Angiotensin II (AngII) causes vascular hypertrophic remodeling which is associated with H2O2 generation. The aim of this study is to investigate the role of cytosolic SOD (SOD1) in AngII-induced vascular hypertrophy. We employed C57/BL6 mice (WT) and SOD1 deficient mice (SOD1−/−) with the same background. They received a continuous infusion of saline or AngII (3.2 mg/kg/day) for seven days. The blood pressures were equally elevated at 1.5 times with AngII, however, vascular hypertrophy was blunted in SOD1−/− mice compared to WT mice (WT mice 91.9 ± 1.13 µm versus SOD1−/− mice 68.4 ± 1.41 µm p < 0.001). The elevation of aortic interleukin 6 (IL-6) and phosphorylation of pro-inflammatory STAT3 due to AngII were also blunted in SOD1−/− mice’s aortas. In cultured rat vascular smooth muscle cells (VSMCs), reducing expression of SOD1 with siRNA decreased AngII induced IL-6 release as well as phosphorylation of STAT3. Pre-incubation with polyethylene glycol (PEG)-catalase also attenuated phosphorylation of STAT3 due to AngII. These results indicate that SOD1 in VSMCs plays a role in vascular hypertrophy due to increased inflammation caused by AngII, probably via the production of cytosolic H2O2.Superoxide dismutase (SOD) is an enzyme that catalyzes the dismutation of two superoxide anions (O In our bodies, the reactive oxygen species (ROSs) are constantly produced, and they are high reactivity which potentially damages the biological systems. However, an appropriate amount of ROSs is necessary for biological processes including cell signaling ,2 and de2·−) or hydrogen peroxide (H2O2) levels in the vascular smooth muscle cell (VSMC) [2·− moves through anion chloride channel (CLC-3) [2O2 diffuses through aquaporin channels, which changes local oxidation state.Angiotensin II (AngII) is the established pathophysiology of hypertension and vascular hypertrophy. Part of this pathophysiology is caused by ROSs produced by NADPH oxidase (NOX) ,5. NOX il (VSMC)  immediatl (VSMC) ,10. Somel (VSMC)  and the  (CLC-3)  and H2O22·− is slowly dismutated to H2O2 spontaneously (rate constant = 8 × 104/mol/s). In the presence of nitric oxide (NO−), O2− quickly reacts with NO− to form nitrogen peroxide acid (OONO−) (rate constant = 4–16 × 109/mol/s). NO· functions as a vasodilator and vascular protector, however, OONO− does not induce vasodilation and further causes organ damage because of its strong reactivity. Superoxide dismutase (SOD) is one of the antioxidant systems and could rapidly catalyze two O2·− into H2O2 and O2 (rate constant = 2 × 109/mol/s). The resulting H2O2 serves as a substrate for catalases, which ultimately break it down into harmless H2O and O2 [2·− by SOD protects from producing OONO− and is likely to relax vascular tension which is related to blood pressure [2O2 produced by SOD plays a role in signal pathways activated with AngII, which leads to protein and DNA synthesis in VSMCs in vitro [2O2 causes aortic VSMC hypertrophy [2·− and H2O2 local concentration. In other words, SOD helps to relax vascular tension and protect from vascular damage by eliminating O2·−; however, the H2O2 produced by SOD would therefore be an important mediator for AngII-induced vascular hypertrophy. SOD may have both positive and negative effects on vascular pathophysiology.OO and O2 . Therefopressure  and protin vitro ,16 and eertrophy ,18. AngISOD is a metal-containing enzyme, and the catalytic reaction is performed by cyclic oxidation and reduction of metal ions. There are three main types of SOD: copper/zinc-containing SOD (CuZnSOD), iron-containing SOD (FeSOD), and manganese-containing SOD (MnSOD). SOD genes have been relatively preserved during evolution , but the2, and now FeSOD is absent in animals and fungi, and only present in chloroplasts in plants. Instead, MnSOD was used during evolution, and FeSOD and MnSOD have similar structures [The SOD of primitive organisms was FeSOD, but it is thought to have fallen out of use with the rise in atmospheric Oructures . In plan2O2 in AngII-induced aortic hypertrophy has not yet been completely elucidated in vivo.Because of the cytosolic location, SOD1 could be important to cell biology. We aimed to investigate the effects of SOD1 in vascular pathophysiology, which changed the local oxidate state in vivo. To date, some studies reported the role of SOD1 in blood pressure and vascular pathophysiology, however, the result was inconsistent and controversial ,22,23,24−/−) and compared them with wild-type mice (WT). Without any intervention, there was no difference in blood pressure and aortic pathology. By infusing a relatively high dose of AngII (3.2 mg/kg/day) for 1 week, although the response to AngII-induced hypertension was comparable, the aortic hypertrophy was blunted in SOD1−/− mice compared with WT mice.In the present in vivo study, we hypothesized that SOD1 exerts its effects on vascular pathology by altering the local oxidative state in vivo and aimed to investigate its effects. We employed SOD1 deficient mice . These mice were backcrossed to C57BL/6J strain mice for 8 generations as we did previously [+/− mice to obtain wild-type (WT) mice and homozygous SOD1−/− mice within the same litter. Animals were maintained in a temperature-controlled facility on a 12 h light/dark cycle (lights on from 7:00 a.m. to 7:00 p.m.) and fed freely with a normal chow diet  and water. The genotype of each mouse was checked by polymerase chain reaction on DNA isolated from a tail or ear biopsy samples using the following primers:WT 5′-TGAACCAGTTGTGTTGTCAGG-3′WT 5′- TCCATCACTGGTCACTAGCC-3′Mutant 5′- TGTTCTCCTCTTCCTCATCTCC-3′Mutant 5′- ACCCTTTCCAAATCCTCAGC-3′Male SOD1eviously . Next, wAll experimental protocols were approved by National Defense Medical College Board for Studies in Experimental Animals .n = 22~26 per group). The delivery rate of AngII was 3.2 mg/kg/day for seven days as in the study of Wang HD et al. [We implanted 12- to 16-week-old male mice with osmotic mini-pumps  containing AngII  or saline . All surgical procedures in this experiment were performed by aseptic manipulation, and disinfectant solution  was used. Rats were sedated in an anesthesia box filled with 5% sevoflurane. After sufficiently deep anesthesia was obtained, the rats were sacrificed to death by cervical dislocation. Saline reflux was performed from the heart, and the thoracic aorta was removed. After cleaning up the fat tissue around the aorta, the aorta was cut longitudinally and opened. Then the media was peeled off from the adventitia. The isolated media was cut into small pieces and incubated with 0.75 mg/mL collagenase and 0.125 mg/mL elastase for 30 min to 60 min by pipetting to disperse the cells. The collected cells were grown in 1.0 g/L glucose Dulbecco’s modified Eagle’s medium  with 10% FBS and penicillin/streptomycin. To decrease expression of SOD1, we incubated VSMCs with SOD1 siRNA  Invitrogen) and Lipofectamine RNAiMAX (Invitrogen) for 72 h, and after changing the medium to 0.1% FBS and leaving for 24–48 h, performed experiments with or without AngII or Interleukin-6 (IL-6). To inhibit SOD1 activity pharmacologically, we used DETC (diethyldithiocarbamate) which was a copper-chelating compound. 0.1 mM DETC was added to VSMC culture fluid 4 h before the addition of AngII. Catalase was administered 24 h before AngII administration. We used polyethylene glycol-conjugated catalase  . It is expected to be highly stable while maintaining good reactivity by binding polyethylene glycol to catalase .v/v) mixture was added to an aliquot (0.6 mL) of hemolysate. After centrifugation for 10 min at 1600× g, the supernatant was collected for SOD assay. This kit uses the nitro blue tetrazolium (NBT) method to measure SOD activity. O2·− produced by the addition of xanthine oxidase (XO) reduces NBT to form nitroblue tetrazolium diformazan, which shows absorbance at 560 nm and the absorbance was measured by the plate reader . In the presence of SOD, some of O2·− is dismutated to H2O2 and O2, inhibiting the formation of diformazan. The SOD activity in a sample is measured by its inhibition rate as shown in Equation (1) [SOD activities in red blood cells were determined using SOD Assay Kit  following the kit’s instructions. The erythrocytes were washed three times with 0.9% NaCl. The washed erythrocytes (0.1 mL) were lysed using distilled water (0.5 mL) and mixed. To remove the hemoglobin, 1.2 mL of an ethanol/chloroform (1:1 tion (1) .SOD actBL:Abs Absorbance of the blind test (water)BLBL:Abs Absorbance of reagent blind test (water without XO)S:Abs Absorbance of sample testSBL:Abs Absorbance of sample blind test (sample without XO)Systolic blood pressure was measured by the tail-cuff method  without anesthesia between 6:00 a.m. and 8:00 a.m., following the instructions as previous reports ,26. To rThe mice were deeply anesthetized and perfused with 0.9% saline from the left ventricular apex, followed by perfusion fixation with 4% paraformaldehyde. Aortas were fixed in 10% formalin for 48 h. The 2 mm thoracic aorta rings were cut off at 4 mm from the aortic arch and embedded in paraffin. These were sectioned (4-µm thickness) and stained with hematoxylin and eosin (HE). For immunohistochemistry, SOD1 was visualized with anti-human SOD1 polyclonal antibodies  using a Vectastain Elite ABC kit  with diamino-benzidine as the substrate, following the instructions.We cut out the aorta ring 5 mm from the aortic arch and measured the thoracic aorta medial area using a HE Stain; Elastica van Gieson (EVG) stain, and Masson’s trichrome (Masson’s) stained cross-section slides. The media was defined as the area between the internal and external elastic lamina. For each mouse, the average of ten consecutive cross-sections was measured and quantified using the NIH Image software .2O2 indicator. The serum d-ROMs test detects hydro-peroxide (R–OOH) and measurements are expressed as Carratelli units (CARR U), with 1 CARR U corresponding to 0.08 mg/dL H2O2. Oxidized glutathione (GSSG) levels and reduced glutathione (GSH) levels were measured in red blood cells as an intracellular H2O2 indicator using GSH/GSSG Kit , following the instructions.We measured the serum derivatives of reactive oxygen metabolites (d-ROMs) using FREE carpe diem  as an extracellular H® detector  after TCA extraction [Superoxide productions in aortic tissue were visualized by dihydroxyethidine (DHE) staining of aortic frozen samples. Vascular GSH/GSSG ratios were assessed with CoulArraytraction .2EDTA 1 mmol/L, EGTA 1 mmol/L, 1% NP-40, sodium pyrophosphate 2.5 mmol/L, beta-glycerophosphate 1 mmol/L, Na2VO4 1 mmol/L) supplemented with PMSF 1 mmol/L and protease inhibitor cocktail . VSMCs were dissolved in homogenization buffer and collected with a scraper. After brief sonication , these samples were centrifuged at 10,000× g for 10 min at 4 °C. Protein concentrations were assessed by the Bradford assay with BSA as a standard. Aortic and VSMCs lysates were resolved by SDS-PAGE and transferred to PVDF membranes . The membranes were incubated with the following antibodies overnight at 1:1000 dilution: SOD1 , SOD2 , Phospho-STAT3 , STAT3 , Phospho-ERK1/2 , ERK1/2 , Phospho-Akt , Akt  and GAPDH . They were then incubated with peroxidase-conjugated anti-mouse or anti-rabbit antibodies  overnight. The specific protein amount was detected after immersing in Enhanced Chemiluminescence (ECL)  and light emission was detected and captured by a LAS-3000 Imager .Aortas were homogenized with a glass-glass homogenizer on ice and dissolved in homogenization buffer . cDNA was synthesized using SuperScript III reverse transcriptase  according to the manufacturer’s protocol. mRNA expression was assessed quantitatively by real-time PCR with Power SYBR Green PCR Master Mix . Samples were run in duplicate on the ABI PRISM 7700 . The following oligonucleotide primer pairs were used .−/− mice by immunohistochemistry and western blot analysis as previously reported [−/− mice (−/− mice (WT mice 59.1 ± 2.9 versus SOD1−/− mice 17.9 ± 2.0 (n = 22–26))  versus SOD1−/− mice 98.5 ± 2.1 mmHg (n = 12)). AngII infusion increased systolic blood pressure similarly and there was no significant difference in blood pressure between the two strains (WT mice 156.6 ± 2.5 mmHg (n = 12) versus SOD1−/− mice 157.3 ± 4.2 mmHg (n = 12) at 7 days) (We confirmed that SOD1 was deficient in aortas from SOD1reported . SOD2 ex−/− mice A. SOD ac 22–26)) B, althou 22–26)) C. SOD ac 7 days) D.−/− mice compared to WT mice, while AngII-treated mice showed hypertrophy of vascular smooth muscle cells with associated thickening of the tunica media and thickening of the adventitia due to fibrosis. Nuclei were enlarged, nuclear staining by hematoxylin was weakened, and cytoplasmic vacuolation was observed, suggesting that cell death occurred. The above findings with AngII administration were more pronounced in WT but were attenuated in SOD1−/−. Quantitative analysis of aortic vessel wall thickness showed that Ang II-induced aortic wall thickening, which was observed in WT, was attenuated in SOD1−/− (−/− mice 68.4 ± 1.41 µm p < 0.001). Vascular tunica media area was also weakened in SOD1−/− (WT mice 0.223 ± 0.0051 mm2 versus SOD1−/− mice 0.186 ± 0.004 mm2p = 0.001)  C.2O2 is produced from O2·− by SOD and we hypothesized that the intracellular H2O2 would be specifically low in SOD1−/− mice because SOD1 is localized in the cytosol and the mitochondrial intermembrane space. We measured both the extracellular and intracellular H2O2 levels in WT and SOD1−/− mice. We used serum d-ROMs levels as an indicator of extracellular H2O2 levels [2O2 levels [−/− mice from 108.7 ± 4.8 to 164.7 ± 11.3 U.CARR (n = 12)) (H2 levels . The ser2 levels . The sern = 12)) A.2O2 levels. The H2O2 in the cytosol is promptly metabolized to H2O by catalase, glutathione peroxidase (Gpx), and peroxiredoxins [2O2, it oxidizes GSH to GSSG. An increase in GSSG/GSH ratio thus reflects an increase in intracellular H2O2 level. GSSG/GSH ratios in red blood cells were increased in WT mice by AngII, while in SOD1−/− mice, the ratios did not increase (WT mice from 0.093 ± 0.016 to 0.20 ± 0.032 versus SOD1−/− mice from 0.080 ± 0.020 to 0.097 ± 0.027 (n = 8)) (−/− but did not differ significantly (WT mice from 0.062 ± 0.017 to 0.12 ± 0.027 versus SOD1−/− mice from 0.059 ± 0.007 to 0.10 ± 0.015 (n = 10–11))  levels and reduced glutathione (GSH) levels in red blood cells as an indicator of cytosolic Hredoxins . When Gp(n = 8)) B. The GS 10–11)) C. We alsoth mice . These r−/− mice  ,10 in or−/− mice E. This wIn the present AngII infusion model, STAT3 was phosphorylated in the aorta in vivo. So, we next examined the phosphorylation of STAT3 by AngII in vitro. We cultured rat VSMCs and incubated them with 100 nM AngII. STAT3 was not activated in a short period of time but its phosphorylation was significantly increased after 24 h A. This mThe effect of AngII has been observed to be polymorphic, with some tyrosine-phosphorylated proteins being phosphorylated more rapidly by AngII . On the Next, we directly administered IL-6 (1 ng/mL) to the VSMCs and examined the phosphorylation of STAT3 by western blot analysis. With down-regulation of SOD1, STAT3 phosphorylation due to incubation with IL-6 was blunted E. These 2O2 and O2 from two O2·− in the cytoplasm. The effect of SOD1 observed above can be considered as a result of the change in the cytoplasmic H2O2 concentration. Catalase is an enzyme that metabolizes H2O2, and since H2O2 can freely pass through the cell membrane, the addition of catalase extracellularly decreases the intracellular H2O2 level. Therefore, we administered catalase into the culture medium 24 h before AngII administration and measured the expression of phosphorylated STAT3 in VSMCs. The administration of catalase (1000 U) attenuated the phosphorylation of STAT3  was decreased in the aorta from SOD1−/− mice. In VSMCs cultured with AngII, inhibition of SOD1 by siRNA or inhibitor suppressed IL-6 secretion, reduced the effect of IL-6 itself, and suppressed the inflammatory STAT3 cascade. PEG-catalase also inhibited STAT3 phosphorylation in the presence of AngII.In this study, we examined the role of SOD1 in vascular physiology and pathophysiology using a well-characterized SOD1 knockout mouse, in particular focusing on aortic hypertrophy and inflammation. We found that knocking out SOD1 alone did not cause hypertension or aortic pathology, going against the common belief that SOD1 has an antioxidant role. Moreover, in the Ang II infusion model, deletion of SOD1 reduced vascular pathology despite a similar increase in blood pressure as in WT. Serum IL-6 levels were increased to a similar extent but the up-regulation in aortic expression of IL-6 mRNA under AngII infusion was markedly blunted in SOD12·− would be upregulated and H2O2 would be downregulated in SOD1−/− mice aortas both under physiological conditions and under AngII infusion. However, under physiological conditions, cytosolic O2·− levels detected by DHE staining and H2O2 levels estimated from the GSSG/GSH ratio were not different in both mice. On the other hand, it has been reported that O2·− levels detected by lucigenin and DHE staining in SOD1−/− mice are higher than in WT mice under physiological conditions [2·− levels in blood vessels are high in old subjects [2·− is mainly produced in mitochondria, it is possible that the O2·− produced in the young cells was rapidly degraded by SOD2, and there was less O2·− leaking into the cytoplasm itself. In AngII-treated mice, the GSSG/GSH ratio in the aorta was increased and DHE staining was enhanced in both mice, but the differences were not significant. On the other hand, in RBCs that lack SOD2, the GSSG/GSH ratio, i.e., the increase in H2O2, was attenuated in SOD1−/− mice by Ang II treatment. This suggests that the presence of SOD2 compensated for the effect of SOD1 deficiency in the cells of the aortas as a whole, and that the change in ROS in the whole vessel might be masked. Since some reactive thiols on proteins  are susceptible to H2O2  [2O2 which may not be detected by GSSG/GSH in cytosol, could have significant effects on local signaling molecules related to hypertrophy in VSMCs.SOD1 is mainly localized in the cytosol and mitochondrial intermembrane spaces. We postulated that intracellular Onditions . One possubjects , and O2·radical) ,43,44. T−/− mice, suggesting that aortic local O2·− production was also reduced by SOD1 deficiency. However, ROSs detected by DHE staining was no difference between WT and SOD1−/− mice  [AngII behaves as a vasopressor hormone via G protein-mediated pathways . In addi2 by SOD . Furtherr (EDHF) .2·− by SOD3 is important to the bioavailability of NO and blood pressure.The role of SOD in the hypertensive response may vary with its individual isoforms as well as their localization. For example, in extracellular SOD (SOD3)-deficient mice, the AngII-induced hypertensive response was enhanced  and brai−/− mice. The Ang II dose was varied depending on study design (0.4–5mg/kg/day 7–14days) [−/− mice as in previous studies [2·− level was observed as described above. Our results showed there was no difference in basal blood pressure and a similar increase was observed in the presence of AngII for seven days.SOD1 is localized in the cytosol and mitochondrial intermembrane spaces and its role in the pressor response is complex and controversial; one study suggested that SOD1 had no remarkable role in blood pressure in the presence of AngII  and othe–14days) ,53,54,55 studies  but no dSome studies used radiotelemetry to measure blood pressure, however, we used the noninvasive tail-cuff method as previously ,22,26. T−/− mice exhibit baseline vascular hypertrophy in cerebral arterioles and renal afferent arterioles [−/− mice was similar to that of WT mice. In our study, SOD1−/− mice revealed less aortic hypertrophy under AngII infusion in spite of similar pressor responses. Wang et al. reported that in mice overexpressing human SOD1, the aortic wall thickened to the same extent as in WT mice, even though the Ang II-induced pressure increase response was attenuated [Some studies have shown that SOD1terioles ,23. We dtenuated . From th2O2 production is an important mediator of protein synthesis in VSMCs in vitro [2·− produced by Ang II is metabolized to cytosolic H2O2 in the presence of SOD1, and that this H2O2 causes VSMC hypertrophy, leading to aortic thickening in vivo.In vitro studies have shown that ROS induced by AngII plays an important role in VSMC hypertrophy ,17,18,57in vitro ,16. In ain vitro . Zhang ein vitro . These s2O2 from NOX in vitro [AngII is a multifunction hormone, and activates a complex series of intracellular signaling pathways for VSMC growth and proliferation , includiin vitro ,37. Howe2O2 generation [−/− mice. Marrero et al. reported that activation of STAT3 by AngII was observed after 60 min [2O2 generation by SOD1, leading to aortic hypertrophy. Cheng JF et al. reported that AngII induces proliferation of aortic VSMCs through activation of the JAK2/STAT3 signaling pathway [AngII promotes the inflammatory JAK/STAT pathway . In otheneration ,62, and neration . Therefor 60 min  and ther pathway , which i−/− mice, whereas the up-regulation of aortic IL-6 RNA expression was blunted in SOD1−/− mice. Under AngII infusion, the aortic expressions of other cytokines, such as MCP-1 and IL-1β were also increased but were blunted in SOD1−/− mice. In a study using cultured VSMCs, AngII had increased IL-6 release into the culture fluid in control VSMCs at 24 h, however, the release of IL-6 was reduced in VSMCs in which SOD1 was down-regulated. The rapid phosphorylation of STAT3 due to IL-6 seen at 5 min was also blunted in VSMCs with down-regulated SOD1. These results indicate that SOD1 was necessary to induce the synthesis and secretion of IL-6 and that it also enhanced the phosphorylation of STAT3 , which is in the active center, and inhibits SHP activity [2O2 production through deletion of SOD1 in VSMCs might activate SHP and dephosphorylate and inhibit the JAK/STAT pathway. Further studies will be required to clarify precise mechanisms for IL-6 production and STAT3 activation due to AngII in a SOD1-dependent manner.As Htivation  and H2O2larified ,70. H2O2activity ,72,73. T−/− with WT mice, we found that SOD1 function in the vasculature is limited under physiological conditions, but in the presence of Ang II, it causes aortic hypertrophy independent of the pressure increase response. Because of the presence of mitochondria in vascular cells, it was inferred that SOD2 compensated for SOD1 deficiency, and no changes in net ROS were observed in the aorta as a whole. However, the elevation of cytoplasmic local H2O2 produced by SOD1 was thought to cause aortic hypertrophy by targeting signaling molecules such as Ras and phosphatases [In conclusion, by comparing SOD1phatases ,43,44 vi


SUVmax was 4.1 ± 0.7, 4.6 ± 0.9 and 3.8 ± 0.7 , and SUVratio was 2.0 [1.9 to 2.2], 2.0 [1.8 to 2.6], and 1.9 [1.7 to 2.0]  for groups 1, 2, and 3, respectively.In total, 37 patients  (mean age 66 ± 8 years) were prospectively included. Perivalvular 18F-FDG uptake with similar SUVmax and SUVratio at 5, 12, and 52 weeks after implantation.Non-infected aortic PHV have similar low to intermediate perivalvular The online version of this article (10.1007/s12350-019-02025-y) contains supplementary material, which is available to authorized users. The ESC guidelines suggest using 18F-FDG PET/CT only if the PHV was implanted > 3 months prior to the scan because it was assumed that the normal healing response after aortic PHV implantation and its associated 18F-FDG uptake would cause false positive results and misinterpretations within this time window.18F-FDG uptake patterns and cut-off values for abnormal uptake have been obtained from retrospective assessment of a limited number of patients with a PHV who underwent 18F-FDG PET/CT for indications other than suspected endocarditis.18F-FDG uptake patterns around PHV’s was published showing no significant differences between 18F-FDG uptake around PHV’s at 1, 6 and 12 months after surgery.18F-FDG uptake at three different time points within the first year following aortic PHV implantation, in order to obtain normal 18F-FDG uptake reference values.Diagnosing prosthetic heart valve (PHV) endocarditis remains difficult.2In this prospective multi-center cross-sectional study, we included patients (age ≥ 50 years) from two different hospitals in the Netherlands  who had undergone an uncomplicated aortic PHV implantation. An uncomplicated PHV implantation was defined as a PHV implantation without any surgical complication during or after the operation as well as the absence of signs of infection as mentioned in the surgical reports and the electronic patient files. The inclusion and exclusion criteria are detailed in Table 18F-FDG PET/CT at either 5 (± 1) weeks (group 1), 12 (± 2) weeks (group 2), or 52 (± 8) weeks (group 3) following valve implantation. The assignment of patients to each group depended on logistic factors such as availability of time slots on the PET/CT scanner and patient availability of one of the three time intervals after surgery.Patients were divided into three groups and received an 18F-FDG PET/CT.Included patients had undergone uncomplicated valve implantations and did not have any clinical signs of prosthetic valve infection  at the time of the 18F-FDG injection of 2.0 MBq/kg. Patients were hydrated with 1000 ml of water 1 hour prior to image acquisition. Blood glucose levels were checked before 18F-FDG injection and the limit was set to 8.9 mmol/L. Approximately 1 hour after 18F-FDG injection, the PET/CT was performed using a Biography Sensation 16scanner . Before the PET acquisition, a low dose CT scan was performed for attenuation correction. A PET-scan of the heart was then obtained with 3-minute acquisitions per bed position using a 3-dimensional acquisition mode. Attenuation-corrected PET images were reconstructed with an ordered-subset expectation-maximization iterative reconstruction algorithm.To induce free fatty acid metabolism and suppress myocardial glucose metabolism, patients followed a 24-hour low carbohydrate diet, of which the last 12 hours were spent fasting.18F-FDG around the PHV was scored both qualitatively and quantitatively by an experienced nuclear medicine physician. For qualitative analyses, the Qualification Visual Score for Hypermetabolism (QVSH) was used, scoring the uptake as “none” , “low” , “intermediate” (more than liver uptake), or “high” (intense uptake). Mediastinal uptake was defined as the mean uptake in the blood pool of the descending aorta at the level of the left atrium. Additionally, the location (former left coronary cusp (LCC)/right coronary cusp (RCC)/non coronary cusp (NCC), circular, PHV struts only or ascending aorta) of this uptake was specified. Quantitative analyses were performed by measuring the maximum Standardized Uptake Value (SUVmax) and target to background ratio (SUVratio) on standardized European Association of Nuclear Medicine Research Ltd. (EARL) and non-EARL reconstructions using commercially available software . SUVmax was measured in an automated volume of interest (VOI) around the PHV, which was visually verified to include the whole valve region. The SUVratio was then calculated as the ratio of the SUVmax and the mean SUV in the blood pool of the descending aorta, taking care not to include the vessel wall.Uptake of Myocardial suppression was scored as “fully suppressed” (no uptake), “low” , “intermediate” (more than liver uptake), “high focal” , “high diffuse” .P = 0.05 and 95% confidence intervals (CI) were used.Descriptive statistics were used for analysis of the outcomes. For continuous variables, means and standard deviations (SD) were used in case of normal distribution. In case of non-normal distribution, medians and interquartile ranges (IQR) were used. The IQR and confidence interval (CI) were denoted in square brackets. Comparisons between groups were made using the Chi-square test for categorical variables. For continuous variables One-way Analysis of Variance (ANOVA) was used in case of normal distribution and Kruskal Wallis test in case of non-normal distribution. A significance level of P = 0.87) and most of the patients were male  . There were 25 (68%) biological and 12 (32%) mechanical prosthetic valves, equally distributed between groups (P = 0.99). Surgical adhesives such as BioGlue that are known to be FDG-avid, were not used during any of the implantations. No patient was suspected of having endocarditis prior to the PET/CT scan. Patients were included in either group 1 (n = 12), group 2 (n = 12), or group 3 (n = 13). Due to logistic problems, 8 patients  underwent the scan outside the time interval originally set-out for each group. The 2 patients in group 1 were scanned 2 and 5 days later than the maximum adjusted days (5 ± 1 week) for group 1. The 3 patients in group 2 were scanned 15, 22, and 38 days later and the 3 patients in group 3 were scanned 15, 23, and 36 days later than originally planned. Baseline characteristics for the overall population and the three groups are summarized in Table A total of 38 patients were initially included after signing written informed consent. One patient was excluded after failure to undergo the PET/CT scan due to scanner malfunction. Age (mean ± SD) of the 37 patients finally included in this study was 66 ± 8 years . Median 18F-FDG dosage was 166 [IQR 145 to 183] MBq and not significantly different between the groups (P = 0.16). Preparation according to carbohydrate diet protocol was followed by 36/37 (97%) patients. Three patients had fasted less than 12 hours prior to the scan, 1 patient failed to follow the low carbohydrate diet and 1 patient inadvertently received a double amount of 18F-FDG activity. Myocardial 18F-FDG uptake was scored as “fully suppressed” in 18/37 (49%) and as intermediate or less in 29/37 (78%) patients. One patient was scored as focal high and 7 patients as diffuse high myocardial uptake. The interpretation of one scan was hampered due to the diffuse high myocardial FDG uptake.The median time between PHV implantation and P = 0.91). The distribution of 18F-FDG uptake was circular in most cases (78%) and not significantly different between the 3 groups (P = 0.50). The 18F-FDG uptake around the PHVs on a reconstructed view in the PHV plane of attenuation-corrected images, non-attenuation-corrected and fused attenuation-corrected images with CT of all patients is shown in Figure The QVSH around the PHV was scored as follows for group 1: low in 8/12 (67%) and intermediate in 4/12 (33%) patients; group 2: 7/12 (58%) low and 5/12 (42%) intermediate and for group 3: 8/13 (62%) low and 5/13 (38%) intermediate. Comparison between groups showed no significant difference in QVSH (max of 4.1 ± 0.8 (mean ± SD) and a median[IQR] SUVratio of 2.0 [1.8 to 2.2] for all included patients. The SUVmax around the PHV was 4.1 ± 0.7, 4.6 ± 0.9, and 3.8 ± 0.7 (mean ± SD) in group 1, 2, and 3 respectively, with no significant difference between the 3 groups (p = 0.08). The median[IQR] SUVratio around the PHV was 2.0 [1.9 to 2.2], 2.0 [1.8 to 2.6], and 1.9 [1.7 to 2.0] with no significant difference between the three groups (P = 0.81) (Table max and SUVratio of 3.6 ± 0.5 and 1.8 ± 0.3 (mean ± SD), respectively. SUVmax around the PHV was 3.6 ± 0.5, 3.8 ± 0.5 and 3.3 ± 0.6 (mean ± SD) in group 1, 2 and 3 respectively, with no significant difference between the 3 groups (P = 0.14). Likewise, the SUVratio around the PHV was 1.8 ± 0.2, 1.8 ± 0.3, and 1.7 ± 0.3(mean ± SD) with no significant difference between the three groups either (P = 0.41). The minimum and maximum measured SUVratio in the study population was 1.4 and 2.5, respectively. EARL SUVratio was < 2.3 in 97% and < 2.1 in 92% of the cases. The distribution of non-EARL and EARL SUVmax and SUVratio are demonstrated in Figure Quantitative analyses on the non-EARL attenuation-corrected images showed a SUV18F-FDG uptake elsewhere in the body was seen in 21/37 (57%) of patients and was not significantly different between the 3 groups (P = 0.18). This elevated 18F-FDG uptake was mainly seen in the thoracic lymphnodes 9/21 (38%) and considered physiological. Other areas of elevated uptake consisted of costal fractures 3/21 (14%), pleural uptake (possible pulmonary nodule) 2/21 (10%), acromioclavicular joint (due to degeneration) 2/21 (10%), thyroid (possible hyperthyroidism) 1/21 (5%), arytenoid  1/21 (5%), possible pathological oesophageal uptake 2/21 (10%), diffuse in muscles 1/21 (5%), and focal uptake due to a surgical clip.Elevated 18F-FDG uptake around the PHV at 5, 12 and 52 weeks after implantation and a mean ± SD SUVmax of 4.1 ± 0.8 and a median[IQR] SUVratio of 2.0[1.8 to 2.2].The present study shows that patients with non-infected aortic PHV have similar low to intermediate mostly circular 18F-FDG PET/CT is an important diagnostic method in suspected PHV endocarditis, especially in cases where the diagnosis cannot be confirmed with transthoracic (TTE) or transesophageal echocardiography (TEE). However, in patients with a recent PHV implantation (< 3 months), the use of 18F-FDG PET/CT is not advised due to possible false positive findings caused by post-surgical inflammation.18F-FDG PET/CT findings could have major inappropriate therapeutic consequences. Patients may be treated while this is not necessary and counterwise not be treated while this is obligatory. Therefore, caution with the interpretation of 18F-FDG PET/CT in the early weeks after PHV implantation is advised, especially in cases of complicated surgery. In such cases, the inflammation response due to the complications could be severe and cause non-diagnostic or false positive 18F-FDG PET/CT results. It is therefore crucial to be able to recognize normal 18F-FDG distribution patterns and establish a quantitative cut-off value for pathological 18F-FDG uptake around the PHV.Nowadays, 18F-FDG uptake around the PHV in our study demonstrated a median[IQR] SUVratio of 2.0 [1.9 to 2.2] for patients at 5 weeks after surgery, with no statistically significant difference compared to 3 months and 1 year . These results corroborate the scarce known literature about this matter. Mathieu et al.max of 3.6  and a median SUVratio of 1.9  on non-EARL attenuation-corrected images. No significant difference in SUVmax and SUVratio between the PHVs implanted < 3 months and those that were implanted > 3 months prior to the PET/CT scan was found.ratio of 2.7 in the patients with vasculitis compared to the other groups.18F-FDG uptake at 3 different time points in the first year after PHV implantation. The study method had similarities with our study, but there were some differences. Roque et al. included also patients post mitral valve implantation, and each patient received 3 times a PET/CT scan in the time periods of 1, 6, and 12 months after valve implantation. Despite these differences, their results also showed no significant difference in 18F-FDG uptake between scans made in the three different time periods and their conclusion that the three months safety period should be reconsidered is in line with our conclusion.Quantitative measurements of nd 1.9 1. to 2.0, nd 1.9 1. to 2.0, nd 1.9 1. to 2.0, ratio was > 2.0.ratio was 2.5 and 97% of scans had an EARL SUVratio of less than 2.3, indicating that the cut-off value might be slightly higher than the > 2.0 reported earlier by Swart et al. in the first year after PHV implantation7Recently, in a retrospectively collected cohort of 243 patients, we found that the optimal diagnostic cut-off value to diagnose PHV endocarditis for the EARL-standardized SUV18F-FDG uptake around the PHV with mostly a circular pattern  and without focal enhancement. The distribution of 18F-FDG can differ widely and its definition is still unclear; however, some of the uptake patterns  have been associated with physiological uptake after PHV implantation.18F-FDG PET/CT can mask adjacent abnormal 18F-FDG uptake around the PHV. Therefore a preparatory low carbohydrate diet that may be supplemented by an intravenous injection of heparin is necessary for reducing myocardial 18F-FDG uptake in order to avoid false positive 18F-FDG PET/CT results.18F-FDG uptake making correct measurement of the SUV values more difficult . Excluding obese patients and patients with diabetes mellitus could also be seen as a limitation to the applicability of our results. Both conditions can affect the healing process following surgery and could therefore potentially impact 18F-FDG uptake. However, in order to prevent inadequate glucose levels prior to the PET and restrict the radiation exposure to patients, the exclusion of these patients was necessary. In total 51% of the patients did not have fully suppressed myocardium and this could be seen as a potential confounder to the qualitative and quantitative 18F-FDG measurements.Our study has some limitations. Eight patients  received the scan somewhat later than the time frame adjusted for each group. This was due to logistic reasons. Another limitation of this study was that the scan was performed once in every patient and not multiple times in the same patient to actually see a change over time in the uptake patterns and SUV values. This approach was not deemed feasible due to the high radiation dose of multiple PET/CT scans in individual healthy patients this would imply. Furthermore, our study population only included patients with an aortic prosthetic valve, and hence we cannot draw any conclusion regarding normal Although the measurements done by the nuclear medicine physicians were carefully done not to include myocardial uptake, this could not always have been prevented. Thus, this could be seen as a limitation of our study.18F-FDG uptake at 5, 12, and 52 weeks after implantation and an average SUVmax of 4.1 ± 0.8 and a median[IQR] SUVratio of 2.0 [1.8 to 2.2]. These normal 18F-FDG uptake values and patterns provide further evidence that 18F-FDG PET-CT can be used as a diagnostic tool for the detection of endocarditis even shortly after aortic PHV implantation and the recommendation to not perform PET-CT within the first three months after PHV implantation in the 2015 ESC guidelines for the management of infective endocarditis should be reconsidered.In conclusion, non-infected aortic PHV have similar low to intermediate mostly circular perivalvular 18F-FDG uptake within the first year after PHV implantation and showed no significant difference in 18F-FDG uptake at 5 weeks, 12 weeks, or 52 weeks after implantation. These findings may help clinicians to differentiate between normal and pathological perivalvular 18F-FDG uptake and suggest the use of 18F-FDG PET/CT as an extra imaging tool in the diagnostic workup of patients with recent aortic PHV implantation that are suspected of PHV endocarditis.Our study supports previous observations on the normal perivalvular Electronic supplementary material 1 (PPTX 156 kb)Electronic supplementary material 2 (M4A 8025 kb)Below is the link to the electronic supplementary material.


Pentacyclic triterpenoids are well-known phytochemicals with various biological activities commonly found in plants as secondary metabolites. The wide range of biological activities exhibited by triterpenoids has made them the most valuable sources of pharmacological agents. A number of novel triterpenoid derivatives with many skeletal modifications have been developed. The most important modifications are the formation of analogues or derivatives with nitrogen-containing heterocyclic scaffolds. The derivatives with nitrogen-containing heterocyclic compounds are among the most promising candidate for the development of novel therapeutic drugs. About 75% of FDA-approved drugs are nitrogen-containing heterocyclic moieties. The unique properties of heterocyclic compounds have encouraged many researchers to develop new triterpenoid analogous with pharmacological activities. In this review, we discuss recent advances of nitrogen-containing heterocyclic triterpenoids as potential therapeutic agents. This comprehensive review will assist medicinal chemists to understand new strategies that can result in the development of compounds with potential therapeutic efficacy. The above-mentioned advantages of heterocyclic derivatives give them great potential for use in pharmaceutical practice. These compounds have been used to develop various organic synthetic procedures and have a wide range of therapeutic applications -3,23-dihydroxyurs-12-ene-28-oate (61) were synthesized by Zhao et al. [60 and 61 exhibited excellent anticancer activity than the reference drug, Gefitini [N-methylpiperazinylamide through a series of transformations  of N-methylpiperazinylamide. They evaluated their in vitro cytotoxicity against some cell lines, namely LNCAP, PC3 22RW1, Huh7, and VA13. Compounds 62 and 63  in Dimethylformamide (DMF) for 24 h. The obtained compound was introduced with piperazine in DMF in the presence of Potassium carbonate (K2CO3) at 80 ˚C, and then reacted with an aromatic to obtain the targeted compound. The newly synthesized compound was evaluated for anticancer activity against human gastric cancer (MGC-803) and breast cancer (Bcap-37) cell lines using standard MTT assay in vitro. The compound showed a cell apoptosis-inducing effect higher than the positive control, HCPT, and UA [66  ratio, disrupted mitochondrial potential and induced apoptosis, and suppressed the growth of Hela xenografts in nude mice [67  [The ursolic acid derivative , and UA . Wang et, and UA  designedd UA [66 , Table 3ude mice . Li et aude mice  indicatemice [67 ), along n et al.  suggestin et al. . Kahnt en et al.  preparedrcinoma) .69–71) with aminoquinoline heterocyclic scaffold. Sommerwerk et al. [69) was the most promising compound with IC50 values of 0.19 µM for melanoma (518A2), 0.22 µM for colorectal adenocarcinoma (HT29), 0.54 µM for breast (MCF7), 0.29 µM for lung adenocarcinoma (A549), 0.08 µM for ovarian (A2780) and 3.23 µM nonmalignant mouse fibroblast (NiH3T3). This compound was further examined by apoptosis through propidium iodide/acridine orange staining, fluorescence spectroscopy, and cell cycle investigation [70 was the most cytotoxic and therefore showed lower EC50 values of 1.48 ± 0.1 µM for melanoma (A375), 4.65 ± 0.5 µM (MCF7), 2.16 ± 0.2 µM (A2780), 2.26 ± 0.2 µM for hypopharyngeal carcinoma (FaDu) and > 30 µM (HT29) compared to > 91.2 µM (NiH3T3). On the other hand, doxorubicin was used as a reference drug with EC50 values of > 30 µM (HT29), 1.1 ± 0.3 µM (MCF7), 0.01 ± 0.01 µM (A2780), not detected for A375 and FaDu compared to 1.3 ± 0.6 µM (NiH3T3). Therefore compound 62 can be further investigated as a antitumor agent [71 was the most promising hybrid with IC50 values of 1.27 ± 0.06 µM (C-32), 2.43 ± 0.68 µM (MDA-MB-231), 6.67 ± 1.30 µM (Colo-8), 14.9 ± 2.30 µM (MCF-7). However, its cytotoxicity against SNB-19 and T47D was not impressive with IC50 values of 41.99 ± 0.11 µM and 99.39 ± 6.98 µM, respectively. Compound 71 reduced the proliferative activity of all tested cells during enzymatic mechanism of action. This compound was tested for its transcriptional activity of gene encoding of cell-cycle (p21 and p53), proliferation maker (H3-histone) and apoptosis. Furthermore, this compound interacted with hydrophobic matrix of the active site of the enzyme near FAD, Trp105, Phe178, and Tyr128 cofactor, which was observed by an increase in gene expression (TP53) [The principle of molecular hybridization is efficient as it is centered on the combination of two different distinct pharmacophores by linking covalently two or more structural entities with distinct biological functions. The positive synergistic effects of these compounds can also occur along with expected pharmacokinetic and pharmacodynamic profiles. In addition, by enhancing bioavailability and distribution through cell membranes of cell organelles, this process can solve a variety of common problems associated with drug molecules and shielding active compounds from enzyme degradation . Quinonek et al.  synthesitigation . Hoenke tigation  synthesior agent . Kadela-or agent  synthesin (TP53) .18, (36a-36d), 43, 44, 45, 46, and 68. Some hybrid compounds have been reported to be the modulators for multiple targets at different stages of cancer progression, such as proliferation, angiogenesis, metastasis, and apoptosis. Almost all the reported hybrid compounds displayed enhanced anticancer activity compared to the parent PTs. The above-mentioned significant points identify the enormous potential of these triterpenoid-based nitrogen-containing heterocyclic moieties in pharmaceutical applications suggesting a massive scope for these promising compounds because of their diverse molecular targets.The growing incidences of cancer are one of the major issues in the developing world. Novel therapeutic agents that would be useful for the treatment of cancers that have acquired resistance to the existing drugs are desperately needed. PTs have the advantage of creating various derivatives with potential pharmacological properties. This review summarized the synthesized PTs derivatives with fused nitrogen-containing heterocyclic scaffolds, which have displayed significant anticancer activity. We noted that recent studies had been done on triterpenoid with nitrogen-containing heterocyclic scaffolds to understand the mechanism of action of these hybrids. The introduction of various nitrogen-containing heterocyclic moieties in PTs brings new mechanism of actions that lead to high anticancer activity. The hybrid compounds exhibited high anticancer activity such as


Although this overall transformation can be classified as an electrophilic fluorination of an aromatic ring, it is a novel type of fluorination that results in construction of tertiary carbon–fluorine bonds. The present protocol can be applied to a range of bicyclic azaarenes, tolerating azines and a variety of functional groups. Additionally, mechanistic studies and enantioselective fluorination have been examined.We have discovered a ring-opening fluorination of bicyclic azaarenes. Upon treatment of bicyclic azaarenes such as pyrazolo[1,5- tert-C–F bonds.A ring-opening fluorination of bicyclic azaarenes was developed. Although this overall transformation can be classified as an electrophilic fluorination of aromatics, it is a novel type of fluorination that results in construction of  Nucleophiles used in electrophilic fluorinations can be broadly classified into carbanions , electron-rich unsaturated bonds , and aromatics.5 However, in these existing methods, fluorination proceeds while retaining the carbon skeleton of the starting material, and fluorinations involving skeletal transformations are rare.Fluorine is one of the most important elements that could be installed onto hydrocarbon frameworks in pharmaceuticals, agrochemicals, and materials science.6 Fluorinations involving bond cleavage in a ring size ≥5 are rare. The Lectka, Leonori, and Ma group reported ring-opening fluorinations via C–C bond cleavage of carbocycles –F bond) instead of a fluorine-substituted heteroarene (C(sp2)–F bond). In other words, we thought that this would be a novel type of fluorination reaction with accompanying skeletal transformation.In the heteroatom–heteroatom (X–Y) bond paradigm, the Yao group reported a ring-opening fluorination using isoxazoline cleavage .11 Howeva]pyridine (1A) as the model substrate  to examine electrophilic fluorinating agents and reaction conditions  were used in MeCN at 80 °C, ring-opening fluorinated product 2A was successfully obtained, albeit in low yields (entries 1–3).12 The use of other non-pyridinium based electrophilic fluorinating agents such as NFSI and Selectfluor® gave fluorinated products in high yields (entries 4 and 5).13First, we selected 3-phenylpyrazolopyridines were examined: Methyl (1B), tert-butyl (1C), and phenyl (1D) at the para-position on the aryl group gave the ring-opening fluorinated products 2B–2D in moderate yields. It should be noted that fluorination of these aryl groups was detected. When using trimethylphenyl (1E) and naphthyl (1F) starting materials, the corresponding products 2E and 2F were obtained in moderate yields, and occurred decomposition of 1E or fluorinated the aryl group of 1F (less than 10% yields). The reaction showed good functional group tolerance in the presence of formyl (1G), acetyl (1H), cyano (1I), trifluoromethyl (1J), nitro (1K), and chloro (1L) groups, as the reaction worked to give the corresponding products 2G–2L in excellent yields. Next, 3-alkylpyrazolopyridines were investigated. The fluorination using bicyclic azaarenes bearing alkyl groups (1M and 1N) or acetal (1O) proceeded smoothly to give the corresponding fluorinated products 2M–2O in moderate to excellent yields. Pyrazolopyridines with alkyl acetate (1P), cyano (1Q), and ethylcarboxylate (1R) afforded the corresponding products (2P–2R) in good yields. Pyrazolopyridine carboxylates were also examined. Substrates with alkyl groups including alkene (1U) and alkyne (1W) remained intact to give products 2S–2X in high yields. Carboxylic acid 1Y also reacted well, but the product was difficult to purify, resulting in a low yield of 2Y. In the case of compounds with amides such as 1Z, deamidation occurred to give 3-fluoropyrazolopyridine as a byproduct. Therefore, the fluorinated product 2Z was obtained in moderate yield (39%) by reacting at a lower temperature (−30 °C). Furthermore, azaarenes 1AA and 1AB derived from probenecid and estrone also gave fluorinated compounds 2AA and 2AB in high yields. Of note, in the case of an unsaturated ester or iodine at the C3 position, the desired fluorinated product could not be obtained, giving a complex mixture.As for the reaction temperature, the yield of 2A was 68% even at 50 °C. The yield increased as the temperature was increased, and the fluorinated product was obtained quantitatively at 80 °C (entries 6–8 stigated . Various4 (1.0 equiv.) was effective for increasing yields 2PHAL, can be prepared by mixing (DHQD)2PHAL and Selectfluor® at room temperature.16 A reaction using stoichiometric amounts of these agents with 3-phenyl-6-bromopyrazolopyrimidine 1AJ in MeCN at 50 °C gave the corresponding product in 68 : 32 e. r., albeit in a low yield. However, when (DHQD)2PHAL was reduced to catalytic amount, enantioselectivity was dropped whereas the yield was increased. The substrate without a bromo atom at the C6 position (1AG) gave the fluorinated product in moderate yield (44%) and 65 : 35 e. r. By lowering the temperature, changing the fluorinating agent, and changing the solvent, we finally succeeded in obtaining the fluorinated compound 2AJ with an enantioselectivity of 84 : 16 e. r.We then studied the enantioselective version of this fluorination reaction .15 We at PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 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 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O bond of amide 10 is align antiparallel to the C–F bond, a conformation in which the amide dipole opposes the C–F dipole due to a dipole minimization effect. The enantioselectivity of this asymmetric fluorination could be explained using the proposed transition state model. Although the direction in which the substrate reacts with the chiral fluorinating agent determines the enantioselectivity, we believe that the transition state of the desired compound has a π–π interaction between the substrate and the methoxyquinoline moiety of (DHQD)2PHAL, which fixes the conformation.16 Finally, the obtained fluorinated compounds were derivatized into various compounds  were condense with amines to give amide 11 in 41% yield. Palladium-catalyzed decarboxylative allylation and removal of allyl esters proceeded to give derivatives 12 and 13 in high yields. Furthermore, we attempted to convert the cyano group of the product of the fluorination reaction. Fluorinated product 2A (R = Ph) was converted to methyl ester 14 by methanolysis. 2A was also converted to amides 15 and 16 by hydrolysis and Ritter reaction.18 Furthermore, borane reduction gave amine 17. In this way, we have succeeded in synthesizing a variety of fluorine-containing compounds by orthogonal functional group transformations following ring-opening fluorination.The absolute stereochemical configuration was determined by derivatization to give optically pure amide 10, recrystallization, and then X-ray structural analysis. In the crystal structure, the Compounds . The rin3-fluorinated compounds via N–N bond cleavage. Studies revealed that the electrophilic fluorinating reagent functioned not only as the fluorine source, but also as the base required for ring opening. Expanding the range of substrates and other electrophiles for this type of transformation is currently underway in our laboratory.19In summary, we developed a ring-opening fluorination of bicyclic azaarenes leading to spAll experimental data is available in the ESI.†B. S. and J. Y. conceived and designed the study. M. K., A. S. and H. K. performed the chemical experiments and analyzed the data. K. K. performed the X-ray crystallography experiments and analyzed the obtained data. H. T. performed the preliminary experimental studies. J. Y. wrote the manuscript and all authors discussed the results and commented on the final manuscript.There are no conflicts to declare.SC-013-D1SC06273E-s001SC-013-D1SC06273E-s002


Ovarian cancer (OC) is one of the most lethal gynecological malignancies. However, the molecular mechanisms underlying the development of OC remain unclear. Here, we report that loss of Ras GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) inhibits the progression of OC cells. Analysis of databases and clinical specimens showed that G3BP1 is upregulated in OC. The Kaplan–Meier plot results showed that G3BP1 is highly expressed in OC with a poor clinical outcome. Moreover, loss-of-G3BP1 suppresses the proliferation, migration, and invasion of OC cells. Protein–protein interaction network analysis and immunoprecipitation assay showed that ubiquitin-specific protease 10 (USP10) interacts with G3BP1. We next found that USP10 coordinately promotes tumor progression with G3BP1. Moreover, loss of USP10could restore the G3BP1-induced proliferation, migration, and invasion of OC cells. These data indicate that G3BP1 coordinated with USP10 to facilitate the progression of OC cells, and that G3BP1 may become a treatment target for OC. Ovarian cancer (OC) is one of the most lethal gynecological malignant tumors on a global scale . One of Ras GTPase-activating protein SH3 domain‐binding protein 1 (G3BP1) is one of the members of Ras GAP binding protein family ,5. This Recently, the role of deubiquitinating enzymes in tumor has received increased attention . UbiquitIn this research, we intend to explore the potential roles and possible mechanisms of G3BP1 in OC progression. We found that G3BP1 is negatively associated with the prognosis of OC patients through Kaplan–Meier analysis. Moreover, we reported that G3BP1 played an oncogenic role in OC and produced an effect on proliferation, migration, and invasion of OC cells. Through searching for protein–protein interaction (PPI) network and Gene Expression Profiling Interactive Analysis (GEPIA) website, we found that USP10 interacted with G3BP1, and that there was a positive correlation between G3BP1 mRNA expression and USP10 mRNA expression in OC. Hence, we hypothesized that G3BP1 might facilitate OC progression by coordinating with USP10. Furthermore, we found that G3BP1 in collaboration with USP10 contributed to OC cells progression. To sum up, our data identified G3BP1-USP10 complex play an oncogenic role in OC cells.www.oncomine.org). OC microarray data were obtained from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/), including GSE14407, GSE27651, and GSE10971. The UALCAN database  was used to investigate the protein expression levels of G3BP1 in OC and the expression of G3BP1 protein in different stage of OC. The Kaplan–Meier plotter OC database  was used to evaluate the progression-free survival (PFS) and overall survival (OS) of OC patients based on G3BP1expression. the progression-free survival (PFS) and overall survival (OS) of OC patients based on G3BP1expression. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) network from the GeneCards website (https://www.genecards.org/) was used to determine whether a key protein was related with G3BP1. The GEPIA online database was used to analyze the mRNA expression correlation between G3BP1 and USP10 in OC tissues.The gene expression profile and clinical profile in the Cancer Genome Atlas (TCGA) were obtained from Oncomine website  at 4°C in a humidified chamber. Then, the tissues were reacted with polymer enhancer for 30 min at 37°C, followed by a biotin-labeled secondary antibody. Antibody staining was carried out by DAB kit. The final score was calculated by multiplying the staining intensity and the percentage of stained-positive cells. The staining intensity was scored according to the intensity of immunoreactivity as follows: 0, negative; 1, weak; 2, moderate; 3, strong; the percentage of stained-positive cells was scored as follows: 0, 0%; 1, 1%~25%; 2, 26%~50%; 3, 51%~75%; 4, 76%~100%. All slides were scored by two consultant clinical histopathologists in a double-blinded manner .OC cell lines, including OVCAR3, ES2, A2780, SKOV3, and IGROV1, and the normal ovarian epithelial cell-line HOSEpiC were purchased from Chinese Academy of Medical Sciences & Peking Union Medical College . The A2780 cell line was cultivated in RPMI-1640 medium  containing 10% fetal bovine serum (FBS) , 100 U/mL penicillin, and 100 mg/mL streptomycin . The remaining above-described cells were cultivated in complete DMEM  medium.The pLenti-CMV-G3BP1-GFP-Puro plasmid and the vector plasmid were purchased from PPL . Cells were transfected using jetPRIME transfection reagent . 2 µg of the plasmid was added to 200 µL of jetPRIME buffer and 2 µL of jetPRIME to 6-well plates at 60% confluence of OVCAR3 cells. Cells were processed for subsequent experiment after transfection 48 h. Targeted G3BP1-specific small-interfering RNA (siRNA), targeted USP10-specific siRNA, and the Negative control (NC) siRNA were purchased from GenePharma . OVCAR3 cells were incubated in 6-well plates at 60–80% confluence, and siRNA was used to inhibit the expression of G3BP1 or USP10 by using Lipofectamine 3000 as a transfecting agent . All siRNAs were used at a final concentration of 75 pmol and transfected into cells with 7.5 μl Lipofectamine 3000 reagent according to manufacturer’s instructions (Invitrogen). The cells were treated with siRNAs for 48 hours before subsequent experiments. The sequences for NC were as follows: UUCUCCGAACGUGUCACGUTT. The sequences for G3BP1 were as follows: CCACACCAAGATTCGCCAT and CCTGAAGAAAGACAGCAAA. The sequences for USP10 were as follows: CCAUAAAGAUUGCAGAGUUTT and CCACAUAUAUUUACAGACUTT.The Cell Counting Kit-8 (CCK-8) assay  was used to evaluate the proliferation ability of OVCAR3 cells. After the cells were transfected, 2500 cells were plated in 96-well plates and cultured. Cells were treated with 10 μL CCK-8 reagent at the specified time and incubated at 37°C for 2 h. Absorbance at 450 nm and 630 nm was measured by enzyme labeling instrument .4 cells/well in 200 μL FBS-free growth media were sowed in the upper chamber. As a chemoattractant, 600 μL normal culture medium was added to the lower chamber. After incubating for 24 hours, the cells on the upper membrane were washed with PBS and wiped off with cotton swabs. The transwell inserts were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet solution. Five transwell fields  were randomly selected and images were photographed under an optical microscope . To assess the cell invasion capability, the transwell chambers were first coated with Matrigel Basement Membrane Matrix . Next, the upper chamber was seeded with 8 × 104 cells/well, and culture medium was added to the lower chamber as previously described for the migration assay. After incubation, the number of cells in the lower membrane was counted [For the migration assay, 8.0-μm pore Transwells  were put into 24-well plates. Cells at a density of 5 × 10 counted .Cells were subjected to RNA extraction with TRIzol . cDNA was synthesized from RNA samples (1 μg) with All-in-One cDNA Synthesis Supermix . For mRNA analysis, cDNA was analyzed using ChamQ Universal SYBR qPCR Master Mix  for quantification by qRT-PCR on an Applied Biosystems QuantStudio Dx instrument . The oligonucleotides used for amplification were as follows: GAPDH Forward, 5′-GACAGTCAGCCGCATCTTCT-3′; Reverse, 5′-GCGCCCAATACGACCAAATC-3′; G3BP1 Forward, 5′-CTATCCTCGGTGCTGTGGTG-3′; Reverse, 5′-GGGGGAAAAGAGTCAAATATGTCC-3′; USP10 Forward, 5′-CCTGGGCCATGATCCCATTT-3′; Reverse, 5′-TCAAGACGGGACAGAATGGC-3′.Cellular proteins were extracted in cell lysis buffer and quantitated. The equal amounts of protein (50–70 µg) were subjected to 10% SDS-PAGE gel and transferred to 0.22-µm polyvinylidene difluoride membranes . The membranes were incubated for overnight at 4°C with the required primary antibodies and incubated with enzyme-labeled secondary antibody for 1 hour at room temperature. The protein bands were then captured using ECL-Plus reagent . The specific primary antibodies used for Western blotting including anti-GAPDH , anti-G3BP1 , anti-USP10 .7 OVCAR3 cells were gently washed twice with cold PBS. Then, cells were harvested in IP lysis buffer on ice for 30 min. The IP lysis buffer included 300 mM NaCl, 0.5% NP-40, 50 mM Tris-HCl pH 7.4, protease inhibitor cocktail (1×). Incubated the lysate supernatant with 2 µg antibody at 4°C overnight. Next, 20 µl of Protein A/G  was transferred to each mixture and rotated for 2 hours at room temperature on a gentle-rotating mixer. Proteins were loaded on a 10% SDS-PAGE gel and visualized by standard procedures [1 × 10ocedures .The association between the expression of G3BP1 and clinicopathological features was analyzed using a chi-square test. Statistical Package for the Social Sciences version 25.0  was used to conduct this analysis . The othIn this study, we hypothesized that G3BP1 might play an important role in OC progression via combing with USP10. We first performed a bioinformatics analysis using several public clinical databases, found that G3BP1 was significantly upregulated in OC with a poor clinical prognosis. We further confirmed that G3BP1 was upregulated in OC clinical specimens. We next conducted siRNA to knockdown the expression of G3BP1 in OVCAR3 cell line. The results showed that genetic inactivation of G3BP1 significantly decreased proliferation, migration, invasion of OVCAR3 cells. To investigate the underlying mechanism, we used the PPI network analysis and IP assay confirmed that USP10 interacted with G3BP1 in OVCAR3 cells. Further, we found that USP10 coordinately promoted proliferation, migration, invasion with G3BP1 in OVCAR3 cells. These results suggested that G3BP1 might serve as a target for the treatment of OC.To analyze the expression of G3BP1 in OC, we analyzed the expression level of G3BP1 in OC and normal tissues from several public clinical databases. The TCGA OC dataset from the Oncomine website showed that G3BP1 was higher expressed in OC than in normal ovarian tissue . We alsoTo verify these results, we performed IHC to evaluate the expression profile of G3BP1 in clinical samples by three tissue chips, which included 134 serous epithelial ovarian cancers, 16 ovarian surface epithelium, and 25 fallopian tube specimens. IHC analysis illustrated that G3BP1 was primarily located in the cytoplasm of OC tissues and manifested as light brown and brown particles . CompareTo determine the functional roles of G3BP1 in cancer progression, we first performed a loss-of-function study by means of siRNA. Based on the result in To explore the mechanisms by which G3BP1 promotes the progression of OC, we used the STRING network from the GeneCards website to determine whether a key protein was related with G3BP1. Analysis of G3BP1 through the PPI network showed 25 correlated proteins . The topTo determine the role of USP10 in OC progression, we investigated cell proliferation after disruption of USP10 by siRNAs , suggesting that it might affect the expression of USP10 at the transcript level. Interesting, G3BP1 knockdown do not influence the USP10 protein expression . We speculate that USP10 is a long half-life protein, G3BP1 mRNA degradation in very short time might not affect the protein level of USP10. As we all know, the protein concentration is determined by the balance between protein synthesis and degradation. It seems reasonable to speculate that if G3BP1 represses the production of USP10 protein together with preventing USP10 protein degradation, the expression USP10 might not change significantly. G3BP1 has been reported to suppress p62&USP10 complex ubiquitination , indicatIn our research, we showed that knockdown G3BP1 plus USP10 could further inhibit the progression of OC in vitro. Our study indicates that the G3BP1-USP10 complex is important for tumor progression. Overall, the G3BP1-USP10 complex may modulate biological processes in several ways: (i) the G3BP1-Caprin1-USP10 complex is involved in stress granule condensation and binds with 40S subunits; (ii) the deubiquitinase complex inhibits the ubiquitination of ribosome 40S subunits via lysosomal degradation at the translational level; (ii i) G3BP1 affects the expression and function of target proteins through changing ubiquitination modification of USP10.Our findings suggest that the G3BP1 might be a promising target for OC therapy and the G3BP1-USP10 complex play vital roles in OC. Although our study implicate the G3BP1-USP10 complex promote the progression of OC, future in-depth investigations are strongly recommended to explore the underlying mechanisms. Moreover, further research involving animal studies and clinical specimens is needed to investigate the precise role of G3BP1-USP10 in OC.In conclusion, we used a combination of computational, biochemical, and functional approaches to demonstrated G3BP1 increased in OC tissues and cell lines, and high G3BP1 expression facilitated malignant biological properties of OC cells. It is the first time for our research to reveal that G3BP1 silencing can suppress the proliferation, migration, and invasion capabilities of OC cells. Also, we identified the role of G3BP1-USP10 complex in OC development. These findings offer a novel therapeutic target for OC treatment.


Aloe vera products, both in food and cosmetics, are becoming increasingly popular due to their claimed beneficial effects, which are mainly attributed to the active compound acemannan. Usually, these end products are based on powdered starting materials. High temperatures during the drying process to obtain the starting materials have several advantages, like shortening the drying time, eliminating toxic aloin and reducing bacterial contamination. Nevertheless, there are two major drawbacks: first, at temperatures of 80 °C or higher, structural changes in acemannan, especially its deacetylation (>46%), are triggered, which does not happen at lower temperatures (14% at 60 °C); secondly, a toxic principle is formed at higher temperatures, resulting in a higher cytotoxicity. Thus, two temperature-dependent but opposing effects cause with a median cytotoxic concentration of CC50 = 0.4× a peak of cytotoxicity at 80 °C; at 60 °C this cytotoxic substance is not formed and at 100 °C aloin is more readily eliminated, resulting in a CC50 = 1.1× and CC50 = 1.4×, respectively. The cytotoxic substance generated by dry heat at 80 °C is not a modified polysaccharide because its polysaccharide-enriched alcohol-insoluble fraction is with CC50 = 0.9× less cytotoxic. Moreover, this substance is polar enough to be washed away with ethanol. Additionally, when Aloe gel is heated at 80 °C under humid conditions (pasteurization), the cytotoxicity does not increase (CC50 = 1.6×). Finally, to produce powdered starting materials from Aloe gel, it is recommended to use temperatures of around 60 °C in order to preserve the acemannan structure  and the low cytotoxicity. Aloe vera have an outer waxy cuticle and below a single cell epidermis, a thin layer of chlorenchyma cells. The vascular bundles produce a bitter tasting exudate (Aloe latex). The inner part consists of a thick watery parenchyma (Aloe fillet), which can be extruded obtaining the Aloe gel [Aloe vera acemannan, the major (50%) storage constituent [Aloe vera gel, the acemannan may undergo important structural modifications [The succulent leaves from Aloe gel . The AloAloe gel ,3. ThereAloe gel . PhysiolAloe gel ,6. Aloe stituent ,7, is costituent ,8,9,10. ications ,12,13,14Aloe vera gel is dehydrated for commercial purpose at temperatures above 60 °C, the average molecular weight of acemannan increases from 45 kDa (fresh Aloe) to 75–81 kDa, which is attributed to losses of galactosyl residues and, also, to deacetylation processes, which, in turn, could contribute to hydrogen bonding between mannose chains, resulting in mannose-rich chains of higher molecular weight [When r weight ,14. Any r weight . Also afr weight .Staphylococcus aureus, Enterobacter faecalis, Escherichia coli, Pseudomonas aeruginosa) decreased when deacetylation of acemannan increased. Generally, Aloe gel is not considered to be toxic or cytotoxic [Aloe vera gel sample, containing much fiber, a cytotoxic concentration for HeLa cells of CC50 = 0.27 g/L (~0.05×) , a cytotoxic concentration of ~1 g/L (0.2×) for HeLa cells [The acemannan polymer may exhibit several biological activities in humans like antitumour, anticancer, antioxidant and immunomodulatory activities . In ordeytotoxic , but forytotoxic  reported [Aloe vera gel, dried at different temperatures. As control was used commercially available 200× lyophilized Aloe inner leaf gel  from Aloe Jaumave .An US enterprise provided nt (ILF) ,22. ThisAloe vera plants grown on the campus of the University in Ocotlán  were cut, which were washed and trimmed, then peeled and extensively washed with distilled water to remove the exudate from their surfaces (fillet). Then, 100 g of fillet were heated under humid conditions (pasteurized) in an autoclave at 60, 80, or 100 °C during 30 min  before the still humid samples were ground using an electrical grinder . On the other hand, 100 g of fillet were dried in an electrical oven at 60, 80, or 100 °C during 15–20 h  achieving a yield of 2.9%, 2.2%, and 1.9%, respectively. Afterwards, the dry samples were ground too, and passed through a sieve with 30 mesh.Fresh whole leaves from DNA barcoding was used for the authentication of plant material used in this work. This method has been widely applied for the rapid and accurate identification of plants using DNA sequences . ApproximatK gene, primers 3F_KIM_f (5′-CGT ACA GTA CTT TTG TGT TTA CGA G-3′) and 1R_KIM_r (5′-ACC CAG TCC ATC TGG AAA TCT TGG TTC-3′), and for amplification of rbcL gene primers rbcLa_R (5′-GTA AAA TCA AGT CCA CCR CG-3′) and rbcLa_F (5′-ATG TCA CCA CAA ACA GAG ACT AAA GC-3′) were used. Amplification reactions were performed in a final volume of 50 µL containing 20 mm Tris-HCl (pH 8.4), 50 mm KCl, 2 mm MgCl2, 0.2 mm dNTPs mix, 1 µM each primer, 20 ng of genomic DNA and 2.5 units of Taq DNA polymerase . Amplifications were performed in a Multigene Thermal Cycler  using the program: 1 step at 94 °C for 5 min, 35 cycles consisting of 30 s at 94 °C, 1 min at 52 °C and 1 min at 72 °C, with a final extension step of 5 min at 72 °C. The presence of single amplification products for each gene was verified by electrophoresis in 0.8% agarose gels and stained with EvaGreen . PCR products were purified using PEG precipitation to remove residual primers and nucleotides [http://www.boldsystems.org/ (accessed on 1 June 2021)) and were submitted to the public sequence repository GenBank [The quality of the extracted genomic DNA was verified by agarose gel electrophoresis and the quantification was achieved by UV spectrophotometry using a Q5000 Spectrophotometer . Primers and PCR conditions were as those reported by the Consortium for Barcode of Life (CBOL) Plant Working Group . For ampleotides . The DNA GenBank .v/v), homogeneized with an Ultra Turrax homogenizer T25 Digital   and boiled for further 5 min as previously described in Waldron and Selvendran (1990) [v/v) ethanol. Then the white residues were washed with absolute ethanol followed by acetone and air-dried overnight. Finally, the AIRs were milled using a laboratory type grain mill  and passed through a 0.5 mm aperture sieve. The AIRs samples were AIR of Inner Leaf Fiber (AILF) and AIR of dehydrated Aloe Gel . The control ILL was completely soluble; thus, it was not possible to prepare AIRs from this sample.Alcohol insoluble residues (AIRs) were prepared from commercial (ILF) and from dehydrated  samples: the samples were suspended in boiling ethanol (85% n (1990) . After cg for 15 min at 20 °C. The supernatants, containing the water-soluble polymer acemannan, and the precipitates, containing the cell wall polysaccharides , were collected and lyophilized. All extracts were stored in a desiccator under anhydrous conditions until later analysis [Dehydrated samples and AIR preparations (100 mg) were suspended in 200 mL distilled water and stirred for 2 h at room temperature. The suspensions were then centrifuged at 13,000× analysis .Aloe vera samples (5 mg) were suspended in 12 M H2SO4 for 3 h, followed by dilution to 1 M and hydrolysed at 100 °C for 2.5 h  [2SO4 (100 °C for 2.5 h) was hydrolysed. Thus, the cellulose content could be estimated by the difference in glucose obtained by Saeman hydrolysis and this milder hydrolysis method. Then, all the hydrolysed samples were neutralized with ammonia and reduced using NaBH4. Next, the neutral sugars present in the samples were derivatized as their corresponding alditol acetates using acetic anhydride and 1-methylimidazole as a catalyst. Finally, the alditol acetates were isothermally separated at 220 °C by gas chromatography (Perking Elmer-Clarus 400 (CR1)), using a 30 m column DB-225  with an internal diameter of 0.25 µm and a film thickness of 0.15 µm, and analysed with an FID detector. Standard mixtures of neutral sugars were derivatized routinely to check GC response factors. Uronic acids were determined colourimetrically, as total uronic acids [2SO4 [Aloe vera samples after Saeman hydrolysis can be found in the Carbohydrate composition was analysed after acid hydrolysis following a modified version of the method reported by Rodríguez-González et al. (2011) . Aloe vel. 1954) . A seconic acids , using aAloe vera derived proton peaks [1H-NMR spectra at 300.13 MHz were recorded on an Avance 300 spectrometer , equipped with a 5-mm broadband multinuclear z-gradient (BBO) probe head. The area under the curve (AUC) was calculated using the acetyl group signal corresponding to nicotinamide. The relative degree of acetylation of processed samples in relation to the fresh (reference) sample was calculated using the following Equation:The degree of acetylation of the acemannan polymer, as the main component of AIR-samples, was analysed using the method described by Bozzi et al. (2007) . Therefoon peaks . Then, t1H-NMR-spectra from different Aloe vera samples to determine the degree of acetylation of acemannan can be found in the Representative 4 cells/cm2 in growth medium (DMEM medium (ATCC), supplemented with 10% FBS ) in 12-well microplates (when necessary round cover slides have been added into the wells before) and incubated overnight at 37 °C, 4% CO2 and 95% RH for attachment. For the experiments, at a confluence of ~80% growth medium was changed by sample-containing growth medium. The samples  were dissolved in concentration of 30 g/L (3×), 10 g/L (1×), 3 g/L (0.3×), 1 g/L (0.1×) in culture medium; the pH was adjusted to pH 7.3 with NaOH, and to inhibit bacterial growth, 50 µg/mL of gentamycin  were added. The commercial samples (200× ILF and ILL) have been dissolved with 15 g/L (3×), 5 g/L (1×), 1.5 g/L (0.3×), 0.5 g/L (0.1×) in growth medium; otherwise, the treatment was the same as for the other samples. As growth control, cells were just grown in growth medium under standard conditions; for the positive control for necrosis, H2O2 to a final concentration of 20 mm was added to the cells.The human intestinal cell line HT-29  was seeded with 4 × 10To measure the impact of the samples on the metabolic activity, the cells were incubated for 24 h under the above-mentioned conditions. Then the sample-containing medium was removed, the cells were washed twice with PBS and 1 mL growth medium, containing 20 µL/mL 2-(4-iodophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium , was added. Afterwards, the cells were incubated further for 4 h.g) to precipitate possible cell debris before measuring the absorbance at λ = 440 nm and λ = 690 nm (as background) . The absorbance of the growth control was normalized to 100%, all other values are expressed relative to this (relative WST-activity).Finally, for quantitative analysis, 800 µL WST-1 containing growth medium was centrifuged  trypan blue  was added, incubated for 5 to 10 min, the cells grown on cover slides were washed with PBS and then observed under the microscope  .2O2. The parameter CC50 was estimated by interpolating the half-maximal relative cell viability in the Boltzmann equation fitted to the experimental plot of relative cell viability vs. concentration using the software Origin 5.0 . Statistical analysis was performed by one-way ANOVA followed by Tukey’s post-hoc test to determine significant difference (p < 0.05) between multiple means using the software Statistica 13.3 .Values for carbohydrate composition, monosaccharide composition and degree of acetylation are expressed as mean ± standard deviation (SD), cytotoxicity values are expressed as mean ± 1.96 × standard error of mean (1.96 × SEM). To calculate SEM for relative WST-activity the following repetitions have been done: 5 × DAG60, 3 × DAG80, 3 × DAG100, 4 × PAG60, 4 × PAG80, 4 × PAG100, 3 × AAG60, 4 × AAG80, 3 × AAG100, 4 × ILF, 4 × AILF, 3 × ILL, 8 × HAloe vera, were authenticated by PCR amplification and DNA sequencing of regions of rbcL and matK plastid genes, which have been proposed as the core plant barcode [rbcL fragment and of 851 bases for matK in the fresh and industrial (ILF) sample. For similarity searches, these sequences were submitted to the Barcode of Life Data Systems (http://www.boldsystems.org/ (accessed on 1 June 2021)) using the Plant Identification tool and all fragments displayed 100% of similarity (without gaps) with the corresponding regions of Aloe vera, confirming the identity of the plant material. A table with a similarity index and a phylogenetic tree can be found in the rbcL) and MW176075 (matK).The commercial Aloe powder ILF as well as the plants, previously visually identified as  barcode . The DNAThe major polymeric constituents from the dehydrated  and commercial  samples and their respective alcohol insoluble residues  were determined . When AIThe relative polysaccharide composition between the dehydrated samples  is quite similar with ~35–39% cellulose, ~26–29% hemicelluloses, ~22–25% acemannan and ~11–13% pectins A and doeAdditionally, the monosaccharide composition, i.e., glucose, mannose, galactose and uronic acids, of the major components was quantified from the dehydrated  and commercial (ILF) samples as well as their respective alcohol-insoluble residues  .The glucose content does not vary among the dehydrated samples (~48–50%) A, but diAloe vera gel was evaluated by estimating their cytotoxic effects. This was determined by the reduction of tetrazolium salt WST by dehydrogenase activity of the human intestinal cell line HT-29, which represents a cell line of first contact when those products would be ingested. First, the dehydrated Aloe fillets  exhibited a low cytotoxicity for DAG60 with CC50 = 1.1× and for DAG100 with CC50 = 1.5×. Not expected was a higher cytotoxicity for DAG80 with CC50 = 0.4× : In all cases cytotoxicity decreased, for PAG60 to CC50 = 2.8×, for PAG80 to CC50 = 1.6× and for PAG100 to CC50 = 2.5× . Here, too, cytotoxicity was reduced in all cases compared to the dehydrated samples: for AAG60 to CCC50 > 3× C.50 > 1×, do not exhibit cytotoxic effects up to a concentration of 1× (In addition to the metabolic activity (WST test), the cytotoxicity was confirmed by trypan blue staining. The Aloe samples treated at 60 °C or 100 °C, which have CCon of 1× B. This ion of 1× C; the coon of 1× E, while on of 1× D. In genon of 1× F, similaon of 1× G.2O as required, the degree of acetylation could only be determined for their respective AIRs . Freshly lyophilized Aloe gel was used as a reference. Generally, the AIR-fraction is enriched in acemannan, compared to the dehydrated samples—with the exception of the industrial ILF-sample  could not be dissolved completely in DF-sample . This isF-sample . AAG60 hF-sample . The samF-sample .Aloe vera [Aloe vera have a high content of polysaccharides, in particular acemannan, and this is also the main reason why different effects of drying procedures –including method, temperature, pH, O2 and the presence of other compounds—as well as the polysaccharides and their chemical and biological properties—like chain length, monosaccharide composition, emulsifying activity or structural conformation– are widely studied [Polysaccharides are the most abundant components in vegetal tissues. Generally, they can be divided into cell wall polysaccharides, such as cellulose, hemicelluloses and pectins, and storage polysaccharides like acemannan, in the case of loe vera . Acemann studied .Aloe vera gel, the industrial sample ILF, which is especially prepared in order to keep a high fiber content [Although acemannan is due to its bioactivity the most interesting polysaccharide in  content , has wit50 = 1.1× and DAG100 CC50 = 1.5×), but, astonishingly, when dehydrated at 80 °C (DAG80) the sample was, with CC50 = 0.4×, rather cytotoxic—similar to the industrial sample ILF, which has a CC50 = 0.3×. This cytotoxic effect cannot be associated directly with the acemannan concentration of the samples; for example, DAG80 with 161 mg/g or ILF with 135 mg/g were cytotoxic, while DAG100 with 174 mg/g or AILF with 85 mg/g were not  [Another possibility is that the heat may promote deacetylation reactions of the acemannan polymer. In fact, Chokboribal et al. (2015)  describe. (2015)  reportedOn the other hand, applying heat has also advantages: high temperatures are a cost-effective manner to accelerate the drying process and, at the same time, to remove the toxic aloin, which is by regulations limited to 10 ppm in foodstuff . Chang eRespective to the cytotoxicity, two temperature-dependent but contrary processes peaked at 80 °C: (i) the reduction of toxicity with increasing temperature due to the elimination of aloin , and  to not introduce a toxic principle of low molecular weight , and (i


B. chinensis based on the inhibition of pro-inflammatory enzyme, human neutrophil elastase (HNE) and anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage. Three stereoisomers of iridal-type triterpenoids (1–3) were isolated from the roots of B. chinensis and their stereochemistries were completely identified by NOESY spectra. These compounds were confirmed as reversible noncompetitive inhibitors against HNE with IC50 values of 6.8–27.0 µM. The binding affinity experiment proved that iridal-type triterpenoids had only a single binding site to the HNE enzyme. Among them, isoiridogermanal (1) and iridobelamal A (2) displayed significant anti-inflammatory effects by suppressing the expressions of pro-inflammatory cytokines, such as iNOS, IL-1β, and TNF-α through the NF-κB pathway in LPS-stimulated RAW264.7 cells. This is the first report that iridal-type triterpenoids are considered responsible phytochemicals for anti-inflammatory effects of B. chinensis.The aim of this study is to explore anti-inflammatory phytochemicals from  Belamcanda chinensis (L.) DC known as She Gan has been used in Traditional Chinese Medicine (TCM) for the clinical treatment of respiratory diseases including bronchitis, asthma, sore throat and so on ) was estimated using a P-2000 Digital Polarimeter . Enzymatic assays were conducted by a SpectraMax M3 Multi-mode Microplate Reader . All chemicals for analyses were of first grade. The root of B. chinensis [imported from China with permission of the Korean Food and Drug Administration (KFDA)] was purchased from a Korean pharmaceutical market .Open column chromatography was conducted using octadecylsilanized (ODS) silica gel . Preparative recycling high pressure liquid chromatography (HPLC) was carried out by LC-9130G NEXT  using AQ C18  and Acclaim Polar Advantage II C-18 . B. chinensis (2.4 kg) were extracted with methanol (10 L × 3) for 2 weeks at room temperature. The accumulated filtrate was evaporated to yield a pale yellow residue (54 g), which was suspended in water (0.5 L) and further fractionated successively with hexane (1L × 3) and ethyl acetate (1L × 3). The hexane fraction (8.1 g) was subjected to ODS silica gel (200 g) using a gradient of water to methanol , which provided five fractions (A–E). HNE inhibitory fractions D–E (1.8 g) were portioned using a preparative HPLC with reversed silica gel CC  and eluted using a gradient of methanol in water  at a rate of 10 mL/min, to yield thirty subfractions (D1–D30). Subfractions D6–D11 (180 mg) were further purified by recycling HPLC with reversed silica gel CC  using a isocratic elution with H2O:ACN  to give rise to compound 1 (7.2 mg) and compound 2 (68.3 mg). Subfractions D24–D27 (95 mg) provided 3 (32.1 mg) through equal recycling HPLC with a reversed silicagel CC using an isocratic elution with H2O:ACN .The dried roots of + 474.3706 . H-1) see .+ 474.3739 . H-1) see .+ 500.3514 . H-1) see .p-nitroaniline after the hydrolysis of N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitro anilide at 405 nm. The inhibitors were dissolved in dimethyl sulfoxide (DMSO) and diluted to a few concentrations. In brief, in a 96-well plate, 10 µL of inhibitor solution and 40 µL of 1.5 mM of MeOSuc-AAPV-pNA were added as a substrate in the 0.02 mM Tris-HCl buffer solutions (pH 8.0). Then, 20 µL of human neutrophil elastase (0.2 unit/mL) was added to the mixture. The test mixtures were incubated and mixed for 15 min at room temperature and then screened at 405 nm for 30 min every 30 s. Inhibitory activities were further characterized by determining the concentration required to inhibit 50% of the enzyme activity (IC50), which was calculated using the following Equation (1), where [I] is the concentration of inhibitor.Human neutrophil elastase (EC 3. 4. 21. 37)  activity was measured in accordance with the previous description  with subKm) and maximal velocity (Vmax) were investigated by a Lineweaver–Burk plot. The IK, dissociation constants for inhibitor binding to the free enzyme were calculated using a Dixon plot. Equations (2)–(4) are representatives for deriving the aforementioned parameters.The modalities of HNE inhibition were estimated in experiments using particular concentrations of the substrates and inhibitors, respectively. The Michaelis–Menten constant (KSV) was calculated using Equation (5). All experiments were conducted in triplicate, and the mean values were calculated [F0 and F are the fluorescence intensities in the absence and presence of quencher (Q). KSV is the Stern–Volmer quenching constant [LM−1]. For static quenching, the correlation between the fluorescence intensity and the concentration of quencher for the series of reactions can be estimated by Equation (6) [To measure fluorescence from the HNE enzyme, 10 μL of 0.01 unit/mL enzyme solution with 180 μL of Tris-HCl buffer (0.02 mM) was accurately added into the 96-well black immunoplates. Then, 10 μL of incremental concentrations (7.8–31.2 μΜ) of inhibitors were added into each well. All fluorescence spectra were measured from 280 to 400 nm with emission slits regulated to 2.0 nm, and the excitation wavelength was 260 nm. The Stern–Volmer quenching constant (tion (6) .(6)log[F0 and F are the fluorescence intensities in the absence and presence of inhibitor; KA is the binding constant; n is the number of binding sites of the enzyme; Qf is the concentration of inhibitor.2. The medium was replaced every 2 days. After treatment with chemicals, the cell morphological changes were observed under a microscope and images are captured before cell viability assay.The mouse macrophage cell line RAW264.7 was obtained from the American Type Culture Collection . The cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C with 5% CO4 cells/well) was seeded with RAW264.7, a mouse macrophage cell line. After 24 h of chemical treatment, each well was filled with 20 µL of 5 mg/mL MTT solution (0.1 mg/mL) and incubated for 4 h. After aspirating the supernatants, the formazan crystals in each well were dissolved in 200 µL of dimethyl sulfoxide (DMSO) for 30 min at 37 °C, and the 24-well plates were read at 570 nm with a microplate reader . Data are expressed as a percentage of viable cells compared with the control.Cell viability was determined calorimetrically using a 3--2,5-diphenyl tetrazolium bromide (MTT) reagent , Duchefa Biochemie, Haarlem, The Netherlands). The MTT assay procedures were performed as described previously . Briefly®, Carlsbad, CA, USA) according to the manufacturer’s instructions. First strand cDNA was synthesized from the total RNA (3 μg) using oligo dT  and was used as a template for PCR amplification with G-Taq polymerase . The first-strand cDNA was quantified using a spectrophotometer . The quantified cDNA was used as a template. PCR assay was performed with specific primers for inducible nitric oxide (iNOS), IL-1β, and TNF-α. The primer sequences are listed in TM CL1500 imaging system . The DNA fragments were directly sequenced with the ABI PRISM® 3100-Avant Genetic Analyzer .Total RNA was isolated from cultured RAW264.7 cells using TRIzol reagent (Ambion4 cells/well) were cultured in a 24-well plate. The cells were pretreated with triterpenoids (1 or 3 µg/mL) 2 h prior to lipopolysaccharides  treatment for 16 h. Supernatants were collected and centrifuged at 2451× g for 10 min. The (50 μL) of supernatants were mixed with equal volumes of Griess reagent , and incubated for 10 min. The optical density was measured at 550 nm, and the nitrite levels were calculated from a standard curve generated from sodium nitrite.Nitrite levels were determined using the Griess method, as described previously . Briefly, RAW264.7 cells (5 × 105 cells/well) were cultured in a 6-well plate for 24 h and treated with compounds (1 or 3 µg/mL) 2 h prior to LPS treatment, followed by LPS (1 µg/mL) for 16 h. The concentration of pro-inflammatory cytokines in the collected supernatant was quantified using an ELISA kit  according to the manufacturer’s instructions. Briefly, 50 μL of assay diluent, sample, and standards were added to the ELISA well plate, which were pre-coated with anti-mouse IL-1β and TNF-α antibody. The plates were then covered with adhesive strip, agitated, and incubated for 2 h at room temperature. The ELISA well-plate was washed five times with a wash buffer, then 100 μL of mouse IL-1β and TNF-α conjugates was added and incubated at room temperature for 2 h. The plate was washed five times, 100 μL of substrate solution was added to each well, and incubated for 30 min in the dark. The reaction was quenched by the addition of 100 μL stop solution to each well, and the absorbance was read 450 nm/570 nm with an ELISA reader .RAW264.7 cell (2 × 10RAW264.7 cells cultured on round cover slips coated with poly-L-lysine were treated with triterpenoids (1 or 3 µg/mL) 2 h prior to LPS treatment, followed by LPS (1 µg/mL) for 16 h. The cells were washed with 1 x phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde for 30 min. After washing the fixed cells with PBS, the cells were permeabilized with a blocking buffer  for 30 min at room temperature, and were incubated with an anti-NF-κB p65 antibody  at 4 °C overnight. After three washes in PBS, the cells were incubated in the dark for 1 h with the mixture of FITC-conjugated anti-rabbit IgG fluorescent secondary antibody diluted at 1:500 in PBS. Finally, the cells were stained with 2 µg/mL Hoechst for nuclei staining. The cells were washed with PBS, wet-mounted on glass slides, and observed using a confocal laser-scanning microscope . The negative control (NC) was analyzed by omitting the primary antibody.g using Eppendorf centrifuge 5424 R at 4 °C for 5 min. The resulting pellets were incubated on ice for 30 min with 2% Triton nuclear isolation buffer and were spun at 12,448× g at 4 °C for 15 min. For the cytosolic fraction, the resulting supernatants in the hypotonic lysis buffer were transferred to a new 1.5 mL tube and spun at 15,401× g for 15 min at 4 °C. The resulting supernatants were centrifuged at 100,000× g at 4 °C for 60 min in an ultracentrifuge . The proteins of nuclear and cytosolic fractions were separated on 8% SDS-PAGE gel and blotted onto polyvinylidene difluoride membranes. Equal amounts of 35 µg were analyzed by Western blotting. Briefly, the membranes were blocked with 5% fat-free dry milk and then incubated with the anti-NF-κB p65 polyclonal antibody , and anti-β-actin monoclonal antibodies (1:5000 dilution). The primary antibody incubation was followed by incubation with a secondary horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibody at 1:5000 dilution . Immunopositive bands were visualized by iBright™ CL1500 Imaging System . The cytosolic and nuclear fractions were verified to be free of contaminating nucleus and cytosol by immunoblotting for the cytosolic marker β-actin and the nuclear marker lamin, respectively.RAW264.7 cells were homogenized in a hypotonic lysis buffer and centrifuged at 142× g for 5 min at 4 °C. The resulting pellets and supernatants were used for the isolation of nuclear and cytosoplasmic fraction, respectively. The pellets were incubated on ice for 5 min with a nuclear isolation buffer and were spun at 142× p < 0.05.One-way ANOVA with post hoc comparisons using the Bonferroni test were used to analyze the difference among groups . Data were presented as the mean ± S.D., and significance was set at B. chinensis has been considered a representative medicinal plant for inflammatory diseases such as bronchitis, asthma, and sore throat. Iridal-type triterpenoids (1–3) were found to effectively inhibit the pro-inflammatory enzyme, human neutrophil elastase (HNE). The Stern–Volmer quenching constant (Ksv) and the binding constant (KA) were in accordance with inhibitory potencies (IC50). The HNE enzyme had only a single binding site for iridal-type triterpenoids. The spiro-type compound 3 showed a 2–3 fold better HNE inhibition and binding affinities than the monocyclic ones (1 and 2). Among them, isoiridogermanal (1) and iridobelamal A (2) effectively suppressed the expression of pro-inflammatory cytokines, such as iNOS, IL-1β and TNF-α through blocking the NF-κB pathway. In summary, iridal-type triterpenoids might be prime phytochemicals for the anti-inflammation potential of B. chinensis.