondrial ROS (mtROS) produces bright-red fluorescence. MG63 cells were treated with manoalide at 0–10 μM concentrations for 4 h and then incubated with MitoSOXTM Red (5 μM) in a medium for 25 min at 37 °C, washed, trypsinized, centrifuged, and re-suspended in 1 mL of PBS. The samples were analyzed using a CytoFLEX LX flow cytometer and histograms of CytExpert analysis software. At least 20,000 cells were analyzed per sample.MitoSOX2O2 using the fluorescence probe chloromethyl derivative 2′,7′-dichlorofluorescin diacetate (CM-H2DCFD-DA), useful as an indicator of ROS in cells. This indicator exhibits much better retention in live cells than H2DCFDA. MG63 cells were treated with manoalide at 0, 0.1, 1, 5, and 10 μM concentrations for 4 h, incubated with 5 μM DCFH-DA in a medium for 25 min at 37 °C, washed, trypsinized, centrifuged, and re-suspended in 1 mL of PBS. The samples were analyzed using a Beckman CytoFLEX LX flow cytometer and histograms of CytExpert analysis software. At least 20,000 cells were analyzed per sample.Intracellular ROS (iROS) was evaluated by determining the level of H® Green reagent is a new fluorescent probe for measuring cell cytosol and nuclear oxidative stress in live cells. In six-well dishes, a density of 3 × 105 cells/well was plated and left to attach overnight. After treatment with manoalide at a concentration of 0, 0.1, 1, 5, and 10 μM for 4 h, the cells were washed with PBS. The cells were then loaded with CellROX® Green (5 mM) in media at 37 °C for 25 min staining, washed, trypsinized, centrifuged, and re-suspended in 1 mL of PBS. The samples were analyzed using a Beckman CytoFLEX LX flow cytometer and histograms of CytExpert analysis software. At least 20,000 cells were analyzed per sample.CellROX4 cells were seeded on Seahorse XF24 microplates. After overnight incubation at 37 °C, cells were treated with 0, 0.1, 1, 5, and 10 μM manoalide for 6 h. After washing the cells with 0.5 mL of Seahorse XF medium, 700 μL of Seahorse XF medium was added to each well and placed into the machine for further examination. Basal OCR was measured and plotted as a function of cells under basal conditions followed by the sequential addition of oligomycin (1 μM), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone , and rotenone (1 μM). At the end of the recording, cells were harvested and the amount of protein was measured using the BCA assay; then, OCR and ECAR values were calculated after normalization with the amount of protein (mg).The Seahorse XF24 Extracellular Flow Analyzer  measures the OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) in living cells, which are direct real-time quantitative indicators of mitochondrial respiration and glycolysis. For comparison between experiments, the data are expressed as OCR of pmoles/min/mg protein and ECAR of mpH/min/mg proteins. At the start, 5 × 10m). In six-well dishes, a density of 3 × 105 cells/well was plated and left to attach overnight. After treatment with manoalide at a concentration of 0, 0.1, 1, 5, and 10 μM for 4 h, the cells were washed with PBS. The cells were then loaded with DiOC6 (5 μM) in media at 37 °C for 20 min staining, washed, trypsinized, centrifuged, and re-suspended in 1 mL of PBS. The samples were analyzed using a Beckman CytoFLEX LX flow cytometer and histograms of CytExpert analysis software. At least 20,000 cells were analyzed per sample.The cationic dye 3,3′-dihexyloxacarbocyanine iodide (DiOC6) is also a type of green fluorescent dye. It is well known as a mitochondrial membrane probe. DiOC6 can pass through the cell membrane and detect the mitochondrial membrane potential  in media at 37 °C for 20 min staining, washed, trypsinized, centrifuged, and re-suspended in 1 mL of PBS. The samples were analyzed using a Beckman CytoFLEX LX flow cytometer and four-quadrant of CytExpert analysis software. At least 20,000 cells were analyzed per sample.The positively charged mitochondrial dye JC-1  was employed to measure ΔΨ6 cells and different concentrations of drugs were added for 24 h. The proteins were dissolved in a protein extraction reagent after the cells were lysed with buffer . The total protein concentration was quantified by the Bradford method , and the molecular weights of the proteins in the samples differed in size, which were then separated using 8–15% SDS-PAGE electrophoresis gels, followed by transfer to PVDF  membranes. The membrane was blocked with 5% nonfat milk and then incubated overnight at 4 °C with the primary antibodies shown in In 10 cm plates, culture medium containing 3 × 10t-test was used to compare statistically significant differences between groups, where ** p < 0.01 or * p < 0.05 was considered statistically significant. Experiments were performed at least three times to verify reproducibility.Data for this study were created using Microsoft Excel and plotted with GraphPad Prism 5.0 software for graphics processing. Results are expressed as the numerical mean ± standard error (SE). Student’s 50  values for manoalide in MG63 cells were 8.88 ± 1.10 and 8.66 ± 1.12 μM after 24 and 48 h, respectively. The IC50 values for manoalide in 143B cells were 15.07 ± 1.54 and 10.93 ± 1.28 μM after 24 and 48 h, respectively (−/PI-PE−), necrosis , early apoptotic cells , and late apoptotic cells  [The study used MTT reagent staining to test the cell toxicity of medical treatment with manoalide for 24 and 48 h on two OS cell lines, and the results revealed that the treatment’s effects were dose-dependent. At manoalide concentrations of 5, 10, 20, and 40 μM, the viability of the human MG63 cell line at 24 h decreased to 83.33% ± 12.38%, 45.13% ± 26.48%, 31.11% ± 20.04%, and 4.40% ± 4.78%, respectively, of that of the control  A left, wectively . The cyt/PI-PE+) . The apo2DCFDA, MitoSOXTM Red, and CellROX® Green were used to detect O2•− and •OH in the cellular components, mitochondria, and nucleus, respectively. mtROS were detected by flow cytometry using MitoSOXTM Red staining; the figure shows a histogram produced using CytExpert analysis software, and we found a considerable shift to the right in MG63 cells treated with different concentrations of manoalide  . Therefoanoalide A. Based anoalide C. Based anoalide D. Similaanoalide E. The quThe Seahorse XF24 extracellular flux bioenergy metabolism analyzer, developed by Seahorse Bioscience in the United States, is the only platform in the world that can evaluate the overall energy metabolism of living samples. To complete the mitochondrial function test, the Cell Mito Stress Test kit was used to first detect the basic oxygen consumption; then, an ATP synthase inhibitor (oligomycin) was added to inhibit the mitochondria’s production of ATP, and the inhibited oxygen consumption indicates how much oxygen is involved in the synthesis of ATP. Then, at the appropriate concentration, FCCP, an uncoupler medication, was added without disrupting the electron transport chain and enabling the mitochondria to remain idle under harsh conditions to determine the mitochondria’s maximum oxygen consumption capacity. Finally, Complex I, an electron transport chain inhibitor, was added. The background value for its detection is determined by the inhibitor rotenone and the complex III inhibitor Antimycin A, which fully shuts down mitochondrial oxygen utilization. As a result, the following mitochondrial respiration parameters can be calculated: basal respiration, ATP-linked production (coupled respiration), maximal respiration, spare respiratory capacity, and non-mitochondrial respiration. MG63 cells were treated with various dosages of manoalide, followed by sequential addition of oligomycin, FCCP, and rotenone/actinomycin, which were found to decrease OCR parameters A. With tm loss and mitochondrial dynamic imbalance [m was detected by flow cytometry using a DiOC6 probe; the figure shows a histogram created using CytExpert analysis software, and we observed a significant shift to the left in MG63 cells treated with various doses of manoalide  at 5 and 10 μM manoalide was considerably greater than that in the 0 μM manoalide group (5.37 ± 0.71%), but the percentage of high ΔΨm  was substantially lower than that in the 0 M manoalide group (94.58 ± 0.74%)  D. Mitoch± 0.74%) F. When M± 0.74%) G. These 2DCFDA dye and analyzing flow cytometry. The findings showed that NAC did not generate iROS and that iROS levels were strongly increased following manoalide therapy, which was substantially reduced by NAC pre-treatment (N-acetylcysteine (NAC) is a reducing agent that functions as an antioxidant by depleting ROS in cells . MG63 cereatment C,D. To ereatment E–G. Thes50 of approximately 8.7 versus 10.9 µM for 48 h. Manoalide has been reported to have antitumor activity in oral cancer studies with an IC50 of approximately 14.0 μM for 48 h, similar to our experiments [50 of MG63 cells was approximately 8.9 µM for 24 h, with very little difference from 48 h. Our study found that the difference in manoalide had a distinct typical anti-viability effect on human OS cancer cells.OS is the most frequent primary bone tumor , resultiMost newly manufactured chemicals are thought to have complex mechanisms that promote apoptosis, and targeting apoptosis signaling is developing as a method for novel cancer therapies ,39,40. T2− to H2O2, and then the antioxidant enzyme protein (TRX) that removes H2O2 is converted into nontoxic H2O because TRX is decreased and it is too late to remove ROS, and ROS are sent to the cytoplasm, resulting in a large increase in intracellular ROS, which induces oxidative stress. The iROS can destroy proteins and DNA to induce pathology, leading to apoptotic cell death. Oral cancer studies showed that manoalide increases ROS [Oxidative stress is a biochemical situation defined by the presence of relatively large amounts of harmful reactive species, primarily made up of ROS, and an imbalance between antioxidant defense mechanisms. ROS are primarily produced in cells as byproducts of regular mitochondrial metabolism and have long been linked to apoptosis induction ,44. NAC ases ROS ,10, but m damage, nuclear condensation, and the generation of apoptotic bodies [m. Mitochondria are active organelles that perform fusion (combination of fragments) and fission . The inner membrane protein OPA1 is required for mitochondrial fusion, and the DRP1 protein is required for mitochondrial fission. For rapid and efficient apoptosis, mitochondria must be expressed in fragments through a highly permeable outer surface, and cristae should be separated for controlling mitochondrial morphology and not allowing content exchange between mitochondria [m [Mitochondria play an important role in eukaryotic cells, where their function is to generate ATP during OXPHOS. Studies have shown that manoalide reduces nonmitochondrial (in the cytoplasm) and OXPHOS (in the mitochondria) respiration, including basal respiratory capacity, ATP production, maximal respiratory capacity, spare respiratory capacity, and nonmitochondrial respiratory. The inner mitochondrial membrane has several folds, among which are components of the respiratory chain or OXPHOS complexes I to V. Complexes I to V are multi-subunit enzymes that can synergistically generate an electrochemical proton gradient on the inner mitochondrial membrane. According to research, manoalide reduces the total number of OXPHOS complex I to V proteins, which combined with complex V (ATP synthase) form the mechanism for ATP generation . It is wc bodies . Studieschondria . As a rechondria . Our finndria [m ,10, therm, and ATP production and affected the down-regulation of the mitochondrial fusion OPA1 protein and the up-regulation of the mitochondrial fission DRP1 protein, resulting in impaired mitochondrial function. Manoalide-induced cytotoxicity and apoptosis via intrinsic apoptosis proteins activated and cleaved caspases-9/-3 and PARP in OS cells. Adding NAC to reverse the effects of manoalide caused changes in apoptosis pathway proteins, cellular ROS, and antioxidant enzymes. This confirms that oxidative stress is a significant issue in the presence of manoalide. In conclusion, manoalide is a PLA2 inhibitor and shows potential as an innovative alternative treatment in OS, and further advancement of this compound into the preclinical phase is warranted.The ROS, mitochondrial malfunction, and mitochondrial (intrinsic) apoptosis pathways of the manoalide-induced apoptosis mechanism in human osteosarcoma MG63 cells are summarized in light of the present findings . Initial