A previous study has revealedthat operating conditions substantially influence removal efficiency.Data points were gathered for the experimental studies that developedour ANN-PSO model. The PSO was used to determine the optimum ANN parametervalues. Based on the two processes tested (Fe(II)/chlorine and H2O2/periodate), the proposed hybrid model (ANN-PSO)has been demonstrated to be the most successful in terms of establishingthe optimal ANN parameters and brilliantly forecasting data for RG12and TP elimination yield with the coefficient of determination (R2)topped 0.99 for three distinct ratio data sets.An efficient optimizationtechnique based on a metaheuristic andan artificial neural network (ANN) algorithm has been devised. Particleswarm optimization (PSO) and ANN were used to estimate the removalof two textile dyes from wastewater  using two unique oxidation processes: Fe(II)/chlorineand H One of the most common occurrences is wastewaterfrom the textile industry. The color is still discernible at low concentrations(less than 1 ppm for some dyes).7 Toxic, carcinogenic, mutagenic, or teratogenic compoundsare often found in textile wastewater. For the most part, the chromophoregrouping of a dye is employed to sort it out. Although anthraquinone,xanthene, phthalocyanine, and sulfur are also utilized, most chemicalsare azo (−N=N−) derivatives.8 Some substances may alter wastewater treatment facilities,leading to more stable and harmful organisms, or they may not changeand remain unchanged. For decades, scientists have been working tocreate advanced oxidation processes (AOPs) that are more environmentallyfriendly.10 Hydroxyl radical •OH,a potent oxidant  and highly reactivespecies to most organic contaminants in situ, is produced by AOPs.AOPs include the Fenton system (Fe(II)/H2O2),UV/H2O2, H2O2/O3, UV/O3, and UV/TiO2.11 An alternative to H2O2 and UV has recentlybeen explored: the UV/chlorine procedure. It has been tried in a pilotor full-scale plant for water treatment, drinking water processing,and groundwater remediation.12 In thisprocess, several free radicals, including •OH, Cl2•–, and ClO•, aregenerated to collectively remove micropollutants at a much fasterrate. Micropollutant elimination is an important goal for environmentalistsand scientists alike. Utilizing multiple free radicals in a novelclass of oxidation processes is one way to achieve this goal.13 As with UV/chlorine, UV/periodate acts as amultifree radical generator, producing radicals such as IO3•, IO4•, •OH, IO3, O(3P), O3, and H2O2 for the removal of a variety of water contaminants.17Toremove organic pollutants, nutrients, and other impurities,a typical wastewater treatment plant uses a variety of physical, chemical,and biological unit processes. Detritus that is solubilized by microorganismsmay only be removed using natural treatment.27 Scientists have put out fuzzy logic (FL) models, artificial neuralnetworks (ANNs), and other ML techniques. The latter have been appliedto processes in several studies.36 Machine learning (ML) offers practical solutions to solve challengingissues in various industrial applications.4 ML includes computer algorithms and statistical methods requiredfor data-driven control, estimation, prediction, classification, orclustering.37To integrate the results of experiments, mathematic modelswereregarded as appropriate. Kinetic modeling models have many constraintsbecause of their complicated nature and nonlinearity, limiting theparameters.38 Several ANNs, such as a multilayerperceptron, are based on the human brain (MLP). This rigorous mathematicalmodel often used in ML may explain any nonlinear relationship betweeninput and output sets. An ANN is a collection of neurons linked bytwo fundamental parameters (connection weights and thresholds).41 Neural network techniques such as Levenberg–Marquardt (LM),scaled gradient descent (SGD), and gradient descent with momentum(GDWM) are among the most often employed (GDM). Adaptation, learning,and generalization may occur even when working with nonlinear functions.Contrarily, the speed of convergence of BPNN algorithms is slow. Somemetaheuristic optimization approaches, such as the genetic algorithm(GA), firefly algorithm (FA), ant colony optimization (ACO), particleswarm optimization (PSO), and differential evolution (DE), may beutilized to solve these problems. They help the ANN find more optimalsolutions faster, increasing its overall efficiency. Techniques likePSO and GA illustrate this trend.41 To cope withthe most complex and complicated issues in optimization, they arethe most promising global optimization approaches.46Although it is notpracticable, complicated problems that weredifficult to describe and analyze may now be appraised using thesemethods.48 These two reactions, Fe(II)/chlorine and H2O2/periodate, have been discovered as multiple sources of free-radicaloxidation of organic contamination. Cl•, ClO•, and Cl2•– chlorineradicals have been implicated in the Fe(II)/Chlorine process,39 while IO3• and IO4• iodine radicals, as well as singlet oxygen (1O2), have been implicated in the H2O2/periodatesystem.49Fe(II)/chlorineprocess2O2/periodateHThey have recently discovered two new oxidationmethods for efficientlyremoving textile colors from wastewater.2O2/periodate processes. Several operationalvariables, including as reagent dosages, solution temperature, andpH, were examined throughout a wide range of reaction times. Industrialapplications need a modeling technique that maximizes the efficiencyof both kinetic processes.To degrade RG12 and TB textile colors fast, we usedFe(II)/chlorineand H2O2/periodate oxidation processes. ThePSO metaheuristic optimization was combined with ANN to constructa feasible model for predicting and optimizing the removal of textilecolors from wastewater effluent using Fe(II)/chlorine and H2O2/periodate, respectively. The model’s adaptabilityand durability were shown using the R2 coefficient and the root mean square error (RMSE) between the predictedand experimental datasets.This work aimed to develop a hybridmodel (ANN-PSO) for the caseof the removal of RG12 and TB dyes from wastewater utilizing Fe(II)/chlorineand H248 developed the ANN-PSO model byassessing the removal kinetics of RG12 and TB dyes from aqueous solutionsunder different experimental settings, utilizing the Fe(II)/chlorineand H2O2/periodate oxidation systems, respectively.The testing methodology and data are summarized in Text S1 in the Supporting Information.Merouani et al.C0: 10–100mg/L), and liquid temperature (20–40 °C). For H2O2/periodate, 169 datasets were collected from the experimentalassessment of TB removal kinetics over time under various experimentalfactors: initial H2O2 concentration (10–200mM); initial periodate dosage (0.5–10 mM); initial solutionpH (3–10.5); initial TB concentration (C0: 5–50 mg/L); and liquid temperature (10–50°C).For the Fe(II)/chlorinesystem, 146 datasets were collected fromthe experimental assessment of the removal kinetics of RG12 over timeunder various experimental factors such as solution pH (3–7.9),chlorine dosage (25–250 mM), Fe(II) initial concentration (5–100M), initial RG12 concentration  are the threearchitectural levels of ANNs.54 Neurons (or nodes) are essential computer components used in parallelcomputing.55 Neurons work together to detect input data sets commonlyseen.56f is thetransfer function; jx is the neuron’sinputs; and ijw is the link between ILand HL (weights) and the HL’s threshold of j neuron.53 Constructing and training neuronal networkshas as its primary goal the minimization of the objective function(or fitness function), which in turn leads to better predictions fornew input.57 The latter compares the outputand experimental data sets to determine how well the network operates.The fitness (error) function may thus be written as follows:N is thenumber of experiments, and iy and oy are the calculated and experimental data,respectively.57Input (IL), output(OL), and intermediate or hidden (HL) layers  are the cognition and social weights,respectively.Arbitraryparticles, also known as solutions, are supported by the PSO algorithm.The search space (or the state space) is transformed into a swarmthat seeks only the most advantageous options. The PSO training iterationuses the experience of individual particles and those around themto adjust their position and speed.w is the inertia weight (or inertiaconstant).60 rand1 and rand2 generaterandom values ranging from 0 to 1. An example of the standard flowchart for the PSO approach may be seen in Figure S3 (Supporting Information).Pbestidn and Gbestidn positions at each iteration.The fundamental idea behindthe PSO approach is that each particleis accelerated toward its k-dimension and m-size ofthe acceleration, each particle may be representedas follows: iX = T; iV =  .When searchingfor the 3.339Analysis of state spaceis carried out by using a collection of particles. Weights and thresholdsare stored in each particle for later adjustment.Figure S5 of the SupportingInformation shows the crucial phases of the ANN-PSO hybrid algorithm.1.Importing experimentaldata.2.Define ANN structure,weights, andthresholds.3.Computethe number of weights and thresholds:A, B, and C are for IL, HL, andOL, respectively.4.Initializeparameters.5.Pbest and Gbest.Calculatethe error, 6.Adjust the location andvelocity 37 and 387.f(Xidn + 1) < f(Pbestidn) Then Pbestidn + 1 = Xidn + 1If Pbestidn + 1 = PbestidnElse 8.f(Xidn + 1) < f(Gbestidn) Then Gbestidn + 1 = Xidn + 1If Gbestidn + 1 = GbestidnElse 9.The ANN parameters’ resultsare shown .3.42O2/periodate system were split into70% training, 15% testing, and 15% validation. Initial solution pH,initial chlorine (or H2O2) concentration, initialFe(II) (or periodate) concentration, initial RG12 (or TB) concentration,and initial liquid temperature are all factors in the IL. The removalefficiency of RG12 (or TP) is saved in the OL  that minimizethe objective function (RMSE) between the anticipated outcomes ofour models and the experimental datasets.4.1The five parametersin the IL were initial solution pH (from 3 to 7.9), initial chlorineconcentration (from 25 to 1000 M), initial Fe(II) concentration (from0 to 100 M), initial RG12 concentration (from 10 to 100 mg/L), andliquid temperature (from 10 to 40 °C). The OL includes RG12 removal(3.59–44.26 mg/L).c1 and c2, of 1.25and 2.5, respectively. w = 0.15; maximum number ofiterations = 4500, and swarm size is 15:ijw (11× 5) correlating IL with HL.Fifty-five weights, wj1 correlatingHL with OL.Elevenweights, j for HL’s neurons.Eleven thresholds, θj forthe OL  is the ultimate architecturalnetwork . After each training iteration,two variables, R2)are 0.99975, 0.99993, and 0.99987. The RMSE for each stage  is 0.00181, 0.00084, and 0.00129, respectively.These statistics demonstrate strong performance across all data sets,with (R2) values closer to unity and anRMSE less than 0.0012. This shows that all data fall along a 45-degreeline with a slope = 1. The network outputs generated by the ANN-PSOhybrid model and relevant experimental data sets have the same linearconnection .4.22O2 concentration (10–200mM), initial IO4– concentration (from0.5 to 10 mM), initial solution pH (3–11), liquid temperature(10–50 °C), and initial TB concentration (5–50mg/L) were the five parameters in the IL. The elimination quantityin the OL is 2.04–22 mg/L.The initialHc1 and c2 equal to 1.25 and 2.5, respectively, w = 0.25, a maximum number of iterations =4500, and swarmsize = 15).ijw (70= 14 × 5) correlatingIL with HL.wj1 (14)correlating HL with OL.j (14) for HL’sneurons.θ1 (1) forOL.θThey are distributed as follows:The final architectural network isthree layers: (5:14:1) network.2O2/period throughout the three stages  using MATLAB software. The practical data sets wereclassified into three types: training data sets (70%), testing datasets (15%), and validation data sets (15%).R2, was 0.99908, 0.9963, and 0.99823 for thethree stages, respectively.The RMSE values for the three stages were 0.00174, 0.00350, and 0.00273, respectively. It is determinedthat the correlation is perfect because R2 is near one and RMSE values are less than 0.0018.The coefficient of determination, 2O2/periodate processes were predictedmore precisely using theANN-PSO model. For new RG12 data sets, Fe(II)/chlorineand HIt can be concludedthat the experimental and simulated findingsagreed very well.52O2/periodate oxidationprocesses. The ANN training function adjusts the weights and thresholdvalues according to the PSO approach. The use of MATLAB software carriedout the results. The coefficient of determination (R2) from the ANN-PSO hybrid mathematical model topped 0.99for three distinct ratio data sets from two independent systems. Theproposed ANN-PSO model effectively predicts new RG12 and TB removaldata sets with high (R2) and low RMSEvalues.Thiswork investigates the use of a new approach based on an ANNand PSO method to remove RG12 and TB dyes from wastewater utilizingFe(II)/chlorine and H


Uric acid is considered the main substrate for peroxidases in plasma. The oxidation of uric acid by human peroxidases generates urate free radical and urate hydroperoxide, which might affect endothelial function and explain, at least in part, the harmful effects of uric acid on the vascular system. Peroxidasin (PXDN), the most recent heme-peroxidase described in humans, catalyzes the formation of hypobromous acid, which mediates collagen IV crosslinks in the extracellular matrix. This enzyme has gained increasing scientific interest since it is associated with cardiovascular disease, cancer, and renal fibrosis. The main objective here was to investigate whether uric acid would react with PXDN and compromise the function of the enzyme in human endothelial cells. Urate decreased Amplex Red oxidation and brominating activity in the extracellular matrix (ECM) from HEK293/PXDN overexpressing cells and in the secretome of HUVECs. Parallelly, urate was oxidized to 5-hydroxyisourate. It also decreased collagen IV crosslink in isolated ECM from PFHR9 cells. Urate, the PXDN inhibitor phloroglucinol, and the PXDN knockdown impaired migration and adhesion of HUVECs. These results demonstrated that uric acid can affect extracellular matrix formation by competing for PXDN. The oxidation of uric acid by PXDN is likely a relevant mechanism in the endothelial dysfunction related to this metabolite. E° = 0.56 V, pH 7.0, HU●−, H+/UH2−) [Uric acid is the end product of purine metabolism in humans since we do not express urate oxidase. Uric acid accumulates in plasma as the mono-anion urate pKa 5.4), and the plasma concentration varies between 50–400 µM in normouricemia, reaching millimolar concentration in hyperuricemia ,2. Due tH+/UH2−) , urate i, and theH+/UH2−) . Urate levels are positively associated with cardiovascular diseases ,10,11,12We and others demonstrated that urate is a substrate for the inflammatory peroxidases myeloperoxidase and lactoperoxidase ,20. The 4 M−1s−1) was around two orders of magnitude lower than that with bromide (5.6 × 106 M−1s−1) [2 M−1s−1), revealing that the complete turnover of the enzyme may be slowed down in the presence of urate. In this sense, urate would rather act as an inhibitor of the enzyme than a substrate, but independently of the exact mechanism, it would disturb basement membrane formation.A recent paper showed that urate can be oxidized by a truncated fully active PXDN (hsPxd01-con4). The rate constant of the reaction of compound I hsPxd01-con4 with urate (1.9 × 10 M−1s−1) ,34. Howe M−1s−1) , being eConsidering the important role of PXDN in extracellular matrix formation and vascular endothelial homeostasis, we hypothesized that the oxidation of uric acid by PXDN could contribute to vascular disease by two mechanisms: (1) the oxidation of uric acid could divert the production of HOBr and collagen IV crosslink by the enzyme; (2) the production of urate free radical and its electrophilic-derived products could contribute to oxidative stress and alter the function of proteins that are targets for oxidation/uratylation ,22,36. BIn this study, we extended previous findings on the reaction of PXDN with urate ,39 by in® Ultra-15 mL and Amicon® Ultra-0.5 mL centrifugal filters 10 kDa cutoff were acquired from Merck Millipore . Plasmids (vector carrying pcDNA3.1) were kindly provided by Prof. Geiszt Miklós from Semmelweis University . Anti-COL4A2 and Anti-V5 antibodies were purchased from Invitrogen , anti-rabbit IgG HRP-linked Antibody from Sigma Aldrich  and Collagenase 1 from Gibco . Small interfering (si) RNAs targeting PXDN (siPXDN) and control siRNA (siRNA scramble) were obtained from Invitrogen . All other reagents were purchased from Sigma Aldrich . HOBr was prepared by adding an equal volume of 50 mM HOCl to 250 mM sodium bromide in phosphate buffer 50 mM pH 7.4 while vortexing. After 10 min, HOBr concentration was determined by measuring absorbance at 329 nm with εHOBr = 335 M−1cm−1 [RPMI medium and fetal bovine serum (FBS) were purchased from Vitrocell . Amplex™ Red Reagent, Lipofectamine™ RNAiMAX Transfection Reagent Lipofectamine™ 3000 Transfection Reagent were obtained from Invitrogen . Solvents were purchased from JTBaker . Mass Spectrometry Grade Trypsin was purchased from Promega . Iodoacetamide and dithiothreitol (DTT) were obtained from Bio-Rad Laboratories . 4-aminobenzoic acid hydrazide (ABAH) was purchased from Fluka Chemicals . Amicon M−1cm−1  after 1:2 atmosphere. For experiments, confluent adherent cells were harvested using a solution containing trypsin (0.1%) and EDTA (0.5 mM) prepared in phosphate-buffered saline solution . After centrifugation and counting, cells were seeded on culture plates in the appropriate concentration. HUVECs were used between passages 6 and 10. Human Embryonic Kidney cells  and PFHR9 , an epithelial cell from mouse embryonal carcinoma, were maintained in high glucose DMEM (Dulbecco’s Modified Eagle’s Medium) containing 10% fetal bovine serum (FBS), streptomycin (100 mg/mL) and penicillin (30 mg/mL) at 37 °C in a 5% CO2 atmosphere. For experiments, confluent adherent cells were harvested using a solution containing trypsin (0.1%) and EDTA (0.5 mM) for HEK293 cells and trypsin (0.25%) and EDTA (0.53 mM) for PFHR9 cells. The plasmids (vector carrying pcDNA3.1/PXDN-V5-His) were transfected into HEK293 cells using lipofectamine 3000 Transfection Kit according to the manufacturer’s instructions. After transfection, cells were treated for 4 weeks with 500 μg/mL of geneticin (G418) as a selection antibiotic. PXDN expression was confirmed by Western blot. Briefly, 20 µg HEK293/PDXN lysate was separated on a 10% reducing SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were incubated with primary antibody to Anti-V5  and subsequently incubated with HRP-conjugated secondary antibody  to detect protein expression by chemiluminescence. A selection of immortalized human umbilical vein endothelial cell line (HUVEC) was kindly provided by Prof. Francisco Laurindo’s group from INCOR (São Paulo—Brazil). HUVECs were maintained in RPMI medium containing 10% fetal bovine serum (FBS), streptomycin (100 mg/mL), and penicillin (30 mg/mL) at 37 °C in a 5% CO5 cells/well) for a further 24 h in 96-well plates without coating in RPMI medium 0.2% BSA. In the migration assay, 24 h after transfection, a scratch was made in the cell monolayer, and cells were washed with PBS and incubated with RPMI medium 0.2% BSA for 24 h. HEK293 overexpressing PXDN were seeded at 50% confluence and transfected with 50 or 100 nM siRNA PXDN (5′-CCUCCAUCCUAGAUCUUCGCUUUAA-3′) or siRNA control/scramble (5′-CCUCCCUCAUAGAUGUUCCCUUUAA-3′) using lipofectamine RNAiMAX Transfection Reagent according to the manufacturer’s instructions. After 24 h, V5 expression was evaluated. Sequences of the siRNAs were provided as described by Péterfi et al. (2009) [In summary, cells were grown until 80% confluence in 100 mm culture plates. To isolate secretome, cells were washed twice with PBS  and incubated for 1 h 30 min (HUVEC), or 36 h (HEK293) with culture media without FBS and phenol red. The culture media were collected after the incubation period, centrifuged at 1400 rpm for 10 min, and filtered through disposable 0.22 μm filters to remove cellular debris and floating cells. Next, the secretome was concentrated 200-fold on Amicon-Ultra 15 mL–10 kDa filters. The ECM isolation procedure was followed as described by Bathish et al. (2018) . Briefly2O2 were added to 10 μg total protein content from cellular fractions  to a final volume of 200 μL  into 96-well fluorescence microplates. Fluorescence  was recorded every 30 s for 90 min in a microplate reader . The results were calculated within the linear fluorescence increase and expressed as the rate of Amplex Red oxidation per minute. To measure peroxidase activity, 50 μM Amplex red and 50 μM H2O2 for 4 h at 37 °C in a thermoblock . Samples were injected into a Prominence HPLC instrument . The products 3-bromotyrosine and 3,5-dibromotyrosine were separated from free L-tyrosine in a Shim-pack VP-ODS reverse phase column  equilibrated with 100% solvent A . L-Tyrosine and its brominated products were eluted at a flow rate of 1 mL/min with a linear gradient generated with solvent B  as follows: 0% solvent B for 5 min, 0% to 100% solvent B over 30 min, and 100% solvent B for 10 min. 3-Bromotyrosine and 3,5-dibromotyrosine were monitored on a diode array detector and quantified at 280 nm against a standard curve constructed with synthetized HOBr and L-Tyrosine [A total of 100 µg protein from cellular fractions was incubated with L-Tyrosine 1 mM, 100 mM NaBr, and 100 µM HTyrosine . 2O2 (100 µM) to a final volume of 200 µL  for 30 min at 37 °C. After incubation, samples were diluted in 2% trichloroacetic acid (TCA) and 60% acetonitrile (ACN) containing 16.6 μM uric acid 1,3-15N2 as internal standard. Samples were centrifuged at 20,000× g . The supernatant was filtered (PTFE filter 0.22 µm) and injected into the LC system coupled to a Q-TOF mass spectrometer , electrospray ionization source (ESI), operating in the negative mode, as described before [15N2. The mass transitions used were m/z 167.0160→126.0201 for uric acid, m/z 169.0151→127.0201 uric acid 1,3-15N2, and m/z 183.0108→140.0102 for hydroxyisourate (HOU) and plotted against a standard curve. The fragmentation of hydroxyisourate from a standard solution as well as its retention time was used to confirm hydroxyisourate detection in the samples.In total, 100 µg total protein from HUVEC secretome or HEK293 cells ECM was incubated with urate (100 and 200 µM) and Hd before  with mod2O2 and 100 µM of NaBr in PBS buffer (pH 7.4).Then, reactions were stopped with 1% sodium azide. The non-collagenous domain (NC1) was obtained after digestion by collagenase 1  (2 mg collagenase/5 mg ECM) overnight. The expression of collagen IV NC1 dimers and monomers was verified by Western blot. Briefly, 100 µg of the experimental samples were separated on a 10% reducing SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were incubated with primary antibody to Anti-ColIVA2 (1:5000) and subsequently incubated with HRP-conjugated secondary antibody  to detect protein expression by chemiluminescence . Band densitometry was calculated using ImageJ software.PFHR9 cells were cultured in DMEM as described above until confluence. Then, the medium was daily changed with the addition of 50 μM PHG (phloroglucinol), a heme peroxidase inhibitor, for 6 days for matrix deposition ,41. ECM 4 cells/tube) containing modified RPMI medium with 0.2% BSA and the indicated treatments: PHG  or urate (25–400 μM). After gentle mixing, cells were seeded in non-coated or 1% gelatin coated 96-well plates and left to adhere for 24 h at 37 °C and 5% CO2. After incubation, the supernatant was removed and adherent cells were washed 3 times with ice-cold PBS, followed by a fixation step with methanol . After washing 3 times with distilled water, cells were stained with crystal violet (0.1% in 25% methanol), for 30 min at room temperature and washed again 3 times with distilled water. Crystal violet was solubilized with 1% SDS and the absorbance was read at 600 nm in a microplate reader . Absorbance was normalized to the control group (100%). To evaluate the capability of cells to attach, confluent HUVECs were harvested with EDTA (0.5 mM in PBS) and distributed in microtubes /initial scratch width × 100%Fifty µg protein from HUVEC secretome was mixed with Laemmli buffer, heated at 95 °C for 10 min, and separated in 10% SDS-PAGE. The gels were stained with Coomassie blue G for 2 h and washed with water overnight.3 pieces, and destained with 50% acetonitrile in water. Samples were reduced with DTT (10 mM) and alkylated with iodoacetamide (50 mM) for 30 min. The reduced and alkylated protein samples were digested with Sequencing Grade Modified Trypsin  (protein/trypsin ratio of 50:1) in ammonium bicarbonate (50 mM) for 4 h. Then, a second aliquot of trypsin (protein/trypsin ratio 50:1 w/w) was added, and the protein samples were further incubated at 37 °C overnight. Digestion was stopped with 5% formic acid in 50% acetonitrile. Peptides were eluted from the gel fragments once with 1% formic acid in 60% methanol and twice with 60% acetonitrile containing 1% formic acid and dried under vacuum. Before analysis, the peptides were suspended in 0.1% formic acid in water and desalted using a Stage Tip protocol [For in-gel digestion, the bands corresponding to 100–250 kDa were excised from the gel, cut into 1 mmprotocol . Samplesw/w for 4 h at 37 °C. A second aliquot of trypsin (50:1 w/w) was added and samples were incubated overnight at 37 °C. After acidic hydrolysis with 2% trifluoroacetic acid, samples were desalted using the StageTip protocol [First, 10 μg protein was solubilized in 100 mM ammonium bicarbonate with 0.4% sodium deoxycholate, reduced with DTT (5 mM), alkylated with iodoacetamide (15 mM), digested with PNGase F Glycan Cleavage Kit  for 1 h at 37 °C and digested with Sequencing Grade Modified Trypsin , protein/trypsin ratio 40:1 protocol . ECM digestion from HUVEC, HEK293, and HEK293 overexpressing PXDN cells were prepared following the in-solution digestion described above but with the replacement of sodium deoxycholate by 8 M urea and 6 M guanidine hydrochloride in the solubilizing step .m/z 200) and the automatic gain control (AGC) target at 4 × 105. The m/z range 400−1600 was monitored. Each full scan was followed by a data-dependent MS2 acquisition with a resolution of 30,000 (at m/z 200), maximum fill time of 54 ms, isolation window of 1.2 m/z, and normalized collision energy of 30.Digested and desalted samples were suspended in 0.1% formic acid  and submitted to MS analyses. Angiotensin (0.2 pmol/μL) was used as a global internal standard to monitor mass variability, and iRT peptides  were used to normalize the retention time of all peptides. An Easy-nLC 1200 UHPLC  was used for peptide separation with a linear gradient of solvent A (0.1% formic acid) and solvent B (0.1% formic acid in 80% acetonitrile). Each sample was loaded onto a trap column  with 12 μL of solvent A at 980 bar. After this period, the trapped peptides were eluted onto a C18 column  at a flow rate of 300 nL/min. Peptides were eluted from the column using a linear gradient of 5−28% B for 25 min followed by 28−40% B for 5 min. Finally, the percentage of solvent B was increased to 95% in 2 min, and the column was washed for 10 min with this solvent proportion. Re-equilibration of the system with 100% A was performed before each injection. Acquisition of the data was performed using an Orbitrap Fusion Lumos mass spectrometer  with a nanospray Flex NG ion source . A full MS scan was followed by data-dependent MS2 scans in a 3 s cycle time. Precursor ions selected for MS2 were excluded for subsequent MS2 scans for 20 s. The resolution for the full scan mode was set as 120,000 (at www.maxquant.org accessed on 30 May 2022) with fixed Cys carbamidomethylation, variable Met oxidation, Asn-Asp exchange, and N-terminal acetylation. MaxQuant default mass tolerance was used for precursor and product ions. Trypsin/P was selected as the enzyme, and two missed cleavages were allowed. The results were processed by label-free quantification.Tandem mass (MS/MS) spectra were searched against the reviewed UniProt human database, using the MaxQuant search engine .All data are expressed as mean ± standard error of the mean (SEM) of at least 3 independent experiments. The difference among groups was calculated by one-way ANOVA with Bonferroni’s post hoc test and was considered significant when Initially, peroxidase activities from HUVEC cellular fractions, secretome, lysate, and extracellular matrix (ECM) were compared. Secretome had the highest activity A, indicaPeroxidase activity was also measured in the secretome and ECM from HEK293 overexpressing PXDN cells. HEK293/PXDN lysates, as well as in HUVEC, did not have considerable peroxidase activity. ECM fraction in these cells presented higher peroxidase activity than secretome A. In add4 M−1s−1 and with compound II at 5.8 × 102 M−1s−1 . This near two orders of magnitude difference among rate constants holds the enzyme as the intermediate compound II, preventing the complete cycle and the return to its native form [In accordance, recent data using a truncated PXDN showed that urate reacts with compound I PXDN at 1.9 × 10ive form . The ratIn an attempt to understand why HUVEC has higher peroxidase activity in secretome, whereas HEK293 cells in the ECM fraction, we identified protein content from ECM in both HUVEC and HEK293 cells by proteomics. Laminin and type IV collagen are the largest class of proteins present in the basement membrane interacting with each other and with other ECM proteins. This laminin/type IV collagen colocalization consequently places PXDN. A recent study showed that PXDN binds to laminin in the basement membrane . Thus, wThe effect of urate on Amplex Red oxidation could be a non-specific effect upon any peroxidase present in ECM or secretome. However, bromination is a specific result of heme-peroxidases, such as PXDN, catalysis ,39. Hypo2O2 was omitted, demonstrating that endogenous H2O2 is enough for PXDN catalysis in HUVEC secretome. Of relevance, products of urate oxidation by heme-peroxidases can form adducts with lysine residues in proteins [Since urate can be a substrate for PXDN , we idenproteins ,48, whicTo evaluate the effect of urate on collagen IV crosslinking in isolated ECM, we initially treated PFHR9 cells with the heme-peroxidase inhibitor, PHG, for 6 days to prevent the formation of collagen IV crosslinks. As illustrated in To access whether PXDN inhibition would affect HUVEC migration, we performed an in vitro scratch assay . The treThe discovery of PXDN catalysis in sulfilimine bond formation was a breakthrough event to unveil the mechanism of collagen IV crosslink. It was also the first evidence of bromine’s role in mammals . HoweverBy using a DDA proteomics approach that included an extra digestion step with PNGase, we identified 22 unique peptides for PXDN in HUVEC ECM and secretome. One of these peptides, the AGEIFER, was also identified in a previous study that used MS to quantify PXDN in cellular extracts . AdditioThe heme-peroxidases from the PXDN family, myeloperoxidase and lactoperoxidase, are known to oxidize the endogenous substrate uric acid, yielding urate free radical and urate hydroperoxide ,19,20. U4 M−1s−1), but it is a poor substrate to the compound II (5.8 × 102 M−1s−1) [In agreement with the oxidation of urate by PXDN, Sevcnikar and collaborators demonstrated that urate is a good substrate for the truncated PXDN intermediate compound I (1.9 × 10 M−1s−1) . Therefo M−1s−1) . Here, wWhereas HEK293/PXDN overexpressing cells had the highest peroxidase activity in ECM enriched fraction, HUVEC had it in secretome. PXDN interacts with laminin in the ECM , and we We also showed that urate decreases the brominating activity of PXDN. As a result, there was a decrease in the formation of sulfilimine crosslinks. The decrease in tyrosine bromination could be by the competition between urate and bromide by PXDN compound I, as well as by a direct reaction of urate with hypobromous acid. In the former, the one-electron oxidation forms urate radical. Two urate radicals can dismutate yielding dehydrourate and urate. In the latter, the two-electron oxidation of urate also forms dehydrourate . This coIncubation with urate decreased HUVEC migration and adhesion. Both events are essential for tissue repair, angiogenesis, and vascular integrity maintenance ,63 and mHyperuricemia has been extensively studied for triggering inflammation and endothelial dysfunction ,66,67,68Oxidation of urate is also relevant in endothelial cell adhesion impairment. Our group recently showed that the product of urate oxidation, urate hydroperoxide, disrupted HUVEC adhesion by oxidizing thiol proteins that are key to cell adhesion . HoweverIn summary, this study provides additional information on the role of PXDN in ECM formation. It shows, for the first time, that inhibition of PXDN is enough to impair adhesion and migration of vascular endothelial cells in absence of another injury. The purine metabolite, urate, can be oxidized by PXDN, producing the unstable urate free radical, decreasing hypobromous acid formation and disrupting ECM formation and HUVEC adhesion and migration. Therefore, this study supports a harmful effect of physiological concentrations of uric acid upon vascular endothelial cells, reinforcing the relevance of this metabolite in cardiovascular disease.


Smilax glabra Roxb (SGR) has been widely applied alone or in combination with other Chinese herbs in heart failure (HF), but its mechanism and protective effect have not been investigated. We aimed to explore the mechanism and protective effect of SGR on the treatment of HF. Network pharmacology analysis predicted that SGR was involved in the regulation of cell proliferation, oxidation–reduction process, apoptotic process, ERK1 and ERK2 cascade, MAPK cascade, etc. Its mechanism was mainly involved in the MAPK signaling pathway, calcium signaling pathway, cardiac muscle contraction, etc. Subsequently, SGR was proved to improve cellular viability, restore cellular morphology, suppress cellular and mitochondrial ROS production, improve H2O2-induced lysosome inhibition, attenuate mitochondrial dysfunction, and protect mitochondrial respiratory and energy metabolism in H9c2 cells. SGR activated the p38MAPK pathway by decreasing the mRNA expression of AKT, PP2A, NF-KB, PP2A, RAC1, and CDC42 and increasing the mRNA expression of Jun, IKK, and Sirt1. SGR also decreased the protein expression of ERK1, ERK2, JNK, Bax, and Caspase3 and increased the protein expression of p38MAPK and Bcl-2. In addition, Istidina at the highest degree was identified in SGR via the UHPLCLTQ-Orbitrap-MSn method, and it was suggested as anti-heart failure agents by targeting SRC with molecular docking analysis. In conclusion, SGR has a protective effect on HF through cellular and mitochondrial protection via multi-compounds and multi-targets, and its mechanism is involved in activating the p38 MAPK pathway. Istidina may be possible anti-HF agents by targeting SRC.  Smilax glabra Roxb (SGR) on heart failure was predicted by network pharmacology analysis(1) For the first time, the mechanism of (2) For the first time, SGR was proved to exert a protective effect on cellular injury and mitochondrial dysfunction with vitro verificationvia the UHPLC-LTQ-Orbitrap-MSn method and molecule docking analysis(3) For the first time, SGR was proved as a multi-target agent with multi-compounds for heart failure (4) Five compounds with potential protective in total glycosides from SGR were identified, and Istidina was first considered as possible anti-heart failure agentHeart failure (HF), a multifactorial and systemic disease, is responsible for more than 17.3 million deaths each year, and its mortality ranks top in cardiovascular diseases . Mitochovia regulating the activity of cellular proteins, enzymes, and transcription factors , one of the most important downstream signaling pathways of ROS, are involved in cellular responses to external stimuli  factors . The mam factors . ERK1/2  factors . Oxidatiade H2O2 . Oxidatiade H2O2 .Smilax glabra Roxb (SGR) has been applied in the treatment of cardiovascular diseases clinically for more than 2,000 years in China , the Encyclopedia of Traditional Chinese Medicine Database , and the TCMID Database (http://119.3.41.228:8000/tcmid) were used to collect the chemical compounds of SGR, the candidate compounds with oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 were identified as active compounds through the Symmap Database (https://www.symmap.org/) indicating the compounds had chemical stability and solubility.The Traditional Chinese Medicine Systems Pharmacology Database  to search and predict the targets of each active compound.SGR active compounds were screened after duplication, and they were imported into the Batman Database (https://www.genecards.org/) with a relevance score no less than 5. Then the intersection targets between HF diseases targets and predicted targets of SGR compounds were screened by Venn analysis. The intersection targets were identified as targets of SGR compounds that had a potential pharmacological effect on HF.The original HF disease targets were obtained from the Genecards Database , with the species limited to “Homo sapiens,” PPI network was then imported into Cytoscape 3.1.1 software for network analysis of the core subsystem. Key targets (Hub-Target) were screened according to the connection degree after network topology analysis.Protein–Protein Interaction (PPI) network of the intersection targets was obtained from Search Tool for the Retrieval of Interacting Genes/Proteins  (via DAVID Database (https://david.ncifcrf.gov), including cellular component (CC), molecular function (MF), and biological process (BP). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted via the “org.Hs.eg.db” package in R software (Version 3.6.3). Enrichment bubbles were constructed through the OMICShare tool online platform (http://www.omicshare.com/tools).Gene Ontology Annotation enrichment analysis (GO) of screened hub targets was performed Reference standards of Istidina, resveratrol, shikimic acid, epicatechin, and dihydrokaempferol were accurately weighed and dissolved with 80% methanol to obtain standard solutions at a concentration of 0.153, 0.110, 0.068, 0.065, and 0.098 mg/ml, respectively. Also, the standard solutions were filtered with a 0.22 syringe filter before analysis. A UHPLC system coupled with LTQ Orbitrap XL high resolution mass spectrometric conditions  was applied for LC-MS analysis. The chromatographic separation was performed on an ACQUITY UPLC HSS T3 Column  at a flow rate of 0.2 ml/min. The mobile phases were composed of solution A (water) and solution B (methanol) and the gradient elution was set as follows: 10% B (0–8 min), 10–35% B (8–15 min), 35–90% B (15–40 min), and 90–10% D (46–50 min). The injection volume was 4 µl. MS analysis was performed on a mass spectrometer equipped with an ESI source and the parameters were as follows: spray voltage, 3.8 KV; capillary temperature, 350.05°C; capillary voltage, −18.00 units; tube lens voltage, −67.68 V; sweep gas, 0.24 units; sheath gas and auxiliary gas were set as 29.95 and 4 units, respectively. Orbitrap mass analyzer was set up with the scan range of m/z 90–1,500 and the resolution of 30,000. The samples used negative ion mode analysis.https://www.rcsb.org/) and were preprocessed by PyMol software. The structural files of the key active compounds of SGR were downloaded from the PubChem Database (https://pubchem.ncbi.nlm.nih.gov/) and were saved in *mol2 format. The abovementioned structures were added hydrogens, and the charges were calculated by autodocktools1.5.6 software, respectively; then, they were saved in a PDBQT format. Finally, molecules and proteins were docked through the Autodock Vina 1.1.2 software, the model with the smallest binding energy value was selected, and their structural visualizations were conducted by PyMOL tools.The protein structures of screened targets were downloaded from the PDB Database (Smilax glabra Roxb was purchased from Kangmei Industry (LOP: 160505781). H9c2 rat cardiomyocytes (ATCC) were purchased from American Type Culture Collection . Dulbecco’s Modified Eagle’s Medium, fetal bovine serum, 100  U/ml penicillin, and 100 μg/ml streptomycin were purchased from Gibco . Reactive oxygen species assay kit, mitochondrial permeability transition pore assay kit, Lyso-Tracker Red, and antifade mounting medium with DAPI were purchased from Beyotime . Cell Meter™ Mitochondrial Hydroxyl Radical Detection Kit was purchased from AAT Bioquest. TMRE Mitochondrial Membrane Potential Assay Kit was purchased from Solarbio . 3- -2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich , Seahorse XF Assay Kit was purchased from Agilent . Primers were ordered from Invitrogen (Thermo Fisher). Antibodies including β-actin (ab182651), Bcl-2 (ab32124), HRP-conjugated goat anti-rabbit (ab6721), and anti-mouse IgG (ab6789) were purchased from Abcam Co. P38 MAPK , SAPK/JNK , and p44/42 MAPK  were purchased from Cell Signaling Technology. Goat Anti-Rabbit IgG AF 488  was purchased from Abmart .2 incubator. Cells in the H2O2 group were exposed to 600 μM H2O2 for 3 h. In the SGR group, cells were pretreated with 200, 400, and 800 μg/ml SGR for 24 h, then they were exposed to 600 μM H2O2 for another 3 h. Cells in the control group were cultivated with a normal medium.H9c2 cells were cultured in Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine serum, 100  U/ml penicillin, and 100 μg/ml streptomycin in a 37°C, 5% CO4/well) were seeded into 96-well plates. After treatment, 10 μl of 5 mg/ml MTT solution was added to each well and then cells were incubated in the dark for 4 h. After removing the cultured medium, the crystals were dissolved in 150 μl of DMSO. The absorbance was measured at 490 nm with a BioTek plate reader (BioTek).Cell viability was detected by MTT assay. In brief, H9c2 cardiomyocytes (1  ×  10Lactate dehydrogenase (LDH) production was measured by using LDH cytotoxicity Assay Kit for detection of cytotoxicity in H9c2 cells, according to the manufacturer’s protocol. Absorbance was measured at 490 nm using a BioTek plate reader (BioTek), and the values were directly proportional to the enzyme activity. In cellular morphology observation, cellular morphology was imaged by using a fully automatic inverted fluorescence microscopic analysis system .Superoxide Dismutase (SOD) activity was measured by using Superoxide Dismutase Assay Kit for the measurement of SOD inhibition rate in H9c2 cells, according to the manufacturer’s protocol. Absorbance was measured at 450 nm using a BioTek plate reader (BioTek), and the values were directly proportional to the SOD Inhibition rate.The level of cellular ROS production was determined by using Reactive oxygen species Assay Kit, according to the manufacturer’s protocol. After treatment, the H9c2 cells were stained with DCFH-DA (1 μM) and incubated in the dark for 30 min, and then were washed three times with PBS. The cellular ROS fluorescence intensity was determined using Flow Cytometry Assay .Cell Meter™ Mitochondrial Hydroxyl Radical was detected by using MitoROS™ OH580. After treatment, cells were stained with MitoROS™ OH580 probe for 1 h at 37°C and washed twice with pre-warmed PBS, and then 100 µl Assay Buffer was added into each group and 5 μl DAPI was added for 5 min in the incubator subsequently. The intensity of fluorescence was imaged by using a laser scanning confocal microscope . Cell Meter™ Mitochondrial Hydroxyl Radical was represented as red fluorescence.Mitochondrial membrane potential (ΔΨm) was detected using TMRE. In brief, after the indicated treatments, cells were stained with a TMRE probe (0.2 µM), for 30 min at 37°C and washed once with pre-warmed DMEM without serum. The fluorescence intensity of cells was detected by using a laser scanning confocal microscope . The degree of TMRE fluorescence was considered as an indication of the change of mitochondrial membrane potential.2 (×100)] were added to each sample, and cells were incubated for 30 min in the dark. After incubation, Calcein AM staining solution was aspirated, cells were washed twice with PBS, and then 500 µl detection buffer was added to the sample and the opening of mPTP was analyzed by using a BD FACSCalibur cytometer .A mitochondrial permeability transition pore assay kit was used to detect the opening of mPTP. After treatment, the culture medium was aspirated and cells were washed twice with PBS, 1 ml Calcein AM staining solution [containing 1 µl Calcein AM , 10 µl Solvent promoting (×100), and 10 µl CoClThe level of lysosome was determined using Lyso-Tracker Red according to the manufacturer’s protocol. After treatment, the H9c2 cells were stained with Lyso-Tracker Red (1 μM) solution and incubated in the dark for 10 min, then the Lyso-Tracker Red (1 μM) solution was replaced with fresh DMEM. The lysosome fluorescence intensity was determined using Flow Cytometry Assay .4 cells/well). Before the assay, 1 mmol/L pyruvate, 2 mmol/L glutamine, and 10 mmol/L glucose were added into Seahorse XF DMEM for preparation. After treatment, the cell medium was replaced with 500 μl prepared Seahorse XF DMEM, and then cells were incubated at 37°C without CO2 for 1 h. Thereafter, cells were exposed to oligomycin , FCCP , and rotenone plus antimycin A  by using the XF Cell Mito Stress Test kit (Seahorse Bioscience) to measure the parameters of OCR. Finally, the OCR value was normalized by CCK8 analysis. Respiration rates were calculated as the mean of all determinations at a certain state.Cellular oxygen consumption rate (OCR) indicating the mitochondrial respiration was measured by using the Seahorse XF24 Extracellular Flux analyzer and software . Firstly5 cells/ml (200 μl per dish). After treatment, the cell culture medium was removed, cells were washed three times with DPBS and fixed with 4% paraformaldehyde for 15 min. Then, paraformaldehyde was removed, cells were washed three times with PBS and blocked with BSA for 15 min, subsequently, cells were incubated with primary antibody Bcl-2 overnight at 4°C. Thereafter, cells were incubated with fluorescent secondary antibody  for 1 h after washing three times with PBS. 5 μl DAPI was added for 5 min in the incubator before imaging by using a fully automatic inverted fluorescence microscopic analysis system .H9c2 cardiomyocytes were seeded in a laser confocal dish at a density of 1 × 10Real-time quantitative polymerase chain reaction (qRT-PCR) was used to evaluate the mRNA expression level of pathway genes. Total RNA was isolated from H9c2 cells using Trizol reagent, according to the standard protocol, and RNA concentration was determined by using a NanoDrop spectrophotometer . 3 μg of total RNA was employed for reverse-transcription to yield cDNA. Subsequently, the synthesized cDNA was amplified using a 10 μl reaction system by the SYBR master Mixture . Finally, the threshold cycle (CT) value and the relative mRNA expression of the genes were calculated. The sequences of primers were shown as follows After the intervention, H9c2 cells were harvested and the total protein was extracted by using RIPA Lysis Buffer  with pierce protease inhibitor tablet  and pierce phosphatase inhibitor tablet . Then total protein was quantified and mixed with 5X SDS-PAGE protein buffer . After electrophoresis, the separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. Later, the membranes were incubated with 5% non-fat milk in TBST  for 2 h at room temperature, washed three times with TBST, and then the membrane with target proteins was incubated overnight at 4°C with primary antibody  against Bax, Caspase-3, p38 MAPK, JNK, ERK, β-actin, and Tubulin overnight at 4°C. Thereafter, the membrane was incubated with a respective HRP conjugated secondary antibody at 1:3,000 for 1 h after washing three times with TBST. BioRad imaging system  was applied to detect the fluorescent signal and the signal strip was quantified using Image Lab Software .The experimental data, obtained from at least three independent experiments, were presented as the mean ± SEM. Statistical analysis was performed by one-way ANOVA analysis followed by the Bonferroni test for multiple comparisons by using GraphPad Prism software, version 8.0 for Windows . A probability of less than 0.05 was defined as statistically significant.via numerous edges . The results of this network suggested that these five hub components may be the potential compounds of SGR in the treatment of HF.According to the TCMSP, ETCM, and TCMID Database, 77 chemical ingredients in SGR were collected, then 55 active ingredients of SGR were screened through the Symmap Database with the condition of OB ≥ 30%.Then, 32 active compounds were selected after removing ethylene ingredients, volatile oil ingredients, and duplicate ingredients , cellular component (CC), and molecular function (MF) using the DAVID Database  is the most important source of intracellular ROS. The results to explore the effect of SGR on the MitoROS production showed that H2O2 induced the elevation of MitoROS production, while SGR pretreatment suppressed this elevation  presented by Mito-tracker live-cell tag probe and the opening of mitochondrial permeability transition pore (mPTP). mPTP opening assay results showed that mPTP was opening because of the damage of mitochondria induced by H2O2, but the excessive opening of mPTP was inhibited in the SGR groups, as To determine whether SGR protected mitochondria damage in response to H2O2 group, the OCR values of basal respiration, maximum respiration and ATP production of the SGR treatment group increased significantly. It revealed that SGR was capable of protecting mitochondrial respiratory and mitochondrial energy metabolism in rat H9c2 cardiomyocytes against H2O2-induced mitochondrial dysfunction.2O2-induced cellular damage and mitochondrial dysfunction, aiming to verify the pathway predicted by network pharmacological analysis, we explored the SGR modulation on the p38MAPK pathway. It indicated that compared with the H2O2 group, SGR decreased the mRNA levels of CDC42, PP2A, RAC1, Akt, NFkB , epicatechin (7 in degree), and dihydrokaempferol (5 in degree) were also identified by the UHPLC-LTQ-Orbitrap-MSn method. SRC, JUN, CTNNB1, MAPK1, IL6, TNF, NFKB1, IL1B, INS, ESR1, PPARγ, CCL21, PPARA, IGF1, SIRT1, FGF2, IL10, PRKCA, SP1, and SCN5A were the key targets of SGR, and they were docked with the identified component of SGR to clarify the interaction relationships between the key targets and SGR active compound. The results showed that Istidina, Trans-resveratrol, Shikimic acid, Epicatechin, and Dihydrokaempferol had higher docking scores with SRC, JUN, TNF, PPARα, and Sirt1, respectively , indicatCardiovascular diseases are still a challenge for physicians and researchers in the world , and theIn this study, network pharmacological analysis results predicted that the important BP of SGR participated in the treatment of HF included regulation of DNA and RNA transcription, cell proliferation, oxidation-reduction process, apoptotic process, ERK1, and ERK2 cascade, and MAPK cascade, etc. Oxidative stress results in DNA damage and worsens mitochondrial function, leading to organ dysfunction and even aggravating the development of heart failure . RNA and2O2-induced damage. It restored cardiomyocyte morphology injury and inhibited LDH excretion. SGR inhibited cellular oxidative by increasing the level of SOD, improving lysosomal dysfunction, and reducing intracellular ROS levels. In addition, SGR protected mitochondrial function, it protected mitochondrial membrane potential, inhibited opening of mPTP, reduced mitochondrial ROS content, and restored mitochondrial respiration function. These results demonstrated that SGR did effectively inhibit cardiomyocyte oxidative damage, promote cardiomyocyte antioxidant capacity and improve mitochondrial function.In verification, we found that SGR did protect cardiomyocyte viability from H2O2 induction. SGR decreased the mRNA expression of AKT, PP2A, NF-KB, PP2A, RAC1, CDC42, and the protein expression of ERK1, ERK2, JNK, Bax, Caspase3. SGR also increased the mRNA expression of IKK, Jun, Sirt1, and the protein expression of p38MAPK and Bcl-2. Thus, SGR exerted protection on H9c2 cells by activating p38MAPKs signaling.Pathway enrichment analysis results suggested that the mechanisms mainly included MAPK signaling pathway, Calcium signaling pathway, Dilated cardiomyopathy, Hypertrophic cardiomyopathy (HCM), and PPAR signaling pathway, etc. MAPK signaling pathway is an important pathway involved in cellular proliferation and migration, regulating myocyte contractility and cell death in cardiomyocytes . ERK andvia reducing the level of triglyceride in the heart of diabetic rats, reducing myocardial mitochondrial damage caused by cerebral ischemia, and inhibiting reactive oxygen species to prevent reperfusion in isolated hearts after ischemia (2O2-induced cellular oxidative damage via JNK/Bax pathway to reduce cellular damage (via a direct antioxidant effect in the cardiovascular system (UHPLC-LTQ-Orbitrap-MSn methods identified that Istidina, resveratrol, shikimic acid, epicatechin, and dihydrokaempferol were contained in SGR, of which Istidina was predicted as active compound of SGR by network pharmacological analysis. Studies have shown that Istidina has cardioprotective effects ischemia . Also, wischemia . Howeverr damage . Epicater system , gastroir system , and resr system . Epicater system . Also, ir system . Moleculvia multi-compounds and multi-targets, its mechanism is involved in activating the p38 MAPK pathway, and Istidina may be possible anti-HF agents by targeting SRC.In conclusion, SGR has a protective effect on HF through cellular and mitochondrial protection


These actions of NPPC were blocked by the cyclic guanosine monophosphate (cGMP)-sensitive cyclic nucleotide-gated (CNG) channel inhibitor l-cis-Diltiazem. Moreover, porcine COCs acquired the ability to promote NPPC expression in the ampullary epithelial cells when the immature COCs were induced to maturation by epidermal growth factor (EGF). Simultaneously, transforming growth factor-β ligand 1 (TGFB1) levels were dramatically increased in the cumulus cells of the mature COCs. The addition of TGFB1 promoted NPPC expression in the ampullary epithelial cells, and the mature COC-induced NPPC was blocked by the transforming growth factor-β type 1 receptor (TGFBR1) inhibitor SD208. Taken together, the mature COCs promote NPPC expression in the ampullae via TGF-β signaling, and NPPC is required for the release of porcine spermatozoa from the oviduct isthmic cells.Porcine spermatozoa are stored in the oviductal isthmus after natural mating, and the number of spermatozoa is increased in the oviductal ampulla when the mature cumulus-oocyte complexes (COCs) are transferred into the ampulla. However, the mechanism is unclear. Herein, natriuretic peptide type C (NPPC) was mainly expressed in porcine ampullary epithelial cells, whereas its cognate receptor natriuretic peptide receptor 2 (NPR2) was located on the neck and the midpiece of porcine spermatozoa. NPPC increased sperm motility and intracellular Ca In many mammalian species, a subpopulation of the ejaculated spermatozoa migrates to the oviduct isthmus to form the “functional sperm reservoir”. The spermatozoa are held in the isthmic reservoir from a few hours to several months, depending on species , resultiDuring oestrus, luteinizing hormone (LH) from the pituitary acts on mural granulosa cells to induce the expression of epidermal growth factor (EGF)-like growth factors. Then, EGF receptor (EGFR) signaling is activated in cumulus cells, resulting in the maturation of COCs ,11. The 2+ levels of spermatozoa via cyclic guanosine monophosphate (cGMP)-sensitive cyclic nucleotide-gated (CNG) channels, and increases mouse and human sperm motility [Npr2 mutant mice are unable to reach the ampullae for fertilization [It is reported that the mature COCs can alter mRNA and protein expression pattern in bovine and porcine oviduct epithelial cells, respectively ,16. Our motility ,18. Morelization . Thus, tNPPC can be upregulated by transforming growth factor-β (TGFB) , estrogeNPPC mRNA was higher than that of NPPB and NPPA mRNAs  in porcine ampullae from oviducts at the corpus rubrum phase A using qPA mRNAs B. We furhe heart . In conthe heart . These r isthmus C. Furthe isthmus D,E. Immu isthmus F. Taken NPR2 mRNA could be detected in porcine spermatozoa, whereas NPPC mRNA was hardly detected . The results showed that 1 nM NPPC significantly increased sperm motility . In addimotility . The actmotility . Thus, w to NPPC A. In add to NPPC A,B, conspulation C, consisntensity D,E. Furt+ levels D,E, suggntensity D,E. The ntensity D,E. Thusl-cis-Diltiazem, but not NNC 055-0396, completely blocked NPPC-induced spermatozoa release from the oviduct isthmic cell aggregates . The basic medium used for COC and oviductal ampulla culture was TCM199 medium  supplemented with 0.23 mM sodium pyruvate, 2 mM glutamine, 3 mg/mL lyophilized crystalline BSA, 100 IU/mL penicillin G, and 25 mg/mL streptomycin sulfate.2, 3.1 mM KCl, 1.5 mM MgCl2, 100 mM NaCl, 0.29 mM KH2PO4, 0.36% lactic acid, 25 mM NaHCO3, 0.6% BSA, 1 mM pyruvic acid, and 20 mM HEPES), and 0.6 mL 10× HBS , and centrifuged for 10 min at 800× g. Then, the spermatozoa were pelleted again for 5 min at 500× g after washing with dmTALP medium. Finally, the concentration of spermatozoa was determined with a hemocytometer and adjusted according to the experiment. The spermatozoa were cultured in dmTALP medium for 1.5 h at 39 °C for capacitation. For protein assay, the heads and tails from 3 × 108 capacitated spermatozoa were separated according to our previous study [g for 3 min at 4 °C to precipitate the sperm heads. Sperm flagella were recovered from the supernatant by centrifugation at 10,000× g for 10 min at 4 °C.Porcine semen was provided by Guangxi Yangxiang Co., Ltd. . The semen samples were stored at 16 °C to 18 °C for 24 h before use. The pooled extended semen of 3 mL was washed through a Percoll cushion containing 5.4 mL Percoll, 4 mL dmTALP (2.1 mM CaClus study . BrieflyThe location of NPR2 in spermatozoa was identified by FAM-NPPC (the mono-5- (and 6)-carboxyfluorescein label NPPC. Phoenix Pharmaceuticals, Belmont, CA, USA). The spermatozoa without or with capacitation were incubated with 100 nM FAM-NPPC for 30 min at 39 °C to ensure full reaction. The spermatozoa were then fixed with 2% paraformaldehyde for 5 min, and washed three times with PBS to remove the unbound ligand. The fluorescent-labeled ligand binding was assessed by LSM 800 confocal microscope . The spermatozoa with uneven dye loading were excluded from the analysis. For competition experiments, the capacitated spermatozoa were co-incubated with 100 nM FAM-NPPC and 1 μM unlabeled NPPC for 30 min at 39 °C.l-cis-Diltiazem  for 30 min. The sperm motion was detected using a CASA system . The parameters included straight-line velocity (VSL), amplitude of lateral head displacement (ALH), average path velocity (VAP), curvilinear velocity (VCL), beat cross frequency (BCF), and percentage of linearity . For each experimental condition, five random fields were evaluated for a minimum total of 100 cells in each field.The capacitated spermatozoa were incubated in dmTALP medium, supplemented with different concentrations of NPPC (0.01–10 nM), NNC 055-0396 , 8-Br-cGMP (1 mM), and/or 2+ levels in the capacitated spermatozoa were detected according to the previous study [5 cells/mL) were placed on glass coverslips treated with polylysine at 0.01% in the recording chamber for 5 min, and then the external solution was infused to wash the supernatant. Imaging analysis of Ca2+ response in motile spermatozoa was monitored using a LSM 800 confocal microscope . We used a sample frequency of 1 Hz, and recorded Ca2+ fluorescence intensity for 3–4 min after different treatments. All Ca2+ imaging experiments were carried out at 39 °C. Cells with uneven dye loading were excluded from the analysis. NPPC was used at 1 nM, and the inhibitor l-cis-Diltiazem was used at 50 μM with a pre-incubation of 15 sec. NPPC and the inhibitor were dropped into the recording chamber using pipette tips and the recordings were conducted in the continuous presence of stimuli. Ca2+-free experiments were conducted using Ca2+-free modified Tris-buffered medium (mTBM) without BSA, obtained by omitting Ca2+ and adding 1 mM EGTA. NNC 055-0396, 8-Br-cGMP, BAPTA/AM, and TMB-8 were used with 2 μM, 1 mM, 5 μM, and 10 μM, respectively. The highest level of Ca2+ fluorescence intensity in at least 50 spermatozoa was used for the presentation of Ca2+ level of spermatozoa in different treatment. Each experiment was repeated three times.Caus study . Briefly7 cells/mL) were incubated without or with 1 nM NPPC for 20 min, and then collected and solubilized in 100 μL of 0.1 M HCl on ice for 15 min. The samples were thawed and centrifuged for 5 min at 12,000× g, and the supernatant was collected and dried in an oven at 60 °C for cGMP assay. The levels of cGMP were determined by a cGMP-enzyme immunoassay kit  according the manufacturer’s instruction.The capacitated spermatozoa of 100 μL  were selected for testing sperm release.Porcine oviducts were collected from a local abattoir and transported to the laboratory within 2 h at 4 °C in PBS. Spherical aggregates were prepared as previously described with slightly modification . Briefly5 cells/mL were added into a 50 µL droplet containing 10 oviduct isthmic cell aggregates. Spermatozoa and the oviduct isthmic cell aggregates were incubated at 39 °C for 15 min for sperm binding to form the complexes. Then, the complexes were incubated without or with 1 nM NPPC for 30 min at 39 °C. In some experiments, the complexes were pre-incubated with l-cis-Diltiazem (50 µM), NNC 055-0396 (2 μM), or BAPTA/AM (5 μM) for 5 min at 39 °C, and then incubated without or with NPPC. At the end of incubation, the free and loosely attached spermatozoa were removed from the oviduct isthmic cell aggregates by washing with dmTALP medium. The aggregates were fixed with 4% paraformaldehyde for 10 min and then transferred to a 3 µL droplet in the microscope slide. Images were captured using Axio Vert. A1 microscope . The number of spermatozoa bound to the periphery of each aggregate was enumerated, and the circumference of the aggregate was calculated using Labscope V 3.2 software . The number of spermatozoa bound per millimeter of circumference was calculated for each aggregate, and the average number of spermatozoa from 10 aggregates was used for statistical analysis. All the cultures were incubated at 39 °C under 5% CO2.Assay of sperm binding to and release from oviduct isthmic cell aggregates was conducted according to the previous report . BrieflyPorcine ovaries were collected from a local abattoir and transported to our laboratory within 2 h in PBS at 4 °C. The immature COCs were aspirated from the follicles with 3–6 mm in diameter, and then 100–120 COCs were cultured in 500 μL TCM-199 medium with EGF (10 ng/mL) for 44 h to acquire the mature COCs. The denuded oocytes (DOs) were obtained by removing the cumulus cells from the mature COCs with 1 mg/mL hyaluronidase. One ampulla at follicular phase was cultured in 200 μL TCM-199 medium, supplemented with 150 immature or mature COCs, 150 DOs, 10 ng/mL TGFB1, and/or 1 μM SD208 (TGFBR1 inhibitor) for 3 h. The ampullae were collected at the end of culture, and the epithelial cells were scraped by the edge of a microscope slide. In some experiments, the epithelial cells were isolated from the ampullae at follicular and corpus rubrum phases. The samples were stored at -80 °C for mRNA and protein analysis.2.OOX cumulus cells were produced by microsurgically removing the oocytes, but not the zona pellucida, from the immature COCs. Groups of 100–120 COCs or OOX cumulus cells were cultured in the medium, supplemented with EGF, growth differentiation factor 9 , bone morphogenetic protein 15 , and/or fibroblast growth factor 8B  in 500 μL medium. After 44 h culture, cumulus cells were collected for gene and protein analysis. All the cultures were incubated at 39 °C under 5% CO8 sperm head and tails, 200 oocytes, the cumulus cells from 100 COCs or OOXs, and the ampullary epithelial cells from one ampulla. Total protein was extracted in WIP buffer  with 1 mM phenylmethylsulfonyl fluoride on ice. Normalized protein amounts were separated by 10% SDS-PAGE and then transferred to polyvinylidene fluoride membranes . The membranes were incubated with primary antibodies  according to the manufacturer’s instructions. RNA (no more than 1 μg) was reverse transcribed into cDNA using the QuantiTect Reverse Transcription System . qRT-PCR was conducted using a Light Cycler 96 instrument . Relative gene expression was quantified using the threshold cycle value and normalized using Oviductal ampullae at corpus rubrum phase were fixed in cold 4% paraformaldehyde overnight at 4 °C, dehydrated in gradient alcohol, embedded in paraffin, and sectioned at 5 μm. Oviductal ampulla sections were deparaffinized, rehydrated, and subjected to antigen retrieval with 0.01% sodium citrate buffer (pH 6.0) at high temperature (95–98 °C) for 16 min. The sections were rinsed thoroughly with PBS and blocked with 10% normal donkey serum in PBS for 1 h at room temperature. The COCs were fixed in 4% paraformaldehyde for 2 h at 4 °C and blocked with 10% normal donkey serum in PBS for 1 h at room temperature. After blocking, all the samples were incubated with primary antibodies  overnight-tests, and p < 0.05 was considered significant.All experiments were carried out at least three times. The data were analyzed and graphed using GraphPad Prism software . The statistical significance between two groups was analyzed by two-tailed unpaired Student’s


ECR gene, named CsECR, was cloned from “Newhall” navel orange. CsECR protein has high identities with other plant ECR proteins and contained a conserved NADP/NAD-binding motif and three conserved functional sites. The highest expression of CsECR was observed in leaves, followed by stems, flavedos, ovaries, juice sacs, stigmas, stamens, albedos, and petals. Besides, the expression of CsECR was significantly induced by PEG6000 and ABA treatments. Ectopic overexpression of CsECR increased the contents of total waxes and aliphatic wax fractions  in the leaves and fruits of the transgenic tomato. Furthermore, ectopic overexpression of CsECR reduced the cuticle permeability in the leaves and fruits of the transgenic tomato and increased its tolerance to drought stress. Taken together, our results revealed that CsECR plays an important role in plant response to drought stresses by regulating cuticular wax biosynthesis.Drought stress often occurred in citrus to limit its growth, distribution, and fruit quality. Cuticular waxes play an important role in regulating plant tolerance to drought stress. Plant enoyl-CoA reductase (ECR) is involved in the biosynthesis of cuticular waxes and catalyzes the last step of very long-chain fatty acids (VLCFAs) elongation. In this study, a putative  Afterward, the C16 and C18 fatty acids are released from the acyl carrier protein (ACP) and esterified to CoA to produce C16 and C18 fatty acyl CoAs. The C16 and C18 fatty acyl CoAs are further extended to VLCFAs in the endoplasmic reticulum (ER) and generate cuticular wax components by the decarbonylation and acyl reduction pathways  and their derivatives with carbon chain lengths ranging from C20 to C34, such as alkanes, primary alcohols, secondary alcohols, aldehydes, esters, and ketones. In addition, cyclic compounds including triterpenoids, alkaloids, and phenylpropanoids are also detected in plant cuticular waxes. The biosynthesis of cuticular waxes started with the The elongation of C16 and C18 fatty acids to VLCFAs in plants includes a series of reaction cycles catalyzed by the multienzyme fatty acid elongase (FAE) complex. Two carbons are added to the acyl chain per cycle. Each cycle consists of four consecutive reactions: biosynthesis of 3-ketoacyl-CoA by condensation of malonyl-CoA with a long chain acyl CoA catalyzed by a 3-ketoacyl-CoA synthase (KCS); reduction of 3-ketoacyl-CoA to 3-hydroxyacyl-CoA catalyzed by a 3-ketoacyl-CoA reductase (KCR); dehydration of 3-hydroxyacyl-CoA to trans-2-enoyl-CoA catalyzed by a 3-hydroxyacyl-CoA dehydratase (HCD); and reduction of trans-2-enoyl-CoA to acyl-CoA catalyzed by an enoyl-CoA reductase (ECR)  in maize participate in the biosynthesis of VLCFAs  is involved in VLCFA biosynthesis . Further research showed that many wax-related genes were downregulated on the surfaces of “Ganqi 3” fruits, resulting in a significant decrease in cuticular wax amounts and wax crystal numbers, and finally leading to the glossy surface of “Ganqi 3” fruits  mutants showed a significant increase in cuticular permeability, which was caused by a decline in n-alkanes and an increase in triterpenoids  cultivated in an orchard of Xinfeng county, Ganzhou city, Jiangxi province of China. Yong leaves were used for CsECR cloning. Mature leaves, stems, flavedos, ovaries, juice sacs, stigmas, stamens, albedos, and petals were used for tissue-specific expression analysis.All citrus tissues were sampled from 8-year-old trees of “Newhall” navel orange (Poncirus trifoliata (L.) Raf.) to produce grafted seedlings. After growing to the height of 30 ± 2 cm in soil culture, the grafted seedlings were transferred to Hoagland's solution in a growth chamber under normal growth conditions  for 7 days and then subjected to the Hoagland's solution containing 20% PEG6000 and 100 μM ABA, respectively, for the drought and ABA treatments. For each treatment per biological replicate, the leaves were collected from 15 seedlings at 0, 1, 3, 6, 12, and 24 h. Three biological replicates were performed for each time point of each treatment.For expression analysis after drought and ABA treatments, shoots of “Newhall” navel orange were grafted on trifoliate orange (Solanum lycopersicum L. cv. Micro-Tom) was used to produce transgenic plants ectopic overexpressing CsECR. For soil culture, seeds of the wild-type “Micro-Tom” tomato (WT) and the transgenic tomato lines were germinated on filter paper in Petri dishes at 25°C in darkness. Germinated seeds were transferred to pots filled with nutritional soil and placed in a growth chamber . For in vitro culture, tomato plants were grown on a modified MS basal medium at 25°C under a photoperiod of 16 h light/8 h dark. The details of in vitro culture were carried out as described in the report by Dan et al. . Trace genomic DNA was removed by DNase I . The RNA yield and quality were detected according to our previous report . According to the cDNA sequence of Cs7g29210, a pair of gene-specific primers  were designed to clone CsECR by PCR. The PCR for CsECR cloning was performed by 50 μL volume containing 25 μL PrimeSTAR Max Premix , 1 μL sense primer, 1 μL antisense primer, 1 μL cDNA, and 22 μL ddH2O. The PCR protocol was 5 min at 94°C, 35 cycles of 30 s at 94°C, 30 s at 55 °C, 1.5 min at 72 °C, and finally 10 min at 72°C. The PCR product was cloned into pMD18T vector , transformed into Escherichia coli DH5α, and sequenced at Shanghai Sangon Biotech as described in our previous report  of http://us.expasy.org/tools/protparam.html) was used to analyze the theoretical isoelectric point and mass values of CsECR protein. The ECR proteins from other plants were obtained by BLAST search in the National Center for Biotechnology Information Server (http://www.ncbi.nlm.nih.gov/). The ClustalW program was used to conduct the multiple sequence alignment. The phylogenetic tree construction was performed by the Minimum-Evolution algorithm of Mega X. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test  are shown next to the branches.The ExPASy ProtParam tool . The CsECR-specific primers  were designed for qRT-PCR analysis. The qRT-PCR was performed on the CFX96 Touch Real-Time PCR Detection cycler  using the SYBR®Premix Ex Taq™ kit . The qRT-PCR was performed by 20 μL volume containing 10 μL SYBR®Premix Ex Taq, 0.5 μL sense primer, 0.5 μL antisense primer, 1 μL cDNA, and 8 μL ddH2O. The qRT-PCR program was 30 s at 95°C for pre-denaturation, 40 cycles of 5 s at 95°C for denaturation, and 30 s at 60°C for annealing. Melting curve analysis of the qRT-PCR products was performed to confirm the amplicon specificity. The relative gene expression levels were normalized relative to the expression of the Citrus β-actin gene  and calculated by the 2ΔCT−Δ method.Expression profiles of CsECR-specific primers with Nco I and Bgl II restriction sites  were used to clone the coding sequence of CsECR by RT-PCR. The PCR product was digested by Nco I and Bgl II and ligated into the pCAMBIA1301 vector to produce the expression vector pCAMBIA1301-CsECR under the control of the CaMV35S promoter. The freeze-thaw method was used to introduce this overexpression vector into Agrobacterium tumefaciens strain LBA4404. “Micro Tom” tomato ectopic overexpressing CsECR was generated by Agrobacterium-mediated cotyledon transformation according to the report of Dan et al. . The genomic PCR was performed by a TransDirect® Plant Tissue PCR Kit . The expression levels of CsECR in leaves and fruit peels of the WT and T2 transgenic lines were examined by qRT-PCR with the CsECR-specific primers as described in the above Section Expression Analysis of CsECR in Navel Orange. Tomato actin gene  was used as the endogenous control to normalize the expression levels between different samples.A pair of n et al. . The regn et al. . The pos2, freeze-dried, and sputter-coated with gold film in a sputter coater . The SEM  was used to observe the coated disks at ×1,000 and ×5,000 magnification.The cuticular wax morphology on the surfaces of the fully expanded leaves and red ripe fruits in the WT and transgenic tomato lines was observed by SEM. The samples were prepared as described in our previous report  was added to the cuticular wax solution as an internal standard. The cuticular wax solution was filtered, concentrated by a rotary evaporator at 35°C under reduced pressure, and dried under a gentle stream of nitrogen. Prior to the gas chromatography–mass spectrometry (GC–MS) experiment, the samples were treated with bis-N,N-(trimethylsilyl)-trifluoroacetamide (BSTFA) in pyridine for 40 min at 70°C to transform the carboxyl and hydroxyl compounds to their corresponding trimethylsilyl ethers and esters. After derivatization, the residual BSTFA was removed under a gentle stream of nitrogen and dissolved in 1 ml of chloroform for GC-MS analysis.Three biological replicates were carried out for cuticular wax extraction and chemical analysis. A total of 20 fully expanded leaves and 15 red ripe fruits in each line per replicate were used for cuticular wax analysis. The leaves and fruits were immersed three times in chloroform at room temperature for 1 min each time to extract the cuticular waxes. Then, 100 μg of −1. GC was performed with an on-column injection temperature of 50°C, held for 2 min at 50°C, increased by 40°C·min−1 to 200°C, held for 2 min at 200°C, increased by 3°C·min−1 to 320 °C, and held for 30 min at 320°C. The MS was performed in positive electron ionization mode at 70 eV, obtaining spectra with an m/z range of 50–850. The wax components were identified by matching their electron ionization mass spectra with those from the NIST 14 MS library. The quantitative analysis of the wax components was performed by the same GC system equipped with a name ionization detector (FID). The same chromatographic conditions as above were used to quantify the wax components, but with H2 as carrier gas. The wax components were quantified by peak area comparisons with the internal standard. The leaf area was determined using a LI-3100C area meter . Tomato Analyzer version 3.0 was used to assess the fruit surface area . The leaf chlorophyll concentration was calculated using the following equation: total micromoles chlorophyll = 7.93 (A664) + 19.53 (A647)  and Duncan's multiple range tests (for one-way ANOVA) in SPSS version 22.All data in this paper are shown as the means ± standard deviations of three biological replicates. Statistical analyses were carried out using Student's in silico cloning and RT-PCR strategy, a novel gene CsECR was isolated from the “Newhall” navel orange and deposited in GenBank under accession No. ON146445. The cDNA sequence of CsECR is 1,524 bp in length with a 933 bp open reading frame (ORF). The CsECR protein contained 310 amino acids with a predicted molecular mass of 36.11 kDa and an isoelectric point of 9.73.Based on the Citrus clementina CcECR , Populus trichocarpa PtECR , Gossypium hirsutum GhECR , Malus domestica MdECR , Prunus avium PaECR , Glycine max GmECR , Solanum lycopersicum SlECR , Prunus persica PpECR , and Arabidopsis thaliana AtECR . Multiple sequence alignment showed that CsECR contained a conserved NADP/NAD-binding motif (225-GSGGYQIPRG-234) and three conserved functional sites   . To invees CeECR .CsECR was detected in all of the tested tissues in “Newhall” navel orange. The highest expression was detected in leaves, followed by stems, flavedos, ovaries, juice sacs, stigmas, stamens, albedos, and petals  with the highest expression of CsECR in leaves and fruit peels were selected for further research  , followed by triterpenoids , n-fatty acids , n-aldehydes , unsaturated fatty acids , iso-, and anteiso-alkanes , unidentified wax component , alkenes , sterols , and n-primary alcohols . The amounts of n-alkanes increased by 66.67% (5.65 μg·cm−2) and 89.38% (6.42 μg·cm−2), respectively, in the two transgenic lines compared to the WT leaves. Notably, the increases in n-alkane amounts accounted for 70.85 and 67.94% increases in total wax loads, respectively, in the two transgenic line leaves. Compared to the WT leaves, the amount of n-fatty acids increased by 26.67% (0.95 μg·cm−2) and 49.33% (1.12 μg·cm−2), respectively, in the two transgenic line leaves. Similarly, the content of unsaturated fatty acids increased by 106.90% (0.60 μg·cm−2) and 100.00% (0.58 μg·cm−2), the alkenes increased by 155.56% (0.046 μg·cm−2) and 211.11% (0.056 μg·cm−2), and the iso- and anteiso-alkanes increased by 60.00% (0.32 μg·cm−2) and 85.00% (0.37 μg·cm−2) in the two transgenic line leaves compared with the WT leaves. However, no significant differences were observed in the amounts of n-aldehydes, n-primary alcohols, triterpenoids, and unidentified wax components between the leaves of the two genotypes , ectopic overexpression of CsECR increased the amounts of C26:0 fatty acid by 278.26% (0.087 μg·cm−2) and 595.65% (0.16 μg·cm−2), respectively, in the two transgenic line leaves. The unsaturated fatty acids consisted of C18:2 and C18:3 fatty acids in the leaves of both genotypes. The amounts of C18:2 fatty acid in the two transgenic line leaves increased by 136.36% (0.26 μg·cm−2) and 172.73% (0.30 μg·cm−2), respectively, in comparison to the WT leaves (0.11 μg·cm−2). Alkene was composed of C27:1, C29:1, and C33:1 alkene in the leaves of both genotypes. The amounts of C33:1 alkene increased by 218.18% (0.028 μg·cm−2) and 320.45% (0.037 μg·cm−2) in the two transgenic line leaves compared with the WT leaves (0.0088 μg·cm−2). A total of three iso-alkanes (C31–C33) and one anteiso-alkane (C32) were observed in the leaves of both genotypes. The amounts of C31 and C32 iso-alkanes in the two transgenic line leaves were significantly higher than those in the WT leaves. However, no significant changes were observed for the amounts of other wax components  in the two transgenic line leaves  . The mosenotypes , 6A. Thee leaves .−2), the total wax loads increased by 108.43% (14.59 μg·cm−2) and 150.29% (17.52 μg·cm−2), respectively, on the surfaces of the two transgenic line fruits , followed by triterpenoids , iso- and anteiso-alkanes , unidentified wax component , n-fatty acids , unsaturated fatty acids , alkenes , and n-primary alcohols . Ectopic overexpression of CsECR increased the amounts of n-alkanes by 191.94% (10.51 μg·cm−2) and 259.17% (12.93 μg·cm−2), iso- and anteiso-alkanes by 42.55% (1.34 μg·cm−2) and 61.70% (1.52 μg·cm−2), n-fatty acids by 32.76% (0.77 μg·cm−2) and 46.55% (0.85 μg·cm−2), unsaturated fatty acids by 100.00% (0.096 μg·cm−2) and 129.17% (0.11 μg·cm−2), and alkenes by 130.77% (0.030 μg·cm−2) and 161.54% (0.034 μg·cm−2) in the fruits of two transgenic lines. Notably, 91.16 and 88.77% increases in the total wax loads in the two transgenic line fruit were attributed to the increases of n-alkanes  were observed in the fruits of both genotypes. The amounts of all n-alkane components in the two transgenic line fruit were significantly higher than those in the WT fruit. The most abundant wax component in the WT fruit was C31 alkane , which increased by 142.03% (5.01 μg·cm−2) and 186.47% (5.93 μg·cm−2) in the two transgenic line fruits. Notably, the increases of C31 alkane in the two transgenic line fruit accounted for 38.79 and 36.73% increases in the total wax loads. A total of three iso-alkanes (C31–C33) and two anteiso-alkanes  were observed in the fruit of both genotypes. The amounts of all iso-alkanes and C33 anteiso-alkanes in the transgenic line fruit were significantly higher than those in the WT fruit. The n-fatty acids in the fruit of both genotypes consisted of series homologs with even chain lengths ranging from C16 to C28. The amounts of n-fatty acids with an even chain length larger than C22 significantly increased in the two transgenic line fruit compared to the WT fruits. Besides, the amounts of two unsaturated fatty acids (C18:2 and C18:3) and C29:1 alkene in the two transgenic line fruits were significantly higher than those in the WT fruits. However, other wax components including C27:1 alkene, C18:0 primary alcohol, α-amyrin, β-Amyrin, γ-Amyrin, and unidentified wax components shared similar amounts between the fruits of the two genotypes  characterized in cotton GhECR and other plant ECR , BnECR from oilseed rape (Brassica napus), MdCER10 (ECR homolog) from apple  in leaves and stems were significantly higher than those in other tissues  and contributed to the major increases in total wax amounts . Surprisingly, the n-fatty acids, which were the final product of ECR, increased much less than the n-alkanes in both leaves and fruits of the transgenic tomato plants  in leaves were much lower than those in fruits of two transgenic tomato lines   . Thus, nTaCER1-1A gene exhibited significant increases of n-alkanes, decreases in cuticle permeability, and enhanced tolerance to drought stress , MdSHINE2 and MdLACS2 from apple , PpCER1 from Poa pratensis, and so on , the National Natural Science Foundation of China (Grant Nos. 31701896 and 31860544), the Natural Science Foundation of Jiangxi Province (Grant No. 20202BAB205001), and by the earmarked fund for Jiangxi Agriculture Research System (Grant No. JXARS-07-cultivation post).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Next–Generation Sequencing (NGS) has provided a deeper genetic understanding of acute myeloid leukemia (AML) that has been recently incorporated into AML classification and risk–stratification guidelines. Single molecular analysis has become inefficient and molecular testing based on NGS is emerging as an irreplaceable diagnostic tool in clinical settings. The PETHEMA cooperative group has constituted a nationwide NGS network with centralized analysis in seven high–skilled laboratories. The study of molecular profiles in the “real–life” PETHEMA cohort supports the increasing role of NGS on the clinical management of AML patients.ABL1, ASXL1, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA2, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1 and WT1) showed 97% of patients having at least one mutation. The mutational profile was highly variable according to moment of disease, age and sex, and several co–occurring and exclusion relations were detected. Molecular testing based on NGS allowed accurate diagnosis and reliable prognosis stratification of 954 AML patients according to new genomic classification proposed by Tazi et al. Novel molecular subgroups, such as mutated WT1 and mutations in at least two myelodysplasia–related genes, have been associated with an adverse prognosis in our cohort. In this way, the PETHEMA cooperative group efficiently provides an extensive molecular characterization for AML diagnosis and risk stratification, ensuring technical quality and equity in access to NGS studies.Next–Generation Sequencing (NGS) implementation to perform accurate diagnosis in acute myeloid leukemia (AML) represents a major challenge for molecular laboratories in terms of specialization, standardization, costs and logistical support. In this context, the PETHEMA cooperative group has established the first nationwide diagnostic network of seven reference laboratories to provide standardized NGS studies for AML patients. Cross–validation (CV) rounds are regularly performed to ensure the quality of NGS studies and to keep updated clinically relevant genes recommended for NGS study. The molecular characterization of 2856 samples  with standardized NGS of consensus genes ( Introduction of Next–Generation Sequencing (NGS) into routine molecular diagnosis has provided deep molecular knowledge of acute myeloid leukemia (AML). These findings have allowed for the refinement of classification and risk stratification systems based on recurrent genetic abnormalities.In 2016, Papaemmanuil et al. proposed the first genomic classification of AML that identifies 11 molecular classes, each with distinct diagnostic features and clinical outcomes , new IntIn this situation, molecular analysis by single–gene techniques has become inefficient in order to provide a complete characterization of AML. In contrast, NGS represents a more sensitive tool to capture all the relevant molecular markers in one assay and is widely recommended to study the molecular landscape of this disease .NGS implementation to perform accurate diagnosis in AML is currently demanded by physicians and patients. However, introduction of NGS into clinical routine faces novel challenges . NGS reqIn order to provide comprehensive NGS studies to AML patients and to guarantee equity of access, the PETHEMA cooperative group (Programa Español de Tratamientos en Hematología) has established a nationwide network of central laboratories aimed to harmonize NGS results under consensual criteria in newly diagnosed and relapsed/refractory AML patients .This study summarized the NGS–AML project (NCT03311815), reporting quality control assays and the molecular profile of 2668 AML patients reported in the PETHEMA AML registry. We show co–occurring and mutual exclusion relationships among genes and distinct molecular profiles according to disease stage, age and sex and genomic classification in the “real–life PETHEMA cohort”.Implementing NGS studies in the routine molecular diagnosis of AML patients requires specialization, budgetary stability and logistical support. The PETHEMA cooperative group established a nationwide network of NGS studies for fast and standardized molecular diagnosis of AML. This strategy aims to provide coverage of NGS studies to 38.5 million inhabitants distributed in geographical areas ranging from 2.2 to 8.9 million habitants. For this purpose, seven centers with logistical and technical capacity for the management of a high number of samples were designed as reference laboratories for the centralization of samples submitted by PETHEMA institutions in each area. In this way, a large territory and population was covered. The platform was supported by PETHEMA in logistical management, as well as, closer and well–established relationships between the sample referral institution and the assigned central laboratory. The designated reference centers for NGS analysis concentrate a large number of AML samples, allowing for the rapid completion of the sequencing runs and their management by highly specialized staff.We show a prospective, multi–center, non–interventional and translational biomedical research, performed in seven Spanish PETHEMA reference laboratories: Hospital Universitario La Fe , Hospital Universitario de Salamanca , Hospital Universitario 12 de Octubre , Hospital Universitario Virgen del Rocío , Hospital Universitario Reina Sofía , Hospital Universitario de Gran Canaria Dr. Negrín , CIMA LAB Diagnostics .ASXL1, BCOR, CEBPA, EZH2, FLT3, IDH1, IDH2, NPM1, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2 and TP53). Moreover, there was a recommendation for the study of other genes with proven evidence on their relevance in AML pathogenesis .The development of the diagnostic platform required several meetings to coordinate criteria on which genes should be analyzed based on their clinical relevance in AML. After extensive bibliographic revision of current molecular basis of AML, all reference laboratories should assess by NGS the mutational status of genes that define the diagnosis and prognosis as well as guide treatment options  rounds have been performed. As previously reported, in the first and second CV rounds, minimum quality parameters [uniformity (>85%) and mean read depth of 1000X] and consensus recommendations for variant report  were stablished. For both CV rounds, 10 samples harboring 54 variants were distributed. In first CV round, VAF for all variants was >5% while in the second CV round, 5 from 30 total variants had low VAF ranging from 1.8–4.9%. In the third CV round, to explore the accuracy and reliability of low VAF variants (<5%) detection, 4 samples harboring 32 variants (11 VAF < 5%) were analyzed, according to previously established criteria  and “WHO2016 set 2” ; and also novel categories: “TP53 and complex karyotype (CK)”, “sAML1” , “sAML2” (More than one mutations in sAML1 genes including DNMT3A and TET2), “WT1”, “Trisomies”, “DNMT3A + IDH1/2”, “Not class defining mutations (mNOS)” and “No events” category. Since our study excludes acute promyelocytic leukemia, category “t” is not applicable.This new classification categorizes AML in 16 molecular classes with different prognostic values and encompass the established WHO entities: “WHO2016 set 1”  represented the 34% of PETHEMA–cohort.Based on the availability of clinical, cytogenetic and mutational data, 954 patients were eligible to assess the updated genomic classification by Tazi et al. . The mosCEBPA bZIP” (Median OS not reached at 32 months) and “NPM1” [29.0 months (95%CI 19.9–38.0)] had the best outcomes while inv(3) had the worst prognostic value [4.9 months (95%CI 0.8–9.1)]. Among new molecular classes, those patients without driver mutations “No events” (Median OS not reached at 33 months) or “Not class defining mutations” [23.3 months (95%CI 11.0–35.6)] showed the best prognostic value while “TP53/CK” [5.3 months (95%CI 2.9–7.6)], “sAML2” [12.1 months (95%CI 9.9–14.2)] and WT1 [4.0 months (95%CI 0.0–18.4)] classes had the worst OS (p < 0.001) ; multi–hit: 4.1 months (95%CI 2.9–5.3) (p = 0.088)] ” (Median OS not reached at 42 months), “< 0.001) . When we 0.088)] .p < 0.001)] and “TP53/CK” [3.5 (95%CI 2.6–4.6) (p < 0.001)] showed the highest risk of death compared to “NPM1” class. Remarkably, the new established “sAML2” [2.1 (95%CI 1.6–2.7) (p < 0.001)] and “WT1” [2.3 (95%CI 1.1–5) (p < 0.05)] categories were the next with higher risk of death ” [3.9 (95%CI 2.1–7.2) (of death .p < 0.001).We also assessed the integrated risk score based on cytogenetic and gene mutations proposed in the new genomic classification . In the p < 0.001) (p < 0.01) and 2.7  increased risk of death relative to favorable risk group  in the favorable risk group, 18.5 months (95%CI 14.3–22.7) in the intermediate group and 9.4 months (95%CI 7.8–11.00) in the adverse risk group (< 0.001) A. Intermsk group .p < 0.001) (p < 0.001)  months and 20.8 (95%CI 13.2–28.3), respectively (< 0.001) B. In con< 0.001) C.p < 0.001) and 3.5  higher risk of death relative to those patients classified in the favorable risk group (p < 0.001) compared to those classified as favorable risk. No statistically significant results were found in the risk of death of intermediate risk patients: 1.0  (p < 0.001), intermediate: 1.8  and adverse: 2.6 .Regarding risk score, young patients classified in the intermediate and adverse risk group showed 2.6  . In genep = 0.839), intermediate  or adverse–risk patients  according to both classifications  for favorable . Noteworthy, the detection of low VAF (<5%) variants (concordance 41.2%) was also consistent with previous studies, which report that accurate detection of low VAF variants by NGS could be compromised. CV rounds results reflected the improvement of the diagnostic platform performance as the error rate decreased in variants VAF > 5% as a result of the experience gained in NGS studies. Although the second CV round included 5 variants with a mean VAF of 3.3%, in the third CV round we aim to explore the performance of NGS studies in very low VAF variants. For this purpose, we included 11 variants with a mean VAF of 1.2%. Data analysis revealed an increase of the ER between both CV rounds due to the very low VAF variants of the third CV round. Consequently, we established VAF 5% cut–off for variant reporting, although according to other specific studies, variants in hotspot regions were reported up to 1% VAF even at borderline technical quality ,17,18.Our results reflect that a comprehensive NGS approach is suitable for defining the molecular profile of AML as over 96% of patients of the “real–life” PETHEMA cohort harbored at least one mutated gene. Our study also reflects the genomic heterogeneity that encompasses AML  as several gene–to–gene interactions but also co–occurrence and mutual exclusivity patterns across functional categories were described. Recently, published studies have highlighted the impact of co–mutations in modulating treatment response ,20.FLT3 mutations, which showed a stability rate of 43.3% in our cohort [Similarly, distinct mutational profiles were detected among disease stages, reflecting the clonal evolution of AML ,21. In fr cohort .FLT3 mutations [In addition to mutational changes according to disease stages, specific mutational profiles have been associated to patient’s clinical characteristics, such as sex and age, with significant impact on prognosis, therapeutic allocation and disease monitoring . In factutations ,31,32,33utations ,35,36. Iutations ,38.p = 0.018). In this sense, further studies are needed in order to certainly clarify the impact of sAML mutations on prognosis.Molecular diagnosis of AML has shifted towards a comprehensive mutational study driven by NGS, yielding large amounts of data. Updated diagnostic (WHO and ICC) and prognostic (ELN) classifications ,4,5 inclTP53 mutations, in the PETHEMA cohort, 47 patients were included in the mono–allelic group (32.9%), while 96 were included in the multi–hit group (67.1%). Similar results were described in the Tazi et al. cohort: mono–allelic (31.1%) vs. multi–hit: (68.9%). We did not find a distinct outcome between these groups, although the sample size was limited to draw solid conclusions. Our results are concordant with Tazi et al., who concluded that the allelic state of TP53  provide no further prognostic information in AML.Regarding CEBPA should be considered as a favorable prognosis marker [CEBPA bZIP mutations is similar to the CEBPAbi subgroup reported by Tazi et al., (1.2% vs. 1.8%) and in both cases, OS analysis includes them within a favorable risk subgroup. However, CEBPAbi and CEBPA bZIP may be overlapping categories in some patients as several studies suggest that CEBPA double mutated frequently includes mutations in the C–terminal region which allocated bZIP domain [CEBPA as a prognostic biomarker may allow for more homogeneous analysis by reducing the variability in the interpretation of the biallelic character of these mutations [On the other hand, recent studies reported that instead of biallelic mutations, only in–frame mutations in the bZIP domain of s marker . In our P domain . Moreoveutations .This report reflects the efforts of the PETHEMA cooperative scientific group to adopt a nationwide strategy network of reliable and consistent NGS analyses. The establishment of consensus subset genes and the periodic CV rounds have strengthened the diagnostic network by unifying analysis criteria and decreasing reporting variability. Molecular analyses through NGS are routinely performed for AML patients and a comprehensive molecular profile of the disease is offered to clinicians in order to individualize the therapeutic strategy. Moreover, NGS results have provided a large amount of molecular data that has revealed the molecular complexity of the disease. In this cohort, mutual exclusion and mutational co–occurrences among genes and functional categories have been deciphered and a distinct molecular profile between age groups at diagnosis and sex has been detected. Moreover, clinical validation of the current genomic classification in the “real–life” PETHEMA cohort has demonstrated the correlation of the molecular subgroups with clinical prognosis, reflecting the utility of the cooperative NGS studies in routine molecular diagnostics.