PurposePatient-specific quality assurance (QA) by gamma (γ) analysis is an important component of high-precision radiotherapy. It is important to standardize institute-specific protocol. In this study, we describe our institutional experience of patient-specific QA for high-precision radiotherapy from a clinical perspective.MethodsThe planning data of 56 patients treated with intensity-modulated radiotherapy (IMRT)/volumetric modulated arc therapy (VMAT) were included. γ index analysis was done using Octavius 4D IMRT QA phantom  using 3 mm/3% criteria. Local, global, and volumetric gammas were calculated and compared. The relationship of γ index in the transverse, coronal, and sagittal direction and anatomical region of treatment was explored.ResultsGlobal three-dimensional (3D) γ indices in the coronal, sagittal, and transverse axes were 96.73 ± 2.35, 95.66 ± 3.01, and 93.36 ± 4.87 (p < 0.05). The average local two-dimensional (2D) γ index was 78.23 ± 5.44 and the global γ index was 92.41 ± 2.41 (p < 0.005). The average local 3D γ index was 84.99 ± 4.24 and the global 3D γ index was 95.25 ± 1.72 . The average local volumetric γ index was 84.29 ± 4.73 and the global volumetric γ index was 95.96 ± 2.08 (p < 0.005). 3D global gamma index was significantly different in different anatomical regions (p < 0.05).ConclusionOur study shows that γ index analysis is a useful parameter for routine clinical IMRT QA. The choice of type of γ index depends on the context of use and degree of stringency in measurement. Average 2D and 3D global γ were different in anatomical regions. The average 3D γ index was significantly different in axes. No difference was observed with techniques of IMRT/VMAT. Localization of failed points in CT anatomy can be advantageous for clinical decision-making. High-precision radiotherapy techniques like intensity-modulated radiotherapy (IMRT) and volumetric modulated arc therapy (VMAT) can improve the therapeutic ratio through the escalation of tumor dose and superior target coverage simultaneously sparing the normal tissue . In VMATThe movement of MLC produces complex dose distribution and intensity modulation in the IMRT technique. There is a considerable increase in the possibility of dosimetric variation due to the inherent complexity of these treatment techniques. Therefore, a three-dimensional 3D) dose matrix, with a volumetric evaluation of composite fields, is superior to a planar dose value map D dose ma. This isCurrent clinical standards of 3% DD criterion (ΔDM) and 3 mm DTA criterion (ΔdM) are widely considered acceptable for photon beams . A 90% pThe planning data of 56 patients treated with IMRT/VMAT were included. The study was approved by Institutional Human Ethics Committee, AIIMS Bhopal (IHEC-LOP/2021/ IM0343).Octavius 4D IMRT phantom commissioning2 to 26 × 26 cm2 measured at 85 cm source to surface distance (SSD). The Octavius 4D CT used for plan verification was the artificial one provided by the vendor. However, to better model the phantom in the TPS, a CT scan of the phantom was performed in Simulator CT  and the obtained averaged Hounsfield unit (HU) was reported into the TPS. Moreover, the correct distance from the couch was measured and reported in the artificial CT by fusion of images. Static delivery tests included 5 × 5, 10 × 10, 20 × 20 cm2 static fields and a “pyramid” field‐in‐field shape given by superimposition of 5 × 5, 10 × 10, and 20 × 20 cm2 static fields.The commissioning of Octavius 4D was carried out with the Elekta Versa HD medical linear accelerator . Percentage depth dose (PDD) was measured for field sizes ranging from 4 × 4 cm2 arc and the combination of 5 × 5 and 10 × 10 cm2 arcs, with a clockwise 180° rotation for both arcs, which were sequentially delivered to cover a full rotation of the phantom. The effect of different spatial directions was assessed by considering transversal, coronal, and sagittal planes and verifying whether there was a different response or not. Dosimetric data were assessed by the γ analysis with acceptance criteria of 3%/3 mm. To achieve absolute dose, the central chamber of the array was cross‐calibrated with a PTW semiflex ionization chamber , inserted into an RW3 slab replacing the 2D array inside Octavius 4D. The position of the chamber with respect to the isocenter was preliminarily verified by orthogonal kV images obtained in Elekta Versa HD On‐Board Imager (OBI).The evaluation of arc delivery performance was obtained using a 5 × 5 cm2, gantry at 0°, 200 MU corresponding to 1.248 Gy for Versa HD, i.e., the expected value in each measure session for the clinical plan verification) and the dose value was deduced from the chamber signal following the International Atomic Energy Agency (IAEA) Technical Report Series 398 approach, taking into account also the correction for the daily linear accelerator (LINAC) output factor. The comparison with the central chamber measurement at the same conditions allowed deducing the so‐called Kuser factor for each LINAC, useful for validation of Octavius 4D for absolute dose assessment. This result was compared with the Kcross, to evaluate the consistency of the two approaches. Finally, it was evaluated that the dose distribution obtained by two uninterrupted clinical arc deliveries vs. the same arcs delivered with up to four interruptions to understand the performance of the LINAC when an undesirable arc interruption happens and to test the inclinometer reliability.Measurements at the reference condition were carried out  with a 6 MV beam for all fields. The γ index metric was computed using Octavius 4D phantom and VeriSoft software. As a detector, the PTW Octavius Detector 1500 array was used, which has a high resolution (0.1 mGy) with 1405 chambers arranged as a checkboard of size 4.4 × 4.4 × 3 mm (0.06 cm³) in 27 × 27 cm area. The inclinometer setup allowed the phantom to be synchronized with the rotation speed and angle of the gantry of the linear accelerator as in actual treatment delivery. The direction of the beam always remains perpendicular to the detector array, avoiding any additional correction factor for beam direction.The dose distribution of the treatment plan was recalculated on the CT of the phantom performed in the simulator CT to generate the verification plan. The dosimetric verification was carried out by comparing the measured plan with the verification plan. Cross calibration based on the reference value provided by the TPS defrayed the output variations in LINAC. Dosimetric information was independent of TPS due to the application of the correction factor to the whole detector matrix and the conversion of PDD from water to phantom material. The 2D and 3D dosimetric metric called the “γ index” in different axes  were obtained by processing the data in VeriSoft software.Gamma analysisThe 2D, 3D, and volumetric γ were evaluated with a 3%/3 mm acceptance criterion . ResultsStatistical analysisStatistical analysis was performed with IBM SPSS Statistics software . Gaussian distribution of data was tested for normalcy by the Kolmogorov-Smirnov test. A parametric test (t-test) was used for the comparison of the mean and analysis of variance (ANOVA) was studied for the comparison of different groups. For statistical analysis, the significance level was taken as p < 0.05.Octavius 4D commission2 field are shown in Figure The low dose threshold for the inconsistent area of comparison was set at 0.1% of the maximum dose and the cut-off threshold was set at 5%. The Octavius phantom set up in LINAC and the dose profile of the static 10 × 10 cmPatient, tumor, and treatment-related factorsThe mean age of the cohort was 48 years (23-78 years). The mean PTV volume was 496.65 cc (230.34-1065.96). Treatment technique involved IMRT in 11 cases and VMAT in 45 cases. Radiotherapy treatment involved the cranial region (n = 28), thoracic region (n = 14), and pelvic region (n = 14).Comparison between different types of gamma metricLocal 2D γ indices in coronal, sagittal, and transverse axes were 82.48 ± 6.29, 80.12 ± 6.02, and 72.1 ± 9.4, and global 2D γ indices in the respective axes were 94.42 ± 3.63, 93.08 ± 4.05, and 89.74 ± 6.74.Local 3D γ indices in coronal, sagittal, and transverse axes were 88.43 ± 5.26, 86.3 ± 5.14, and 80.25 ± 8.37, and global 3D γ indices in the respective axes were 96.73 ± 2.35, 95.66 ± 3.01, and 93.36 ± 4.87. The average (across three axes) 2D local γ index was 78.23 ± 5.44 and the global γ index was 92.41 ± 2.41 . The average 3D local gamma index was 84.99 ± 4.24 and the global 3D gamma index was 95.25 ± 1.72 . It was normally distributed .Directional dependence of γ indexThe result of concordance of γ index depending on the section of the phantom in three different axes  is shown in Figure Relationship of γ index and anatomical region of treatmentThe average 2D global γ indices in the cranial region (n = 28), thoracic region (n = 14), and pelvic region (n = 14) were 91.85, 94.85, and 94.85, respectively . The average 3D global γ indices in the cranial region (n = 28), thoracic region (n = 14), and pelvic region (n = 14) were 94.68, 97.08, and 94.56, respectively . There was no statistically significant difference in global or local volumetric γ index across the regions  for VMRT plans were reported to be 95.45%, 97.51%, and 96.91% . Our stuPark et al. reported a weak correlation between local and global gamma index at the 3 mm/3% cut-off level . In our study, we found a significant correlation between the local and global gamma index. This could be because the dosimetric verification system and resolution of detectors were different. Gamma analysis varies depending on the type of dosimeter used and variation between different dosimeters could be considerable .Conventional IMRT QA metrics need to be considered together with the anatomical region in which dose errors are overlaid . This isRecently, the integration of the γ index with radiobiological factors to obtain a better clinical perspective had been investigated ,19. The As this is a single institute study with a limited sample size, a multi-centered study involving multiple institutions, including diversified patients with variability achieving a larger sample size, may be required to generalize the clinical applicability in patient-specific quality assurance in high-precision radiotherapy.Our study shows that γ index analysis is a useful parameter for routine clinical IMRT QA. The choice of type of gamma index depends on the context of use and the degree of stringency of measurement required. 2D local and global gamma differed significantly in different directions. Average 2D and 3D global gammas were different in different anatomical regions. Average 3D global and local gammas were significantly different along directions. Local and global volumetric gammas were significantly different though correlated. No difference was observed with techniques of IMRT/VMAT. Though volumetric γ encompassed more “passing points” than 2D γ, it is recommended that failing points of γ analysis are interpreted in a clinical context keeping in mind the anatomical region where the points of failure lie. The development of a radiobiological model with γ index and survival and toxicity data can have prognostic significance and should be further investigated.


L-buthionine sulfoximine (a specific inhibitor of γ-glutamylcysteine synthetase) confirmed that the alleviation of As and Cd translocation and toxicity in wheat by S is mediated by increased PC production. Also, TaHMA3 gene expression in wheat root was not affected by the As+Cd and S treatments, but the expression of TaABCC1 was upregulated by the high As+Cd treatment and further increased by sufficient S supply and high As+Cd treatment. These results indicate that S-induced As and Cd subcellular changes affect As and Cd translocation mainly by regulating thiol metabolism and ABCC1 expression in wheat under As and Cd stress.Accumulation of arsenic (As) and cadmium (Cd) in wheat grain is a serious threat to human health. Sulfur (S) can simultaneously decrease wheat grain As and Cd concentrations by decreasing their translocation in wheat; however, the mechanisms are unclear. We conducted hydroponic experiments to explore the mechanisms by which S modulates As and Cd translocation and their toxicity in wheat. Wheat seedlings were grown in deficient sulfate (2.5 µM) or sufficient sulfate (1.0 mM) nutrient solutions for 6 days and then exposed to zero (control), low As+Cd (1 µM As plus 0.5 µM Cd), or high As+Cd (50 µM As plus 30 µM Cd) for another 6 days. Compared with the control, plant growth was not affected by low As+Cd, but was significantly inhibited by high As+Cd. In the low As+Cd treatment, S supply had no significant effect on plant growth or root-to-shoot As and Cd translocation. In the high As+Cd treatment, sufficient S supply significantly alleviated As and Cd toxicity and their translocation by increasing phytochelatin (PC) synthesis and the subsequent vacuolar sequestration of As and Cd in roots, compared with deficient S supply. The use of  Triticum aestivum L.) is one of the most important staple foods and ranks first in terms of global consumption, accounting for about 28% of cereal consumption annually  and cadmium (Cd) are ubiquitously distributed, highly toxic, and readily transferred from contaminated soil to the food chain . Long-teannually . Compareannually . In alkaannually . As a reannually . Althougannually , high Asannually . Arsenicannually , which iannually . Risks tannually , Pakistaannually , and Argannually . TherefoMost previous studies focused on the uptake and translocation of As and Cd individually in soil-crop systems, and many mitigation strategies have been employed to reduce As or Cd accumulation in food crops. However, farmland contaminated with both As and Cd has been reported worldwide . SimultaOsHMA3  expression  seeds were surface-sterilized in 10% (w/w) hydrogen peroxide for 10 min, washed thoroughly with clean water and soaked in distilled water for 12 h, and germinated in wet quartz sand for 3 days. After germination, uniform seedlings were selected and transplanted into a black plastic beaker (twelve seedlings per beaker) with 1.1 L of 0.5 mmol L–1 CaCl2 solution for 3 days, and then the seedlings with residual endosperm removed were transferred into nutrient solution with deficient sulfur  or sufficient sulfur . The sufficient S nutrient solution (full nutrient solution) contained 1.0 mM Ca(NO3)2, 1.5 mM KCl, 1 mM MgSO4, 0.2 mM KH2PO4, 1.5 mM CaCl2, 1 µM H3BO3, 0.05 µM (NH4)6Mo7O24, 1 µM ZnSO4, 1 µM MnSO4, 0.5 µM CuSO4, and 40 µM FeNa2-EDTA  was added to high As+Cd + deficient S supply and high As+Cd + sufficient S supply. The concentration of BSO used in this study was set according to previous studies , low , or high As and Cd  for another 6 days maintained in deficient- or sufficient-S nutrient solution under the above-mentioned growth conditions. The concentrations of As and Cd selected were based on the results of preliminary toxicity assays. Arsenic at 1–10 µM or Cd at 0.5–5 µM has no visual negative effect on the growth of wheat seedlings after 6 days; and As at 50 µM or Cd at 30 µM both caused about 40% inhibition in root elongation 2, and 5 mM MES (pH 6.0) for 10 min to remove root surface-adsorbed As and Cd  at 125°C  and shoots were measured using a ruler before and after 6 days of As+Cd treatment. After measurement of root and shoot length, wheat plants were harvested. The roots were rinsed in deionized water and immersed in an ice-cold desorption solution containing 1 mM Ks and Cd . Roots aat 125°C . Arsenic× g for 15 min at 4°C; the residue was considered the cell-wall fraction. The supernatant was centrifuged at 20,000 × g for 30 min at 4°C (3–H2O2. Arsenic and Cd concentrations were determined by ICP-MS (Frozen root and shoot tissues were powdered in liquid nitrogen and extracted with 10 mL of extraction solution containing 50 mM Tris-HCl buffer (pH 7.5), 0.25 mM sucrose, and 1.0 mM DL-dithioerythritol. The homogenate was centrifuged at 1500 n at 4°C . The supy ICP-MS .× g for 20 min at 4°C. The supernatant was passed through a 0.22-µm membrane filter, and a 250-µL aliquot of the supernatant was mixed with 20 µL of 1 mM TCEP and immediately derivatized with 10 µL of 25 mM monobrombimane, together with 420 µL of 200 mM HEPES buffer containing 6.3 mmol L–1 DTPA. Derivatization was conducted in the dark for 30 min at 45°C and terminated by adding 300 µL of 1 M methansulphonic acid (MSA). The thiol derivatives were separated and analyzed by reverse-phase ultra-performance liquid chromatography. The details of the procedure are described elsewhere . The tubulin gene was used as an internal standard control gene  based on the manufacturer’s instructions, and RNA quality was examined by agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer. First-strand cDNA was synthesized from total RNA using M-MLV Reverse Transcriptase (Fermentas). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was carried out on a Bio-Rad CFX96 Real-Time PCR system with SYBR Green detection in accordance with the manufacturer’s instructions. The primers used for eviously , and thorol gene . The prin levels .p-value ≤ 0.05 was considered indicative of statistical significance. Figures and tables were generated using Excel 2016 and SigmaPlot 12.5 software. The translocation factor (TF), an indicator of the ability of plants to translocate As or Cd from roots to shoots, was calculated as the ratio of As or Cd concentration in shoots to roots  using SPSS software (v. 25.0). Duncan’s multiple comparison test was used to assess differences among treatments, and a to roots .p ≤ 0.05) inhibition of plant growth, leading to 58.2%, 44.7%, 13.3%, and 15.2% decreases in root elongation, shoot elongation, root dry weight, and shoot dry weight, respectively, under deficient S supply. By contrast, sufficient S supply significantly alleviated the plant growth inhibition induced by high As+Cd, because the four growth parameters were increased by sufficient compared to deficient S supply. The root elongation, shoot elongation, root dry weight, and shoot dry weight in sufficient S supply were 24.7%, 17.8%, 12.7%, and 6.9% higher than those in deficient S supply, respectively. In the zero (control) and low As+Cd treatments, the differences in the four growth parameters between the two S supplies were not significant.We evaluated the impact of S supply and As+Cd treatment on the growth of wheat seedlings in terms of root and shoot elongation and dry weight Figure 1The concentrations of As and Cd in wheat roots and shoots increased significantly with increasing As and Cd concentrations in nutrient solution Figure 2As shown in 4 was only detected in high As+Cd-treated plants. The average (of two S treatments) concentrations of PC2 and PC3 in wheat roots in the high As+Cd treatment were 38.2- and 22.2-fold higher than those in the low As+Cd treatment, and 88.9- and 37.9-fold higher than those in the zero As+Cd treatment, respectively  transporting Cd-PC complexes into vacuoles via a PC-dependent pathway , the Jiangsu Agricultural Science and Technology Independent Innovation Fund (CX(20)1010), and the National Natural Science Foundation of China (41601541).The authors are grateful to Ms. Xin Wang for her technical assistance with ICP-MS analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In this work, watermelon rind was used for extraction of pectin with acetic acid solution. The effects of pH, temperature and extraction time on the pectin yield were investigated. Response surface based on Box-Behnken model was employed to optimize the extraction parameters. The model shows an optimum pectin yield of 18.21%, which is in agreement with the value confirmed through experiment (18.20%). The moisture content, ash content, degree of esterification, degree of methylation, equivalent weight, methoxy content, and anhydrouronic acid of the extracted pectin were determined. The values of the moisture content, ash content, degree of esterification, degree of methylation, equivalent weight, methoxy content, anhydrouronic acid are 8.42%, 5.1%, 57.30%, 23.5%, 983.9 mg/mol, 7.3% and 72.36%, respectively. The results show watermelon peel can be an alternative source for pectin production with reasonable pectin yield and pectin quality. Currently, 85.5% of the pectin in the global market is produced from citrus peels, 14.0% from apple pomace and the remaining 0.5% from sugar beet pulp. From the citrus peels, the contributions of lemon, lime and orange peels are 56%, 30% and 13%, respectively . AlthougBiomass residues such as banana peel, coffee pulp, soy hull, papaya peel, sunflower head, mango peel, watermelon rind, etc. have attracted attention from both academia and industry. In the present work, watermelon rind was used for extraction of pectin using acetic acid solution. Wet watermelon rinds contain 19–21% pectin . Moreove22.1°C for 48 h. The dried watermelon peel was ground in mesh size of 100 μm and packed in polyethylene bag at ambient temperature in addition to that stored for extraction process.All chemicals used in this work were analytical grade. Acetic acid, ethanol, sodium hydroxide, sodium chloride, hydrochloric acid and phenolphthalein were used for extraction and characterization of pectin. The red watermelon was supplied by Debre Zeit Agricultural Research Center, Bishoftu, Ethiopia. The watermelon rinds were separated from the fruit, cut into pieces, washed and dried in a vacuum oven at 45 2.2Pectin extractions were carried out using 1 M aqueous acetic acid with solid to liquid ratio (1: 30), at pH (1.0–5.0), extraction temperature (60–120 °C) and extraction time (50–110 min). Watermelon rind powder (15 g) was added to a 500 mL flask and 450 mL 1.0 M aqueous acetic acid was added to the flask. The solution was then heated to the required temperature under constant mixing for a given time. Sodium hydroxide was used to adjust pH of the solution. The solution was separated by applying muslin cloth. Then, ethanol was added to the solution at ethanol to solution ratio of 2:1 (v/v) and the mixture was stirred for 10 min and kept overnight to allow pectin coagulation. The coagulated pectin was separated by muslin cloth and washed three times using ethanol. The separated pectin was dried in a vacuum oven at 45 °C for 24 h and then cooled at room temperature. Finally, the dried pectin was ground into fine particles and stored in a moisture-proof airtight bag until further use.2.32.3.1Ch, Ms, Cp, Cf and As are the carbohydrate, moisture, crude protein, crude fat and ash contents, respectively.The proximate analyses of watermelon rind were carried out according to Association of Official Analytical Chemistry . Moistur(1)2.3.2Moisture and ash contents of the watermelon rind pectin were determined using the method developed by Association of Official Analytical Chemistry . The equ(2)To determine the methoxy content, 25 mL (0.25 M) sodium chloride was added to the solution and the solution was stirred and allowed cool at ambient temperature for 30 min. Then, 25 mL (0.25 M) hydrochloric acid was added and titrated again with 0.1 N of sodium hydroxide to the same endpoint as equivalent weight. The percent methoxy was determined using equation (3)V1 is the volume of titration from equivalent weight and V2 is volume titration from methoxy determination.Degree of esterification (DE) was calculated using equation (4)V1 is sodium hydroxide used in determination of equivalent weight content and V2 is the sodium hydroxide used in the determination of methoxyl content.The anhydrouronic acid content (AUA) was determined using equation (5)2.4−1 wavenumbers. The morphology of the watermelon rind and its pectin were investigated using SEM . The crystal structures of the samples were analyzed using XRD (XRD-7000). A differential thermogravimetric analyzer (DTG60H) was used to analyze the thermographic properties of watermelon rind powder and extracted pectin.Fourier-transform infrared spectroscopy (FTIR), Scanning electron microscope (SEM), X-ray Powder Diffraction (XRD) and Thermogravimetric Analysis (TGA) were used to analyze the raw watermelon rind and extracted pectin and the changes observed during extraction of pectin. The results were also used for comparison of the characteristics of the current pectin with pectin obtained from other sources. The FTIR spectrum was taken using Fourier transform infrared spectroscopy (Thermo Scientific iS50 ABX). The FTIR spectrums of both dried watermelon rind and extracted pectin were taken over 4000 and 400 cm2.5X, is the factors and βi is the coefficient. The developed model was evaluated by analyzing the values of regression coefficients, ANOVA, F- and P-values. Finally, three-dimensional response surface plots were drawn to visualize the individual and the interaction between the effects of the independent variables on the response. RSM design was carryout with a Design Expert . The proposed model was validated by performing new tests under optimal condition. The pectin yield was calculated using equation (7)m1 and m2 are the weight of dried watermelon rind and dried pectin.A 3- factor- 3- level Box-Behnken design (BBD) based response surface methodology (RSM) was employed to investigate the effect of three factors  on the response (pectin yield). A total of 15 experimental runs were conducted with three center points. A second-order polynomial model was used to fit the response variable with the factors Equation .(6)Y=β0+33.13.1.1The proximate analysis of the watermelon rind was determined to mainly see the composition of its ash, carbohydrate, protein, fiber and fat contents. These parameters affect the quality and quantity of the extracted pectin. The moisture content of the dried watermelon rind is 6.20%, which is lower than the values reported for other red watermelon rinds . The ash3.1.2Hylocereus polyrhizus peels [The physicochemical properties of the pectin extracted from the watermelon rind are shown in us peels . The lowThe equivalent weight shows the total content of free galacturonic acid in pectin. The equivalent weight of the extracted pectin is about 984 mg/mol which is higher than the minimum value (400 mg/mol) recommended by the international pectin producer association . The equMethoxy content shows the degree of methylation and it determines the capacity of pectin to form gel and may suggest strong cohesive and adhesive characteristics of the pectin . Pectin 3.1.3−1 represents the OH stretch of water molecules in the watermelon rind. The very weak around 2900 cm−1 corresponds to C–H stretching vibrations. These peaks became more pronounced in the FTIR of the pectin. The peak observed between 3990 and 2990 cm−1 corresponds to O–H stretching absorption hydrogen bonding of the galacturonic acid  backbone [−1 is the characteristic band for the CH stretching vibration from –CH, –CH2, and –CH3, methyl esters of galacturonic acid in polysaccharide components [−1 correspond to C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="pc-E00C" format="PBM" resolution="300" x-size="12" y-size="9">000000000000000000000000000000000000111111111111000000000000111111111111000000000000000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="20.666667pt" height="16.000000pt" viewBox="0 0 20.666667 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z M0 280 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z"/></g></svg>O of esterified carboxylic groups (-COOCH3) and free carboxylic groups (-COOH), respectively [−1 correspond to the ether R-O-R and cyclic C–C ring linkages of the pectin structure [The Fourier-transform infrared spectroscopy (FTIR) spectrum of the dried watermelon rind and the extracted pectin is shown in backbone . The banmponents . The peaectively . The spetructure . The pea3.1.4X-ray diffraction (XRD) analyses were performed both for dried watermelon rind and the extracted pectin. X-ray diffraction (XRD) analyses were used to study the pectin structure  of the rind and the pectin. XRD spectrum was taken for sample extracted with acetic acid at pH (2.0), temperature (90 °C) and time (100 min) was obtained. XRD pattern of the watermelon rind and the extracted pectin are shown in 3.1.5°C was 18.5%, while the value for extracted pectin is about 10.5%.The thermal stabilities of dried watermelon rind and extracted pectin were investigated using thermogravimetric analysis. The thermogravimetric was performed for sample extracted with acetic acid at pH (2.0), temperature (90 °C) and time (100 min). 3.1.6Scanning electron microscope (SEM) images of dried watermelon rind and extracted pectin are depicted in 3.2Pectin yield depends on various factors, such as pH, temperature, extraction time, types of acid used, and the nature of pectin sources. In this work, the effect of pH, temperature and extraction time were investigated . The eff3.32 value is 0.9955. This implies the model sufficiently predicts the pectin extraction yield from the three extraction parameters. The second-order polynomial model for pectin extraction yield is shown in equation , temperature (93 °C) and extraction time (101 min). The experimental pectin yield value at this condition was 18.20%. Three measurements were made and average value was reported for the experimental pectin yield at the optimum extraction condition. This shows the model can predict the optimum pectin yield under the current extraction conditions.In this work, response surface methodology based on Box Behnken design (BBD) was employed to determine the optimum condition of the three factors . A total of 15 experiments were carried out to generate the response surface. A second-order model was fitted to the experimental data to develop a numerical relationship between the three factors and the response equation . The anaequation . The expThe pectin extraction yield from watermelon yield using acetic acid under the current extraction process conditions is compared with other pectin sources. The current pectin yield is higher than the values reported for papaya powder (2.6%) , banana + concentration in the medium and stimulates the hydrolysis of protopectin and suppress highly hydrated carboxylate groups leading to higher pectin yield [in yield . The eff4In this work, pectin was successfully extracted from dry watermelon rind with acetic acid. Both the watermelon rind and pectin were characterized for morphological and chemical characteristics. The effects of pH of solution, extraction temperature and extraction time on the yield of extracted pectin were found to be significant. The highest extraction yield of 18.20% was observed pH of 2.0, temperature of 90 °C and extraction time of 100 min. Response surface method based on Box-Behnken experimental design was employed to establish the numerical relation between the three pectin extraction parameters  with reasonable accuracy. The model provided 18.21% pectin yield which is comparable is the experimental result. The pectin extracted can be considered as high methoxy pectin (DE 57.30% and Meo 7.30%) and has high quality (AUA >65%). This shows extraction of pectin with acetic acid from watermelon rind can provide reasonable pectin yield with good quality pectin.Dawit Mamiru: Performed the experiments.Girma Gonfa: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Addis Ababa Science and Technology internal research grant (IG 03/2022).This work was supported by the Data will be made available on request.The authors declare no conflict of interest.


As a result, the ionic conductivity of Li5.875Y0.875Zr0.1Ce0.025(BO3)3 (ZC-LYBO) reached 1.7 × 10−5 S cm−1 at 27 °C, which was about 5 orders of magnitude higher than that of undoped Li6Y(BO3)3. ZC-LYBO possessed a large electrochemical window and was thermally stable after cosintering with a LiNi1/3Mn1/3Co1/3O2 (NMC) positive electrode. These characteristics facilitated good reversible capacities in all-solid-state batteries for both NMC positive electrodes and graphite negative electrodes via a simple cosintering process.The ionic conductivity of Li 6Y(BO3)3 (LYBO) is enhanced by the substitution of tetravalent ions through an increase in the conduction pathways etc. Zr,Ce-doped LYBO can be used as an electrolyte for all-solid-state batteries via a cosintering process.Ionic conductivity of Li Oxide-based ASSBs are expected to be applied as on-board power sources for next-generation wireless Internet-of-Things devices because of their long-life performance and thermal stability. However, the discovery of better ion-conductive oxides is not always related to the enhancement of the performance of oxide-based ASSBs. This corresponds to another issue, that is, the difficulty of impurity-free interface formation between the active materials and oxide electrolyte during cosintering.6–10 Therefore, oxide-based ionic conductors that are both highly conductive and thermally stable with respect to the electrode materials are required for the development of electrolytes for sintered oxide-based ASSBs.The steady evolution of new crystal systems with high ionic conductivities has recently heightened expectations for the realisation of all-solid-state batteries (ASSBs).3BO3-related families have been considered as suitable electrolytes for fabricating good contacts at the electrode/electrolyte interface by cosintering. For example, Li3BO3 was used as an interfacial additive between LiCoO2 electrode particles and a garnet-type sintered electrolyte plate, and reversible charge–discharge capacities were demonstrated for the ASSB after cosintering at 700 °C, despite the low conductivity of Li3BO3 (σ < 10−7 S cm−1 at 150 °C).11,12 In a later report, a solid solution of Li2CO3 and Li3BO3, Li2.2C0.8B0.2O3, was described as a superior candidate for assembling the ASSB because of its better conductivity of ≈10−6 S cm−1 at 25 °C.13,14 Two other Li3BO3-related electrolytes—Li3BO3–Li2SO4 amorphous materials15 and LISICON-Li3BO3 amorphous materials16—were also attractive candidates, but the ASSBs were assembled according to their deformability due to their amorphous character rather than their thermal stability.The Li3BO3-related families, Lopez-Bermudez et al. proposed a solid solution of YBO3 and Li3BO3, that is, Li6Y(BO3)3 (LYBO), as a new crystal-type electrolyte candidate.17 The observed ionic conductivity was low at 1.9 × 10−8 S cm−1 at 50 °C because Li+-ion diffusion was permitted only by thermodynamic point-defect formation in the Li6Y(BO3)3. However, based on DFT calculations of the defect-formation energies and Li+-ion diffusion barriers, they suggested the structural superiority of Li+-ion diffusion in the LYBO-type structure, and mentioned the possibility of conductivity enhancement by aliovalent substitution.17Among Li4+ and Ce4+)-doped LYBO-type materials and confirmed an ionic conductivity enhancement to ≈10−5 S cm−1 at 27 °C. Furthermore, the performance of oxide-based ASSBs was successfully demonstrated with the Zr- and Ce-doped Li5.875Y0.875Zr0.1Ce0.025(BO3)3 (ZC-LYBO) electrolyte because of its thermal stability to layered rock-salt positive electrodes, such as the LiNi1/3Mn1/3Co1/3O2 (NMC) positive electrode, and its large electrochemical window.In the present study, we prepared tetravalent ion (Zrx−y6−Yx−y1−ZrxCey(BO3)3, were prepared using Li3BO3 , H3BO3 , Y2O3 , ZrO2 , and CeO2  as starting materials. An appropriate amount of each compound was weighed to obtain the desired molar ratio, and the mixture was ground using a mechanical milling technique . The mixture was pelletized and heated at 650 °C for 12 h. The reacted product was ground and sintered by spark-plasma sintering (SPS) at 600–700 °C for 10 min under a pressure of 30 MPa, to produce the tetravalent ion-containing LYBO compacts.Tetravalent ion-containing LYBO samples with various molar ratios, Li18 where a modified split pseudo-Voigt function19 was selected for fitting to best represent the profile parameters of the samples. To estimate the Li+-ion diffusion pathways in the structure, the refined structural parameters were used as the input file for bond valence site energy (BVSE) calculations, which were performed using SoftBV software.20,21 The structural images and maps were drawn using the VESTA software program.22The structural changes and purities of the samples were evaluated using synchrotron X-ray diffraction (SXRD) analysis (0.5 Å) performed at BL19B2, SPring-8, Sayo, Japan (2019B1881). Rietveld crystal structure refinement of the obtained SXRD patterns was performed using the RIETAN-FP software program,via sputtering) as blocking electrodes. The AC impedance profile was collected with a frequency response analyser  over a frequency range of 0.1 Hz to 1 MHz at 27 °C.For ion conductivity measurements, both sides of the tetravalent ion-containing LYBO compacts were polished and coated with gold  or graphite . The composite electrode powder (10 mg) and a Au plate as a current collector were placed on a ZC-LYBO-electrolyte separator of 30 mg in a 10 mm-diameter carbon die, which was heated at 550 °C with an electric current under a pressure of 30 MPa in the SPS process. Lithium foil was used as the counter electrode. A polyethylene oxide (PEO)-based polymer electrolyte film  was placed between the lithium foil and the electrolyte side of the composite electrode/electrolyte pellet to reduce the interfacial resistance via adhesion.13To measure the charge–discharge performance of the ASSB, a composite electrode powder consisting of 50 wt% active material and 50 wt% ZC-LYBO electrolyte was first prepared by mixing in an agate mortar. The active material was either LiNiTo confirm the reactivity of the composite electrode after cosintering, laboratory XRD patterns were collected on a diffractometer using Cu Kα radiation . All XRD analyses were performed in the Bragg–Brentano geometry mode. The microstructure of the composite electrode was observed using field-emission scanning electron microscopy .To evaluate the electrochemical stability of the ZC-LYBO electrolyte, a metal substrate (Au or Cu) was placed on a ZC-LYBO electrolyte (100 mg) in a 10 mm-diameter carbon die, which was heated at 550 °C with an electric current under a pressure of 30 MPa in the SPS process. Lithium foil and a PEO-based polymer electrolyte film were used as the counter electrode and interfacial connector, respectively.4+ ion-containing LYBO samples, Lix6−Yx1−Zrx(BO3)3 (0 ≤ x ≤ 0.6). The I(1 1 −2) diffraction peak is clearly shifted to a higher angle with an increasing amount of Zr4+ until x ≈ 0.1 in Lix6−Yx1−Zrx(BO3)3 because of the difference in ionic radii: 1.019 Å for Y3+ and 0.84 Å for Zr4+ in dodecahedral coordination.23 In addition, the I(1 1 −2) diffraction peak is broadened with an increasing amount of Zr4+ ions, which corresponds to the lattice distortion of the LYBO-type structure in addition to the aforementioned mismatch of ionic radii, which will be discussed later. Furthermore, the ZrO2 impurity phase begins to appear from x ≈ 0.1.4+ ions. The refined patterns for typical samples and the estimated parameters for all the samples are summarised in Fig. S2(a–c), and Table S1,U for the LYBO-type phases estimated from the refinements. Herein, U is one of the simulated components for representing the FWHMs of the total diffraction patterns based on the Caglioti formula24 under Rietveld refinement.V and W mainly depend on the diffractometer characteristics, which were estimated from the refinements of a CeO2 reference in advance, and θBragg is the diffraction peak angle.Rietveld refinements of the SXRD patterns were performed to clarify the structural changes caused by the ZrU represents the FWHM corresponding to the structural distortion in a sample. It should be noted that the structural distortion is difficult to discuss quantitatively in this study because a modified split pseudo-Voigt function19 was used to refine the profiles. Despite the difficulties in this case, the definite change in U due to the Zr4+ ion is strongly related to the structural distortion until x ≈ 0.1 in Lix6−Yx1−Zrx(BO3)3, which is due to the substitution of smaller Zr4+ ions at the Y3+ sites. The shrinkage of the cell volume until x ≈ 0.1 is also proof of Zr4+-ion doping. When x > 0.1, these changes are suppressed because of the limits of Zr4+-ion doping into the LYBO-type structure; thereafter, the ZrO2 impurity  is observed. Moreover, the lattice parameter b and area of the ac plane are shown separately in 2Y(BO3)3 layer and the Li layer along the b-axis, as shown in 8 dodecahedra upon doping with smaller Zr4+ ions until x ≈ 0.1 directly reduces the area of the ac plane. On the other hand, the lattice parameter b is expanded until x ≈ 0.1 because of the decrease in Coulomb repulsion force at the Li layer due to the lack of Li+ ions under the doping of Zr4+ ions into Lix6−Yx1−Zrx(BO3)3. This tendency is similar to the change in the lattice parameter c during the electrochemical de-lithiation of layered rock-salt Lix1−CoO2.25 Therefore, Li+ ions are likely to be removed from the Li layer rather than from the Li2Y(/Zr)(BO3)3 layer by doping Zr4+ ions into Lix6−Yx1−Zrx(BO3)3, which is supported by previous DFT calculation results in which the Li defect energies at the Li layer are lower than those at the Li2Y(BO3)3 layer in Li6Y(BO3)3.17Thus, +-ion diffusion pathways in the LYBO-type structure were examined from bond-valence site energy (BVSE) maps; the ab and ac planes are shown in +-ion pathways as clouds with low bond valence site energies. Although the mobile Li+ ions form a 2D diffusion pathway network at the Li layer on the ac plane [Fig. S3(b)+ ions to diffuse along the b axis via Li3 or Li5 sites in undoped Li6Y(BO3)3, as shown by the red dashed boxes in 17 As shown in b-axis could be induced by doping Zr4+ ions into Lix6−Yx1−Zrx(BO3)3, which indicates that the structural distortion in the Li2Y(/Zr)(BO3)3 layer contributed to expanding the diffusion pathways. The 2D diffusion pathways in the Li layer also become thin, as can be confirmed from Fig. S3.+ ions are more easily diffused by the structural distortion associated with doping Zr4+ ions into Lix6−Yx1−Zrx(BO3)3. The activation energies for the 2D and 3D pathways estimated from BVSE decrease from 0.57 and 0.69 eV to 0.49 and 0.59 eV, respectively, even by doping with only x = 0.025 Zr4+ ions into Lix6−Yx1−Zrx(BO3)3.The Li4+ ions are incorporated into Lix6−Yx1−Zrx(BO3)3 (0 ≤ x ≤ 0.1). The observed conductivity at 27 °C is drastically enhanced from 5.6 × 10−11 to 5.8 × 10−6 S cm−1 by doping with x = 0.025 Zr4+ ions into Lix6−Yx1−Zrx(BO3)3. Li+-ion diffusion in Li-ion fully occupied Li6Y(BO3)3 occurs only through the formation of a few thermodynamic point defects. In contrast, the substitution of tetravalent Zr4+ ions in the Y3+ sites results in the intentional formation of vacancies at Li sites in the structure, which contributes to the conductivity enhancement. Additionally, the observed conductivities are sensitive to the relative densities of the Lix6−Yx1−Zrx(BO3)3 compacts, which are summarised in Table S2.3.5Ge0.75S0.25O4 compact (79%) was over a hundred times lower than a high density compact (∼90%).26 Therefore, the increase in relative density upon doping with Zr4+ ions, from 77% (x = 0) to ∼90% (x > 0), also contributes to the ionic conductivity enhancement. Moreover, the expansion of 3D diffusion pathways by Zr4+ doping, as estimated by the Rietveld analyses, would also affect the enhancement. To confirm this effect, the structure and conductivity of Ce-doped Li5.975Y0.975Ce0.025(BO3)3 were also examined. The ionic radius for the Ce4+ ion dopant in dodecahedral coordination is 0.97 Å, which is more comparable to that for the Y3+ ion (1.019 Å) than the Zr4+ ion dopant (0.84 Å).23 Therefore, the lattice distortion by doping the tetravalent ion is supressed; the lattice distortion in Ce-doped Li5.975Y0.975Ce0.025(BO3)3 (U = 0.02354 deg2) is closer to that in undoped Li6Y(BO3)3 (U = 0.01488 deg2) than that in Zr-doped Li5.975Y0.975Zr0.025(BO3)3 (U = 0.119 deg2), as can be confirmed in Table S1.4+ doping (Li5.975Y0.975Ce0.025(BO3)3; σ = 6.9 × 10−7 S cm−1 at 27 °C), which is due to the formation of vacancies at Li sites in the structure and the increase in compact densification, as discussed above. However, the conductivity of the Ce4+-ion doped sample is over ten times lower than that of the Zr4+-ion doped one. This difference indicates that the structural distortion associated with the doping of the larger Zr4+ ions certainly affects the enhancement of the conductivity. The differences in the Li+-ion diffusion pathways estimated from the BVSE maps also support the aforementioned results: for the less-structurally-distorted Ce4+-ion doped sample b-axis in the BVSE map is nearly unchanged. Therefore, Zr4+-ion doping exerts three types of effects that enhance the conductivity of the LYBO-type structure: (1) the formation of vacancies at Li sites, (2) an increase in compact densification, and (3) an increase in the Li+-ion conduction pathways in the LYBO phase associated with structural distortion. The highest conductivity in Zr4+-ion-doped Lix6−Yx1−Zrx(BO3)3 at 27 °C is 1.4 × 10−5 S cm−1 at x = 0.10. However, Zr4+-ion doping achieves a limit at x ≈ 0.1 in Lix6−Yx1−Zrx(BO3)3, which would be due to the large mismatch of ionic radii. Therefore, the conductivity decreases for x > 0.1 3 (ZC-LYBO) was also measured, and the obtained value of 1.7 × 10−5 S cm−1 at 27 °C is the highest conductivity of LYBO-type oxides to date. The structural information estimated from Rietveld analysis, Li+-ion diffusion pathways estimated from BVSE maps, and relative densities of the compacts are summarised in Table S1, Fig. S3(d), and Table S2.5.9Y0.9Zr0.1(BO3)3, a slight enhancement in conductivity is confirmed, but the reason is difficult to determine in the present study. The conductivity measurement results indicate the possibility of further enhancement by other aliovalent substitutions. Moreover, the control of structural distortion is one of the keys for enhancing the conductivity of the LYBO-type structure. It should be noted that the activation energies estimated from Arrhenius plots 3 and Zr,Ce-doped Li5.875Y0.875Ce0.025Zr0.1(BO3)3 (ZC-LYBO). Based on its high conductivity, ZC-LYBO was used as the electrolyte in subsequent ASSB studies.The conductivity of Zr,Ce-doped Lis Fig. S5 were 0.41/3Mn1/3Co1/3O2 (NMC) electrode and ZC-LYBO electrolyte after cosintering was evaluated by laboratory XRD patterns .The cross-sectional microstructure of the NMC + ZC-LYBO composite electrode after the 550 °C SPS process was also investigated. As shown in via SPS. A reversible capacity of ∼120 mA h g−1 is observed at 60 °C owing to the impurity-free sintered interface between the NMC and ZC-LYBO. The charge–discharge capacity retentions at various current densities are shown in 3.5Ge0.5V0.5O4 (LGVO), which has also been reported as a thermally stable electrolyte with an NMC electrode.27 This is due to the lower conductivity of ZC-LYBO compared to Li3.5Ge0.5V0.5O4 (i.e. 9.6 × 10−5 S cm−1 at 25 °C).27 However, a broader acceptance of aliovalent substitutions in the less-reported LYBO-type electrolytes could lead to the development of a more highly ion-conductive LYBO family, analogous to the advances achieved with other electrolyte candidates.1vs. Li/Li+ and in the high-voltage range from 2.0 to 5.0 V vs. Li/Li+ with a Cu substrate (red) and an Au substrate (blue), respectively. The ZC-LYBO electrolyte is stable at high voltages. Au is kinetically stable within a wide electrochemical window but produces LixAuy alloys at voltages below 0.2 V vs. Li/Li+ when in contact with Li metal.28,29 Therefore, Cu was used as the substrate to evaluate the low-voltage stability.30 Cathodic and anodic currents corresponding to Li metal deposition (Li+ + e− → Li) and dissolution (Li → Li+ + e−), respectively, are observed at 0 V vs. Li/Li+. Although the ZC-LYBO electrolyte is nearly electrochemically stable in the range from 0.0 to 5.0 V vs. Li/Li+, a slight cathodic peak can be observed below ≈1.0 V vs. Li/Li+. Thus, the CVs under 5 cycles in the range from 3.0 to 0.0 V vs. Li/Li+ were also recorded  assembled by the SPS process are shown in V Li/Li+ .3.5Ge0.5V0.5O4 (LGVO) can also be cosintered with layered rock-salt positive electrodes but decompose at low voltage because of the presence of Ge4+ and V5+ ions.27 In fact, other Li3BO3-related families such as Li2.2C0.8B0.2O3  for the Y3+ sites in the Li6Y(BO3)3 (LYBO)-type structure to enhance conductivity. The ionic conductivities of Zr-doped Li5.9Y0.9Zr0.1(BO3)3 and Zr,Ce-doped Li5.875Y0.875Ce0.025Zr0.1(BO3)3 (ZC-LYBO) were respectively 1.4 × 10−5 and 1.7 × 10−5 S cm−1 at 27 °C, which are some of highest conductivities reported for Li3BO3-related electrolyte candidates. The effects of Zr4+-ion doping on the conductivity of the LYBO-type structure were revealed from the structural information estimated from Rietveld analysis, relative density of compacts, and Li+-ion diffusion pathways estimated from BVSE maps, and included: (1) the formation of vacancies at Li sites, (2) the increase of compact densification, and (3) an increase in the Li+-ion conduction pathways in the LYBO phase associated with structural distortion.We substituted tetravalent ions (Zr1/3Mn1/3Co1/3O2 (NMC) positive electrode and a graphite negative electrode. This was due to the thermal stability of the layered rock-salt oxide and the electrochemical stability of the ZC-LYBO at low voltages. The conductivity of the LYBO-type electrolyte could be further enhanced by other aliovalent substitutions for the practical use of sintered ASSBs. The control of the structural distortion associated with the dopant size, which was revealed in this study, could play an important role in this enhancement.Furthermore, sintered ASSBs using ZC-LYBO as an electrolyte successfully performed with both a LiNiThere are no conflicts to declare.RA-011-D1RA02191E-s001


Because of their dual roles and the complex surface chemical reactions, the mechanism involved in Fe2(MoO4)3-catalyzed PDS activation processes remains obscure. In this study, Fe2(MoO4)3 was prepared via the hydrothermal and calcine method, and photoFenton degradation of methyl orange (MO) was used to evaluate the catalytic performance of Fe2(MoO4)3. Fe2(MoO4)3 catalysts with abundant surface oxygen vacancies were used to construct a synergistic system involving a photocatalyst and PDS activation. The oxygen vacancies and Fe2+/Fe3+ shuttle played key roles in the novel pathways for generation of •O2−, h+, and 1O2 in the UV–Vis + PDS + FMO-6 photoFenton system. This study advances the fundamental understanding of the underlying mechanism involved in the transition metal oxide-catalyzed PDS activation processes.The environmentally benign Fe The difion (2)) , oxygen ion (4)) . Fe3+ caand (7)) ,41,42. T3)3·9H2O; 99.9%), p-benzoquinone , L-histidine (99.5%), and ammonium oxalate monohydrate  were purchased from Shanghai Macklin Biochemical Co., Ltd. . Sodium molybdate dihydrate , ammonia solution (NH4OH), sulfuric acid (H2SO4), methanol (MeOH), ethanol (EtOH), tert-butyl alcohol (TBA), and methyl orange (MO) were purchased from Sinopharm Chemical Reagent Co., Ltd. . All reagents and chemicals used in this study were of analytical grade and used without further purification.Ammonium persulfate , iron(III) nitrate nonahydrate (Fe(NO2(MoO4)3 was synthesized by a simple hydrothermal method. Briefly, 2.9034 g of Na2MoO4·2H2O and 3.232 g of Fe(NO3)3·9H2O were weighed according to obtain a ratio of Mo/Fe 3:2. Then, the compounds were dissolved separately in 36 mL of deionized water, mixed well, transferred to a hydrothermal reactor, and heated to 180 °C for 24 h. After cooling to room temperature, the resulting precipitate was washed 3 times with ultrapure water and alcohol and dried at 60 °C for 12 h. Finally, they were calcined in an air atmosphere at 2 °C min−1 and at different temperatures . The catalyst preparation process is shown in Fe2 adsorption–desorption methods of Bruner–Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH) with a Micrometritics (ASAP 2460) apparatus. Additionally, high-resolution X-ray photoelectron spectroscopy (XPS) was carried out with an ESCALAB 250Xi spectrometer with Al Kα excitation at 280.00 eV to detect surface electronic states and element bonding. Thermogravimetric analysis (TGA) was performed on a TG209F3 system with heating from room temperature to 800 °C in air and a N2 atmosphere using a ramp rate of 10 °C min−1. The surface potentials of the samples at different solution pH values were obtained with a Nano-Z Zeta potential tester .X-ray diffraction  was conducted to investigate phase structures with Cu-Kα (λ = 1.540 Å) radiation. The morphologies were examined by field-emission scanning electron microscopy  and further investigated with high-resolution transmission electron microscopy . FT-IR spectroscopy (Nicolet IS5) detected the presence of and changes in chemical bonds. Raman spectroscopy (Horiba LabRAM HR Evolution Raman spectrometer) was carried out with a Via Reflex instrument with an excitation laser providing radiation at 532 nm. Specific surface areas and pore size distributions were determined with the N2SO4 and NH4OH to adjust the initial pH. Predetermined amounts of Fe2(MoO4)3 catalyst were added to the beakers, and they were left in the dark for 1 h to reach adsorption equilibrium. After this time, reactions were initiated by adding an appropriate amount of PDS. Aliquots of 3 mL were taken at predetermined time intervals, and an equal volume of MeOH was immediately added to stop the reaction. The mixed solution was filtered through a 0.22 μm membrane filter. Finally, the samples were transferred to a cuvette, and MO concentrations were analysed by ultraviolet–visible (UV–vis) spectrophotometry.In the present work, all degradation experiments were carried out at room temperature (25 ± 2 °C). Batch experiments were carried out in a series of 100 mL beakers containing different concentrations of MO solution using a 1000 W xenon lamp as the light source and 0.05 M H2(MoO4)3, which has abundant surface oxygen vacancies, was used as a catalyst to construct a photoFenton system for the degradation of MO. Notably, a new pathway for free radical degradation was generated. This may be attributed to the efficient Fe3+/Fe2+ redox cycle and its simultaneous use as an active site for photocatalysis and PDS activation in the synergistic system. The present work therefore provides new ideas for interpretation of catalytic processes using transition metal oxides.In summary, Fe


Sarcophyton convolutum (Alcyoniidae) a Red Sea–derived soft coral has been shown to be a rich source of macrocyclic diterpenes and cyclized derivatives. Two previously undescribed polyoxygenated cembrane-type diterpenoids, sarcoconvolutums F (1) and G (2), as well as four identified analogues (3–6) together with a furan derivate (7) were isolated from a solvent extract. Compounds were identified by spectroscopic techniques, including NMR, HREIMS, and CD, together with close spectral comparisons of previously published data. Sarcoconvolutum F (1) contains a rare 1-peroxid-15-hydroxy-10-ene functionality. Isolated metabolites (1–7) were screened against lung adenocarcinoma (A549), cervical cancer (HeLa) and oral cavity carcinoma (HSC-2) lines. Compound 4 exhibited an IC50 56 µM and 55 µM against A549 and HSC-2 cells, respectively.Natural products and chemical analogues are widely used in drug discovery, notably in cancer and infectious disease pharmacotherapy.  Sarcophyton, in the order Alcyonacea includes 35 species, six of which were recently recognized as new + ion at m/z 400.2094 , the chemical formula was estimated to be C20H32O8, reflecting a five degrees of unsaturation. Two double bonds and one carbonyl from NMR data are responsible for three of the five elements of unsaturation, giving rise in a bicyclic molecule. An absorption for a hydroxyl (υmax 3450 cm−1), a carbonyl (υmax 1750 cm−1) and an olefin (υmax 1669 cm−1) were confirmed by IR analysis. The 1H NMR spectrum ; and δH 3.64 . Three olefinic protons at δH 5.64 , 5.61  and 5.22 ; four signals at δH 1.80 s, 1.69 s, 1.21 s, and 1.16 s were identified as methyls. Twenty carbon signals were observed in the 13C NMR spectrum , five methylenes , five methines , and six quaternary carbons . The cembrene-based diterpenoid was generated using the 1D NMR analysis described above of 1 + .Colourless oil; ®) were grown as monolayers in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 4 mM l-glutamine, 100 U/mL penicillin, and 100 g/mL streptomycin sulphate Monolayers at 70–90 percent confluence were passed using a trypsin-EDTA solution. In a humidified CO2 incubator with 5% CO2, cell were held at 37 °C. All materials and reagents for the cell cultures were purchased from Lonza .Squamous cell carcinoma of the oral cavity (HSC-2), non-small cell lung adenocarcinoma (A549), and human cervical cancer cell (HeLa) (ATCC1–5) dissolved in dimethyl sulfoxide (DMSO) were screened at concentrations of 100, 10 and 1 µM after a 24 h incubation period at 37 °C with 5 percent CO2, followed by varying concentrations for the generation of concentration-dependent curves of the most cytotoxic compounds with culture medium . MTT solution in PBS (5 mg/mL) was added to each well after 48 h of incubation, and the incubation was continued for another 90 min. A phase contrast microscopic analysis verified the production of intracellular formazan crystals . The medium was withdrawn at the conclusion of the incubation time, and 100 µL of DMSO were added to each well with shacking for 10 min to dissolve the produced formazan crystals (200 rpm). The absorbance at 492 nm (OD) of dissolved crystals was measured on a microplate reader  and utilized as a marker of cell proliferation.A modified MTT  test based on a previously published technique  was used50 values were obtained using GraphPad Prism® v6.0 software and the concentration-response curve fit to the non-linear regression model .ICS. convolutum, the biological activity of key Sarcophyton metabolites has drawn extensive chemical analyses of the genus. Herein a solvent extract of the soft coral afforded seven cembrane-type diterpenoids, including sarcoconvolutum F and G , previously undescribed. Compound 4 exhibited modest cytotoxic activity with an IC50 of 56 μM and 55 µM against lung adenocarcinoma (A549) and oral cavity carcinoma (HSC-2) lines, respectively. Although there are few chemical studies that focus specifically on the species


Melanoma is the deadliest form of skin cancer, with its incidence increasing significantly over the past few decades. The hypoxic microenvironment of melanoma gives rise to the expression of hypoxia-inducible factor-1 (HIF-1), which mediates various signaling pathways that lead to melanoma progression. In this study, we aim to identify an epigenetic regulator of HIF-1α that could be targeted for therapeutic interventions in melanoma. Here, we demonstrate that histone deacetylase 8 (HDAC8) deacetylates HIF-1α and enhances its stability. Accordingly, the pharmacological inhibition or the genetic ablation of HDAC8 suppresses the transcriptional activity of HIF-1 and demonstrates anti-cancer effects. Therefore, we suggest HDAC8 as a novel regulator of HIF-1α and a therapeutic target for melanoma treatment.HDAC8 is correlated with HIF1A expression and poor prognosis in samples from patients with melanoma. These findings uncover a novel epigenetic mechanism that maintains HIF-1α stability and implicates the potential of HDAC8 inhibitors for melanoma therapy.Melanoma is the most lethal type of skin cancer, and it causes more than 55,000 deaths annually. Although regional melanoma can be surgically removed, once melanoma metastasizes to other regions of the body, the survival rate drops dramatically. The current treatment options are chemotherapy, immunotherapy, and targeted therapy. However, the low response rate and the development of resistance necessitate the search for a novel therapeutic target in melanoma. Hypoxia-inducible factor-1 α (HIF-1α) is overexpressed in melanoma and plays a crucial role in driving malignant transformation in cancer cells. Here, we identified that histone deacetylase 8 (HDAC8) enhances the protein stability of HIF-1α. HDAC8 directly binds to and deacetylates HIF-1α, thereby promoting its protein stability. This, in turn, upregulates the transcriptional activity of HIF-1α and promotes the expressions of its target genes, such as hexokinase 2 (HK2) and glucose transporter 1 (GLUT1). The inhibition of HDAC8 suppresses the proliferation and metastasis of melanoma cells. Furthermore,  Melanoma develops from melanocytes, which are highly differentiated cells that produce melanin, a macromolecule that protects against UV-induced damage . Melanom2 leads to increased levels of reactive oxygen species (ROS) that cause irreversible cellular damage [2 availability. HIF-1 is a heterodimeric transcriptional activator that consists of a constitutively expressed β subunit and an α subunit. In normoxic conditions, the α subunit undergoes rapid proteasomal degradation. However, in hypoxic conditions, the hydroxylation of the prolyl residues in the α subunit is inhibited, allowing HIF-1α to escape proteasomal degradation and accumulate. This allows HIF-1α to promote the transcription of target genes by associating with the hypoxia-response element (HRE) [Maintaining oxygen homeostasis is crucial for cell survival . The depr damage ,8. Hypoxnt (HRE) . Intratunt (HRE) . Consistnt (HRE) , and it Epigenetics refers to the study of heritable changes in a chromosome that do not result from altering the DNA sequence . The epiHDAC8 is the latest member of class I HDAC. Various studies have demonstrated the pathological role of HDAC8 in diseases, including cancer. The aberrant overexpression of HDAC8 has been observed in various cancers, such as gastric cancer , hepatocBecause HIF-1α is upregulated in melanoma and contributes to the malignant transformation of melanocytes , we sougHDAC inhibitors  were sol2 at 37 °C in humidified incubators and subcultured every 3–4 days. Hypoxia was induced using cobalt chloride (CoCl2)  or by placing the cells in a hypoxia induction chamber  loaded with mixed gas containing 1% O2, 5% CO2, and 94% N2.The human melanoma cell line and A2058, SK-MEL-2, and SK-MEL-28 were purchased from the American Type Culture Collection (ATCC). The melanoma cells were cultured in Gibco Minimum Essential Media . HEK293T cells were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium . Both media were supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cell lines were maintained in 5% CO5 HEK293T cells were seeded and transfected with 1.4 µg of the gRNA lentiviral vector, 1.2 µg of the packaging vector psPAX2, and 0.4 µg of the envelope vector pMD2.G. To mediate the transfection, 7.5 µL of polyethyleneimine  was added. Then, 24 h after the transfection, the supernatant was collected and transduced into A2058 cells with 4 µg/mL of polybrene . The KO cells were selected by treating the cells with 2 µg/mL of puromycin . To generate the HDAC8 rescue cell lines, the A2058 cells were first transfected with gHDAC8 to generate HDAC8 KO cell lines. Following puromycin selection, the HDAC8 KO cells were transfected with either wild-type (WT) HDAC8 or catalytically dead Y306F-mutant (MT) HDAC8. The gRNAs used are listed in HDAC8 KO stable cells were generated using gRNAs targeting HDAC8. The gRNAs were cloned into the LCV2 lentiviral vector. The lentivirus was made with HEK293T cells. On a 6-well plate, 5 × 103 cells per well in 96-well plates containing 130 μL medium. After overnight incubation, the cells were treated with 0.1% DMSO or PCI-34051 at various concentrations  for 24 h, 48 h, and 72 h. The A2058 HDAC8 KO and overexpression (OE) cells were seeded at 3 × 103 cells per well in 96-well plates containing 130 μL medium and incubated for 24 h, 48 h, and 72 h. Hypoxia was induced using 100 µM CoCl2 or by placing the cells in a hypoxia induction chamber. Then, 13 μL of the CCK-8 reagent was added to each well for 3 h. Absorbance was measured at 450 nm using a multimode microplate reader . The results were quantified relative to the control, and they are presented as percentages. The half-maximal cell viability inhibition concentration (IC50) and half-maximal growth inhibition concentration (GI50) were determined using GraphPad Prism ver. 7.0 software .Cell growth and viability were determined using a CCK-8 assay . SK-MEL-2, SK-MEL-28, and A2058 cells were seeded at 3 × 103 cells per well in a six-well plate. After overnight incubation, the cells were treated with 0.1% DMSO or 20 µM PCI-34051. The A2058 HDAC8 KO and OE cells were seeded at 1 × 103 cells per well in a six-well plate. Hypoxia was induced using 20 µM CoCl2 or by placing the cells in a hypoxia induction chamber. The cells were incubated for another 12 days, and colonies were stained with 1 mL of 0.05% crystal violet solution  for 5 min.A2058 cells were seeded at 1 × 105 cells per well in a six-well plate. After overnight incubation, the cells were transfected with hypoxic response element (HRE)-Luc reporter gene with polyethyleneimine. Hypoxia was induced using 100 µM CoCl2. After 24 h, luciferase activities were measured using the Dual Luciferase Assay System  and a multimode microplate reader.A2058 cells were seeded at 3 × 106 cells per plate in a six-well plate. After overnight incubation, a linear scratch was made using a 200 μL pipette tip. Cell debris was removed by washing the cells with a serum-free medium. Then, the cells were treated with 0.1% DMSO or 20 µM PCI-34051 for 24 h. Hypoxia was induced using 100 µM CoCl2. A2058 HDAC8 KO and OE cells were seeded at 1 × 106 cells per well in a six-well plate. After overnight incubation, a linear wound midline was made using a 200 μL pipette tip. Hypoxia was induced using 100 µM CoCl2. Cell debris was removed by washing it with a serum-free medium. An inverted microscope  was used to observe the wound closure, and ImageJ software  was used for quantification.A2058 cells were seeded at 1 × 104 cells per well in a 400 μL serum-free medium to the upper chamber of a Transwell insert plate . Then, 500 μL of a medium containing 10% FBS was added to the lower chamber. After overnight incubation, 20 μM PCI-34051 was applied to the lower well. A2058 HDAC8 KO and OE cells were seeded at 3 × 104 cells per well in a 400 μL serum-free medium to the upper chamber, and 500 μL of medium containing 10% FBS was added to the lower chamber of the Transwell insert plate. Hypoxia was induced using 100 µM CoCl2. After 48 h incubation, the migrated cells were fixed and stained using 0.1% crystal violet solution containing 20% methanol. To visualize the stained cells, a light microscope with iSolution Lite  was used. For quantification, the stained cells were solubilized with 10% acetic acid. Absorbance was measured at 560 nm using a multimode microplate reader.A2058 cells were seeded at 3 × 105 cells per well in a six-well plate. After overnight incubation, the cells were treated with 0.1% DMSO or 20 µM PCI-34051 for 24 h. A2058 HDAC8 KO and OE cells were seeded at 5 × 105 cells per well in a six-well plate and incubated for 24 h. Hypoxia was induced using 100 µM CoCl2 for 12 h or by placing the cells in a hypoxia induction chamber. The cells were washed with ice-cold 1× PBS and lysed with 100 µL lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 25 mM glycerol phosphate, 25 mM NaF, 5 mM EGTA, 1 mM EDTA, 1 mM DTT, 1% NP-40, 1 µg/mL leupeptin, 1 µg/mL aprotinin, 1 mM PMSF, 1 mM benzamidine). The protein concentrations of the cell lysates were measured using a Bradford assay. Samples were prepared with a 5× sample buffer and boiled for 5 min. The prepared samples were loaded onto a polyacrylamide gel and subjected to SDS-PAGE. Then, the gel was transferred onto a nitrocellulose membrane. After blocking the membrane with 5% skim milk, it was incubated with primary antibodies at 4 °C overnight. Membranes were washed with 0.1% Tween-20/PBS and incubated with an anti-mouse or anti-rabbit secondary antibody coupled to HRP at room temperature for 3 h. The ECL Western blotting analysis system  was used for detection.A2058 cells were seeded at 5 × 106 cells per plate in a cell culture dish. After overnight incubation, the A2058 cells were treated with 100 µM CoCl2 for 12 h to induce hypoxia. HEK293T cells were seeded at 1 × 106 cells per plate in a cell culture dish. After overnight incubation, the HEK293T cells were transfected with 1 µg of FLAG-HDAC8 and 1 µg of HA-HIF-1α. Polyethylenimine was added to mediate the transfection. The cells were washed with ice-cold 1× PBS and lysed with 100 µL lysis buffer. Then, 1000 µg of cell lysates was incubated with primary antibodies and 20 µL of A/G beads  at 4 °C overnight. The lysate mixtures were washed with lysis buffer (300 mM NaCl), prepared with 40 µL of 5× sample buffer, and boiled for 5 min to be subjected to SDS-PAGE.A2058 cells were seeded at 1 × 106 cells per plate in a 60 mm cell culture plate. After overnight incubation, the cells were treated with 0.1% DMSO or 20 µM PCI-34051 for 24 h. A2058 HDAC8 KO and OE cells were seeded at 1 × 106 cells per plate in a 60 mm cell culture plate. Hypoxia was induced using 100 µM CoCl2 for 6 h or by placing the cells in a hypoxia induction chamber. The total RNA from the cells was extracted using a FavorPrepTM Blood/Cultured Cell Total RNA Mini Kit . One microgram of total RNA was reverse-transcribed into cDNA using an EasyScript™ cDNA synthesis kit . One microliter of cDNA was amplified using 10 µL of TOPreal™ qPCR 2X PreMIX SYBR green reagent . A qPCR analysis was performed using the Applied Biosystems 7500 System . The mRNA values were normalized by GAPDH expression levels. The primers used are listed in A2058 cells were seeded at 1 × 10HDAC8 and HIF1A was analyzed using gene expression profiles from the NCBI Gene Expression Omnibus (GEO) database  database (n = 473) using the website http://ualcan.path.uab.edu/index.html accessed on 1 November 2022 [The association between patient survival and the expressions of  n = 214 ) using tber 2022 .p-values < 0.05.All data are expressed as means ± standard deviation (SD) of more than three independent experiments. Statistical significance was determined using an unpaired two-tailed Student’s t-test or a one-way analysis of variance (ANOVA) with a post hoc analysis under Turkey’s multiple comparison test using GraphPad Prism software 7.0 . Differences were considered statistically significant at 2 levels (2)-induced hypoxic conditions , vascular endothelial growth factor A (VEGFA), and hexokinase 2 (HK2), PCI-34051 treatment significantly inhibited this effect (SLC2A1 and HK2 were downregulated upon the genetic ablation of HDAC8 (SLC2A1) in the PCI-34051 treated and HDAC8 KO cells  showed that high levels of HDAC8 significantly correlated with poor disease-specific survival (DSS) . Patients with melanoma with high levels of HDAC8 were associated with poor DMFS  A. Widespoor DMFS B. Next, melanoma C,D. Moremelanoma E. CollecHDAC8 and HIF1A expressions show a positive correlation and the elevated expression of these genes is associated with poor prognosis. These data indicate HIF-1α as a novel HDAC8 substrate and suggest that HDAC8 may be an effective therapeutic target in melanoma.In this study, we delineated an epigenetic mechanism in which the epigenetic eraser HDAC8 stabilizes HIF-1α expression and promotes melanoma progression. HDAC8 interacts with HIF-1α and catalyzes its deacetylation, thereby promoting HIF-1α protein stability and transcriptional activity. Consequently, the depletion of HDAC8 suppresses cell proliferation and migration in melanoma. In patients with melanoma, Previous studies have identified several epigenetic regulators that interact with HIF-1α to regulate HIF-1α protein stability and transcriptional activity . AlthougThe tumorigenic role of HIF-1 is considerable. Various oncogenic pathways are activated by HIF-1. Not only does HIF-1 exert its effects on tumor cell proliferation, but it also affects apoptosis, angiogenesis, metastasis, immune evasion, and metabolism in cancer . In melaHDAC8 plays a critical role in cancer development. It is involved in the enhancement of cell proliferation and metastasis, as well as in the suppression of apoptosis and evasion from the immune system. In melanoma, HDAC8 mediates the escape from BRAF inhibitor therapy, and HDAC8 and BRAF dual inhibition has been suggested as a potential therapeutic strategy for melanoma treatment . In thisTo conclude, we demonstrated the epigenetic regulation of HIF-1α in melanoma. HIF-1α is upregulated in melanoma and drives tumor progression. Therefore, targeting HIF-1α has been an attractive strategy in cancer treatment. However, various efforts concentrated on developing HIF-1α inhibitors have proven to be difficult. Here, we provide an alternative mechanism that regulates the expression and activity of HIF-1α. HDAC8 interacts with HIF-1α and mediates its deacetylation, enhancing the protein stability and transcriptional activity of HIF-1α. Importantly, targeting HDAC8 showed anti-cancer effects, such as suppressed cell proliferation and migration in melanoma. Therefore, HDAC8-selective inhibitors may function as indirect HIF-1α inhibitors and may be a promising strategy in treating patients with melanoma.


MDL72527 reduced LOX activity at passage 3 and 5 (## p < 0.01) and had no effect on LOX protein, and mRNA expression. At the basal level, SSAO activity also decreased with passage, and its protein expression was maintained between passages. MDL72527 abolished SSAO activity (**** p < 0.0001 for 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 5 (** p < 0.01) and 8 (**** p < 0.0001), and Aoc3 mRNA levels at passage 8 (* p < 0.05). βAPN inhibited SSAO activity (**** p < 0.0001 for 5 vs. 3 and 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 3 (* p < 0.05), and Aoc3 mRNA levels at passage 3 (* p < 0.05). Knockdown of the LOX gene (**** p < 0.0001 for Si6 vs. Sictrl and *** p < 0.001 for Si8 vs. Sictrl) and LOX protein (** p < 0.01 for Si6 and Si8 vs. Sictrl) in VSMCs at passage 3 resulted in a reduction in Aoc3 mRNA (#### p < 0.0001 for Si6 vs. Sictrl and ### p < 0.001 for Si8 vs. Sictrl) and VAP-1 protein (# p < 0.05 for Si8 vs. Sictrl). These novel findings demonstrate a passage dependent decrease in LOX activity and increase in SSAO activity in rat aortic VSMCs and show an association between both enzymes in early passage rat aortic VSMCs, where LOX was identified as a regulator of SSAO activity, protein, and mRNA expression.Vascular smooth muscle cells (VSMCs) are the main stromal cells in the medial layer of the vascular wall. These cells produce the extracellular matrix (ECM) and are involved in many pathological changes in the vascular wall. Semicarbazide-sensitive amine oxidase (SSAO) and lysyl oxidase (LOX) are vascular enzymes associated with the development of atherosclerosis. In the vascular smooth muscle cells, increased SSAO activity elevates reactive oxygen species (ROS) and induces VSMCs death; increased LOX induces chemotaxis through hydrogen peroxide dependent mechanisms; and decreased LOX contributes to endothelial dysfunction. This study investigates the relationship between SSAO and LOX in VSMCs by studying their activity, protein, and mRNA levels during VSMCs passaging and after silencing the LOX gene, while using their respective substrates and inhibitors. At the basal level, LOX activity decreased with passage and its protein expression was maintained between passages. βAPN abolished LOX activity (**  Vascular smooth muscle cells (VSMCs) are the main stromal cells in the medial layer of the vascular wall, and are involved in many physiological functions and pathological changes that take place in the vasculature . These c2O2) and ammonia. In VSMCs, SSAO is important for vascular tone regulation, cell differentiation, and extracellular matrix organization [Semicarbazide-sensitive amine oxidase (SSAO) is a vascular enzyme highly expressed in the VSMCs  and endothelial cells . Being a copper amine oxidase, SSAO is encoded by the amine oxidase copper-containing 3 (Aoc3) gene, and distinguishes itself by its highly rich copper content [nization ,5,6. Incnization .Lysyl oxidase (LOX) is another copper-containing amine oxidase which is abundantly present in the vascular smooth muscle cells, where it contributes to the formation and stability of the extracellular matrix (ECM). LOX oxidizes primary amine substrates, such as the epsilon amino group of lysine or hydroxylysine residues in collagens and lysine residues in elastin, and forms peptidyl α-aminoadipic-β-semialdehydes ,9. TheseThe very close similarity in terms of substrates and inhibitors poses a great challenge when studying SSAO and LOX activity in cell culture. Despite sharing common substrates and inhibitors, LOX is considerably smaller; its primary sequence lacks copper-coordinating histidines, and has lysine tyrosyl quinone (derived from the cross-linking of a modified tyrosine residue to the ε-group of a lysyl side chain) instead of topa-quinone  as a cofactor . Previou® Red Monoamine Oxidase assay, which has been previously implemented for the detection of LOX and SSAO-dependent H2O2 production [2O2 in a horseradish peroxidase coupled reaction, using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red reagent), a highly sensitive and stable probe for H2O2 [Here, we examined the activity, protein expression, and mRNA levels of SSAO and LOX in rat aortic VSMCs from passage 3, 5, and 8, using their respective substrates and inhibitors. SSAO and LOX activity were measured with the fluorometric Amplexoduction ,15,16,17for H2O2 . Benzylafor H2O2 . LOX actfor H2O2 ,21.MDL72527 was applied to irreversibly inhibit SSAO. MDL72527 is a propenyl amine-based inhibitor which was initially designed as polyamine oxidases (PO) inhibitor; however, it has also been identified as a mechanism-based, suicide inhibitor of SSAO ,23,24. BIn this study, we provide evidence for association between SSAO and LOX in rat aortic VSMCs while demonstrating LOX as a main regulator of SSAO activity, VAP-1 protein, and Aoc3 mRNA expression in early passage rat aortic VSMCs.Cell culture reagents were purchased from Fisher Scientific . Unless otherwise stated, chemicals and reagents were purchased from Sigma-Aldrich .2 asphyxiation, and the aorta was removed and placed in a DMEM solution supplemented with 10% Fetal Bovine Serum (FBS (v/v)), 1% penicillin (100 units/mL), streptomycin (100 μg/mL), and 2 mM L-Glutamine.Male Wistar rats (180–220 g) were housed in pairs in standard cages (Tecniplast 2000 P) with sawdust (Dates and grade 7 substrate) and shredded paper wool bedding with water and food (5LF2 10% protein LabDiet) in the Biological Services Unit at the University of Hertfordshire. The housing environment was maintained at a constant temperature (21 ± 2 °C) and light–dark cycle (12:12 h). All experiments were carried out in accordance with the University of Hertfordshire animal welfare ethical guidelines and European directive 2010/63/EU, and all tissues collected were naïve shared within teaching/research in accordance with the 3 Rs. Rats were euthanized by COThe VSMCs were selected due to the fact that they expressed high levels of both SSAO and LOX. This study used primary VSMCs, because primary cell cultures most closely represent the tissue of origin. Primary cells require several passages to fully develop receptors and maintain a consistent and fast proliferation rate ; however2 asphyxiation followed by cervical dislocation. The aortas were removed and placed in a DMEM solution supplemented with 10% Fetal Bovine Serum (FBS (v/v)), 1% penicillin (100 units/mL), streptomycin (100 μg/mL), and 2 mM L-Glutamine. In order to characterize the cells, the isolated rat VSMCs were stained for the smooth muscle cell marker SM22α [VSMCs were isolated from the rats’ aortas, as per standard protocol . The rater SM22α . Please 2O2 production) were studied with the Amplex® Red assay [® Red (20 mM), 10 μL horseradish peroxidase (200 U/mL), 10 μL clorgyline (0.5 mM), and 200 μL (0.5 mM) benzylamine, or 1,5 diamino pentane dihydrochloride (cadaverine), respectively. Clorgyline (MAO-A and MAO-B selective inhibitor) was added in the reaction mixture because benzylamine is a common substrate for not only SSAO and LOX, but also MAO-A and MAO-B. SSAO activity (expressed as H2O2 production) was also studied in the presence of MDL72527 and βAPN. Cells were treated with a reaction mixture containing 860 μL of 0.25 M sodium phosphate buffer (pH 7.4), 20 μL Amplex® Red (20 mM), 10 μL horseradish peroxidase (200 U/mL), 10 μL clorgyline (0.5 mM), and 200 μL (0.5 mM) benzylamine, or 200 μL (0.5 mM) benzylamine and 10 μL (100 µM) MDL72527—irreversible SSAO inhibitor, or 200 μL (0.5 mM) benzylamine and 10 μL (200 µM) βAPN—irreversible LOX inhibitor.SSAO and LOX activity , 10 μL horseradish peroxidase (200 U/mL), 10 μL clorgyline (0.5 mM), and 200 μL (0.5 mM) 1,5 diamino pentane dihydrochloride (cadaverine), or 200 μL (0.5 mM) cadaverine and 10 μL (100 µM) MDL72527, or 200 μL (0.5 mM) cadaverine and 10 μL (200 µM) βAPN. Cells containing reaction mixture without benzylamine or cadaverine were considered as controls. SSAO and LOX activity were measured after 6 h incubation with a reaction mixture at 37 °C, at excitation 540 nm and emission 590 nm, on a Clario Star® Microplate Reader . The end-point fluorescence was measured with resorufin standards after 1 h incubation at 37 °C. Then, 2 mM of resorufin stock solution was diluted to a concentration of 1000 μM in a 1X reaction buffer (2 mL of 5X reaction buffer (0.25 M sodium phosphate at pH 7.4), 10 mL distilled water) to yield resorufin standards ranging from 0 to 20 μM. The resorufin readings were used to prepare a standard curve of resorufin fluorescence (RFU) versus concentration (μM). In order to express SSAO/LOX activity in nmol H2O2/mL, the fluorescence readings from different time intervals were multiplied by the slope and added by the intercept . In order to express SSAO/LOX activity in nmol H2O2/mg protein, the nmol H2O2/mL values were divided over the protein concentration (mg/mL), which was calculated with the BCA assay.LOX activity was assessed with cadaverine in the presence of MDL72527 and βAPN, respectively. Cells were treated with a reaction mixture containing 860 μL of 0.25 M sodium phosphate buffer (pH 7.4), 20 μL Amplex® conjugated VAP-1 antibody  at dilution 1:1000 in 5% w/v BSA, 1X TBS, 10% Tween®20, or rabbit monoclonal to Pro-LOX antibody , ab174316) at dilution 1:1000 in 5% w/v BSA, 1X TBS, 10% Tween®20, overnight at 4 °C. After washing, blots were incubated in horseradish peroxidase (HRP) conjugated secondary antibodies  for 1 h at room temperature, apart from blots incubated with VAP-1. A β-actin-HRP-conjugated antibody  was used as a loading control, with blots incubated for 1 h at room temperature. A peroxidase detection system  and myECL Imager  were used for the visualization of immunoreactivity. The protein bands were quantified with Image J software Protein was extracted from VSMCs cultured in control condition (non-treated), and after 1 h pre-treatment with 200 µM βAPN  based on a relative quantification method, also known as comparative threshold method. The QPCRBIO SyGreen 1-Step Go Kit  was used to perform the PCR reaction. The primers for VAP-1, LOX, and β-actin  were desA master mix  was prepared for all reference (non-treated) and target (MDL72527 and βAPN treated) samples . β-actinThe master mix per well for each gene was prepared with: 1X qPCRBIO SyGreen 1-Step Mix (5 μL), forward primer at concentration 10 μM (0.4 μL), reverse primer at concentration 10 μM (0.4 μL), 1X RTaseGo—which contains RNase inhibitor (0.5 μL), 20 ng/μL template RNA (1 μL), and 2.7 μL qPCR grade water. Samples prepared without 1X RTaseGo were used as a negative control. After loading, the plate was centrifuged at 1000 RPM for 1 min, and readings were taken on the QuantStudio™ 7 Flex Real-Time PCR System (Thermo Fisher Scientific). The data were analyzed with the 2-ΔΔCT method of relative quantification .Associations between LOX and SSAO in early passage rat aortic VSMCs were studied with siRNA gene knockdown using reverse transfection and lipofectamine—RNAiMAx transfection reagent . In ordeMOCK was included to observe any phenotypic changes to the cells caused by the transfection. Two siRNAs that target LOX gene, SiRNA_LOX_6 and SiRNA_LOX_8, were selected based on their target sequences .5 cells/mL. Then, 6.650 mL of cells were added to each T-75 flask, followed by the addition of the reaction mixture previously prepared, thus obtaining a final siRNA concentration of 20 nM and 2 × 106 cells/flask. Cells were distributed evenly through the flask by swirling in a figure of eight, and the flasks were incubated at 37 °C/5% CO2. The following day, 6 mL of antibiotic-free DMEM (supplemented with 10% FBS) was added to each flask. When 72 h had passed post-transfection, the cells were harvested by trypsinization and re-suspended in two falcon tubes with 2 mL antibiotic-free DMEM (supplemented with 10% FBS) at a concentration of 5 × 104 cells/mL. One falcon tube with cells was used for extraction of protein samples, and the other for the extraction of RNA. Separate T-75 flasks were used for transfection with individual siRNAs, Si control, and HS death. Four reaction mixtures were prepared with individual siRNAs, Si control, and HS death. In doing this, 8 μL RNAiMAX was added to 8 μL SiRNA_LOX_6 or SiRNA_LOX_8 at a concentration of 20 μM, and then supplemented with 1350 μL OptiMEM. In addition, 8 μL RNAiMAX was also added to 8 μL Si control (20 μM) or 16 μL HS cell death (10 μM), and then supplemented with 1350 μL OptiMEM. The reaction mixtures were incubated for 20 min at room temperature, during which early passage (2 and 3), fully confluent rat aortic VSMCs were harvested and resuspended in antibiotic-free DMEM (supplemented with 10% FBS) at a concentration of 3 × 10In order to extract protein samples, cells were first centrifuged at 2000 RCF for 6 min at 4 °C. The supernatant was removed, and cells were washed with 1X PBS and centrifuged again at 2000 RCF for 6 min at 4 °C. After the removal of the supernatant, the cells were re-suspended with 1mL RIPA buffer , then supplemented with a 10% protease and phosphatase inhibitor cocktail. The samples were then moved to a centrifuge tube, vortexed for 30 s, sonicated in icy water for 5 min, and stored at −20 °C until further use.In order to extract RNA, cells were centrifuged at 300 RCF for 5 min and re-suspended in 1× PBS, after which they were centrifuged again at 300 RCF for 5 min. The supernatant was then removed, and cells were re-suspended in 250 μL lysis buffer . The lysate was mixed well by repeatedly pipetting 7–10 times, and was then transferred to a microcentrifuge tube. Afterward, 85 μL isopropanol (100%) was added to each centrifuge tube and the tubes were mixed by vortexing for 5 s.g for 30 s at 22 °C. After centrifugation, the liquid was discarded and the minicolumn was placed in a different collection tube. A total of 500 μL of RNA wash solution (previously prepared by adding 60 mL of 95% ethanol to a 35 mL concentrated RNA wash solution—RWA) was added to the minicolumn, and the collection tube containing the minicolumn was centrifuged again at 13,000× g for 30 s. After that, 30 μL of DNase I incubation mix  was added to the minicolumn membrane, and the minicolumn was incubated for 15 min at 22 °C. After the incubation, 200 μL of column wash solution (prepared by adding 7.5 mL of 95% ethanol to a 5 mL concentrated column wash solution—CWE) was added to the minicolumn, and the minicolumn was centrifuged at 13,000× g for 15 s; after that, 500 μL RNA wash solution was added again, and the minicolumn was centrifuged at 13,000× g for 30 s. The wash solutions and the collection tube were then discarded. The minicolumn was placed to a new collection tube. Then, 300 μL of RNA wash solution was added again followed by centrifugation at 20,000× g for 2 min. The minicolumn was then transferred from the collection tube to an elution tube. Then, 30 μL of nuclease-free water was added to the minicolumn, and the elution tube containing the minicolumn was centrifuged at 13,000× g for 1 min. The purified RNA was quantified with a NanoDrop Spectrophotometer  and stored at −80 °C until further use.The lysate from each centrifuge tube was then transferred to a ReliaPrep™ minicolumn, placed in a collection tube, and centrifuged at 13,000× The data was analyzed with the statistical software GraphPad Prism 7 . Statistical comparisons were made using one-way or two-way ANOVA, followed by Dunnett’s or Tukey’s multiple comparison tests. Sidak’s multiple comparison test was applied for a set of means, to adjust significance level for multiple comparisons. Probability values < 0.05 were considered to be statistically significant.2O2/h/mg protein) during VSMCs passaging, SSAO activity was measured at the basal level using benzylamine as substrate (0.5 mM), and in the presence of SSAO and LOX respective irreversible inhibitors, MDL72527 and βAPN. In order to investigate basal and enzyme-induced changes in SSAO activity (nmol H2O2/h/mg protein) during VSMCs passaging, LOX activity was measured at the basal level using cadaverine as a substrate (0.5 mM), and in the presence of SSAO and LOX respective irreversible inhibitors, MDL72527 and βAPN. In order to investigate basal and enzyme-induced changes in LOX activity , or after 1 h treatment with 200 µM βAPN (B). β-actin from each cell sample was detected to normalize the level of protein. The protein was quantified using ImageJ software. In order to investigate basal and enzyme-induced changes in VAP-1 and LOX protein expression during VSMCs passaging, cell samples from passages 3, 5, and 8 were lysed without being previously treated (A), or after 1 h treatment with 100 µM MDL72527 (B). β-actin from each cell sample was detected to normalize the level of protein. The protein was quantified using ImageJ software. In order to investigate basal and enzyme-induced changes in Aoc3 and LOX mRNA expression, the RNA was extracted from non-treated and MDL72527 or βAPN treated VSMCs from passages 3, 5, and 8 before performing the quantitative polymerase chain reaction (qPCR). In order to confirm association between SSAO and LOX and to ascertain whether the observed inhibitory effect of βAPN over SSAO activity, protein, and mRNA expression is through direct mechanism, or a consequence of irreversibly inhibited LOX, gene knockdown was performed using two small interfering RNAs (siRNA6 and siRNA8). 2O2 production), VAP-1 expression, and Aoc3 mRNA after βAPN treatment, and was later confirmed by the observed reduction in VAP-1 and Aoc3 mRNA levels after silencing the LOX gene.In order to identify a connection between SSAO and LOX in rat aortic VSMCs, the activity of both enzymes, as well as their protein and mRNA levels, were assessed at the basal level and in the presence of their respective suicide inhibitors MDL72527 and βAPN. In our study, we demonstrated that active LOX regulates SSAO activity, VAP-1 protein, and Aoc3 mRNA expression in early passage rat aortic VSMCs. This effect was first observed from the reduced SSAO activity   A,B. The ssage 8) B but alsssage 8) A–C and Assage 8) A. This iFurthermore, in our study, βAPN demonstrated inhibitory potential over LOX’s activity A; howeveIn our study, we observed suppressed SSAO activity  A, VAP-1Furthermore, we showed a LOX-driven reduction in SSAO protein and mRNA expression . This inOur data show predominant LOX activity in early passage and predominant SSAO activity in late passage rat aortic VSMCs. It also demonstrates association between these two enzymes in early passage rat aortic VSMCs, where LOX was identified as a regulator of SSAO activity, VAP-1 protein, and Aoc3 mRNA expression. Additional studies are needed to further investigate the relationship between LOX and SSAO in rat aortic VSMCs, and to demonstrate how this might contribute to the development of atherosclerosis.