Characterization of a glycosyltransferase from Paris polyphylla for application in biosynthesis of rare ophiopogonins and ginsenosides.

Saponins are bioactive components of many medicinal plants, possessing complicated chemical structures and extensive pharmacological activities, but the production of high-value saponins remains challenging. In this study, a 6'-O-glucosyltransferase PpUGT7 (PpUGT91AH7) was functionally characterized from Paris polyphylla Smith var. yunnanensis (Franch.) Hand. -Mazz., which can transfer a glucosyl group to the C-6' position of diosgenin-3-O-rhamnosyl-(1 â†’ 2)-glucoside (1), pennogenin-3-O-rhamnosyl-(1 â†’ 2)-glucoside (2), and diosgenin-3-O-glucoside (5). The KM and Kcat values of PpUGT7 towards the substrate 2 were 8.4 µM and 2 × 10-3 s-1, respectively. Through molecular docking and site-directed mutagenesis, eight residues were identified to interact with the sugar acceptor 2 and be crucial for enzyme activity. Moreover, four rare ophiopogonins and ginsenosides were obtained by combinatorial biosynthesis, including an undescribed compound ruscogenin-3-O-glucosyl-(1 â†’ 6)-glucoside (10). Firstly, two monoglycosides 9 and 11 were generated using a known sterol 3-O-ß-glucosyltransferase PpUGT80A40 with ruscogenin (7) and 20(S)-protopanaxadiol (8) as substrates, which were further glycosylated to the corresponding diglycosides 10 and 12 under the catalysis of PpUGT7. In addition, compounds 7-11 were found to show inhibitory effects on the secretion of TNF-α and IL-6 in macrophages RAW264.7. The findings provide valuable insights into the enzymatic glycosylation processes in the biosynthesis of bioactive saponins in P. polyphylla var. yunnanensis, and also serve as a reference for utilizing UDP-glycosyltransferases to construct high-value or rare saponins for development of new therapeutic agents.

Effects of n-pentanol/biodiesel blend fuels on combustion and conventional and unconventional emission characteristics of diesel engine.

The use of n-pentanol/biodiesel as a diesel engine fuel is one of the important ways to reduce fossil fuel consumption and lower diesel engine emissions. The objective of this work was to investigate the mechanism of the effect of different n-pentanol blending ratios (0%, 10%, 20%, and 30%) on the combustion and emission performance of a common rail diesel engine. Tests were conducted on a four-cylinder supercharged intercooled diesel engine at 1540 r/min with brake mean effective pressures of 0.289, 0.578, and 0.867 MPa. The results showed that with the increase of the n-pentanol blending ratio, the ignition delay was prolonged, the combustion duration was shortened, and the heat release center was shifted forward. The combustion process at medium and high loads was improved. When the blending ratio of n-pentanol reached 20%, the blended fuel showed better combustion characteristics at all three loads, and the peak in-cylinder pressure of the blended fuel increased by 13.74%, 1.95%, and 5.26% at the three loads, respectively, compared with that of pure biodiesel. With the increase of the n-pentanol blending ratio, HC, CH2O, CH4, and CH3CHO emissions increased at all three loads. Soot emission was reduced by 25.86%, 19.71%, and 31.59% at three loads when the n-pentanol blending ratio was 30%. C2H4 emissions increased with the increase of n-pentanol blending ratio at the low-load condition and showed a decreasing tendency at the medium and high loads. At high load conditions, NOx emissions increased with increasing n-pentanol blending ratio, and CO emissions decreased with increasing n-pentanol blending ratio.

Three-dimensional analysis of mandibular and mental canals corroborating with teeth and mental foramen by cone beam computed tomography.

BACKGROUND: This study aimed to analyse the 3D patterns of the mandibular and mental canals (MDC and MC) referring to the surrounding prominent surgical landmarks such as teeth and mental foramen by cone beam computed tomography (CBCT). METHODS: CBCT scans of 354 patients aged 18-67 years with mandibular first premolar to second molar were included and reconstructed 3-dimensionally (3D) by mimics. The parameters of MDC and MC were measured referring to teeth and mental foramen. RESULTS: From the first premolars to the second molars, the mandibular canals showed a trend of gradually closer to the cementoenamel junction (CEJ) of the adjacent teeth and farther away from the buccal cortical plate. The distance of the MDC with the root apexes (RA) was relatively constant from the first premolar to the first molar, but became much closer to the second molar. About 10.8% of the second molars had MDC-RA distances of shorter than 2 mm, and 1.34% even had the MDC superior to the RA. Moreover, the Type III of MC presented in 66.0% of the subjects and had a relatively longer length. Besides, the existence of Type I MC may be related to the MDC featuring with close distances to the RA and CEJ of the adjacent teeth. CONCLUSION: Dentists and surgeons should know the patterns of mandibular and mental canals. A better understanding of the MDC and MC and their relationship to local anatomical landmarks may facilitate the planning of surgeries and alert potential nerve injuries in the operative procedures.

Legumain inhibitor prevents breast cancer bone metastasis by attenuating osteoclast differentiation and function.

Breast cancer is the main lethal disease among females, and metastasis to lung and bone poses a serious threat to patients' life. Therefore, identification of novel molecular mediators that can potentially be exploited as therapeutic targets for treating osteolytic bone metastases is needed. A murine model of breast cancer bone metastasis was developed by injection of 4 T1.2 cells into the left ventricle and hence directly into the arterial system leading to bone. AEP (Asparagine endopeptidase) inhibitor combined with epirubicin or epirubicin alone was administered by intraperitoneal injection into animal model. The presence of bone metastatic and osteolytic lesions in bone were assessed by bioluminescent imaging and X-rays analysis. The expression of EMT (Epithelial-Mesenchymal Transition) relevant genes were examined by Western blotting. Cell migration and invasion were investigated with a transwell assay. Compound BIC-113, small molecule inhibitors of AEP, inhibited AEP enzymatic activity in breast cancer cell lines, and affected invasion and migration of cancer cells, but had no effect on cell growth. In animal model of breast cancer bone metastasis, compound BIC-113 combined with epirubicin inhibited breast cancer bone metastasis and attenuated breast cancer osteolytic lesions in bone by inhibiting osteoclast differentiation and EMT. These results indicate that compound BIC-113 combined with epirubicin has the potential to be used in breast cancer therapy by preventing bone metastasis via improving E-cadherin expression and inhibition of osteoclast formation.

Polydatin administration attenuates the severe sublesional bone loss in mice with chronic spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is often accompanied by rapid and extensive bone mineral loss below the lesion level, and there is currently no gold standard for treatment. Evidence suggests that polydatin (PLD) may help promote osteogenic differentiation and exhibit anti-osteoporotic activity. However, whether PLD could reverse substantial bone loss in SCI patients, especially those with protracted injury, and the underlying regulatory mechanism have not been investigated. STUDY DESIGN: Male C57BL/6J mice were subjected to either contusion SCI or laminectomy at the T8-9 level. Eight weeks after SCI, PLD (40 mg/kg/day) or vehicle was administrated to the mice via the intragastric route for consecutive eight weeks. Blood was collected after the treatment regimen, and the tibiae and femora were removed. Bone marrow stromal cells were isolated from the long bones for ex vivo osteoblastogenesis and osteoclastogenesis assays. RESULTS: Chronic SCI led to a rapid and significant decrease in bone mineral density (BMD) of the distal femur and proximal tibia, resulting in structural deterioration of the bone tissues. Treatment with PLD largely restored BMD and bone structure. In addition, static histo-morphometric analysis revealed that PLD enhanced bone formation and inhibited bone resorption in vivo. PLD also promoted osteoblastogenesis and inhibited osteoclastogenesis ex vivo, which was accompanied by increased OPG/RANKL ratio, and reduced expression levels of CTR, TRAP, NFATc1 and c-Fos. However, PLD had no marked effect on serum 25(OH)D levels and VDR protein expression, although it did significantly lower serum and femoral malondialdehyde levels, inhibited expression level of matrix metallopeptidase 9 (MMP9), upregulated skeletal Wnt3a, Lrp5 and ctnnb1 mRNAs, and increased ß-catenin protein expression. CONCLUSIONS: PLD protected mice with chronic SCI against sublesional bone loss by modulating genes involved the differentiation and activity of osteoclasts and osteoblasts, abating oxidative stress and MMP activity, and restoring the Wnt/ß-catenin signaling pathway.

Accuracy and Safety of Robot-Assisted versus Fluoroscopy-Guided Posterior C1 Lateral Mass and C2 Pedicle Screw Internal Fixation for Atlantoaxial Dislocation: A Preliminary Study.

Objective: To compare the accuracy, efficiency, and safety of robotic assistance (RA) and conventional fluoroscopy guidance for the placement of C1 lateral mass and C2 pedicle screws in posterior atlantoaxial fusion. Methods: The data of patients who underwent posterior C1-C2 screw fixation (Goel-Harm's technique) in our hospital from August 2014 to March 2021 were retrospectively evaluated, including 14 cases under fluoroscopic guidance and 11 cases under RA. The hospital records, radiographic results, surgical data, and follow-up records were reviewed. Accuracy of screw placement was assessed using the Gertzbein and Robbins scale, and clinical outcomes were evaluated by Japanese Orthopedic Association (JOA) score, visual analogue scale (VAS), modified MacNab criteria, and postoperative complications. Results: Baseline characteristics of both groups were similar. The mean estimated blood loss in the fluoroscopic guidance and RA groups was 205.7 ± 80.3 mL and 120.9 ± 31.9 mL, respectively (p = 0.03). The mean surgical duration was 34 min longer with RA compared to that performed with free-hand (FH) method (p = 0.15). In addition, lower intraoperative radiation exposure was detected in the RA group (12.4 ± 1.4 mGy/screw) versus the FH (19.9 ± 2.1 mGy/screw) group (p = 0.01). The proportion of "clinically acceptable" screws (graded 0 and I) was higher in the RA group (93.2%) than that in the FH group (87.5%, p = 0.04). There was no significant difference in the increase of JOA score and decrease of VAS score between the two surgical procedures. Furthermore, there were no significant differences in overall clinical outcome between the two groups and no neurovascular complications associated with screw insertion. Conclusions: RA is a safe and potentially more accurate alternative to the conventional fluoroscopic-guided FH technique for posterior atlantoaxial internal fixation.

AgNTf2 catalyzed cycloaddition of N-acyliminium ions with alkynes for the synthesis of the 3,4-dihydro-1,3-oxazin-2-one skeleton.

A catalyzed process for the synthesis of the 4,6-substituted 3,4-dihydro-1,3-oxazin-2-one skeleton has been developed through cycloaddition of in situ generated acyliminium intermediates with alkynes. A range of chain N,O-acetals and terminal alkynes were amenable for this mild transformation. As a result, a series of desired cycloaddition products were obtained in moderate to good yields.

TMSOTf-mediated synthesis of skipped dienes through the addition of olefins to imines and semicyclic N,O-acetals.

A novel approach to skipped dienes has been developed through the TMSOTf-mediated one-pot addition-substitution of olefins 2a, 2f and 2g with imines 1a-1g, and a series of aryl substituted skipped dienes 3aa-3gf were accordingly obtained in 62%-94% yields. Moreover, semicyclic N,O-acetals 5 and 7 could also undergo this transformation to produce the corresponding skipped dienes 6aa and 6af-6al and 8ba and 8bf-8bk in moderate to good yields.

A One-Pot Approach to 2-Substituted-2-(Dimethoxyphosphoryl)-Pyrrolidines from Substituted tert-Butyl 4-Oxobutylcarbamates and Trimethyl Phosphite.

A novel approach to 2-substituted-2-(dimethoxyphosphoryl)-pyrrolidines 7a-7o and 9a-9r has been developed, which features a TMSOTf-mediated one-pot intramolecular cyclization and phosphonylation of substituted tert-butyl 4-oxobutylcarbamates. The major advantages of this method include simple operation under mild reaction conditions, the use of cheap Lewis acid, and good to excellent yields with high diastereoselectivities (dr up to 99:1).

Synthesis and in vitro antitumour activity of 4(R)-methyl-3-O-phosphonomethyl-α-l-threose nucleosides.

A series of novel α-l-threose nucleoside phosphonate analogs, 4(R)-methyl-3-O-phosphonomethyl-α-l-threose nucleosides, were synthesized in multistep sequences starting from d-xylose. The synthetic sequence consisted of the following key stages: (i) the multistep synthesis of 1,2-O-isopropylidenyl-4(R)-methyl-3-O-phosphonomethyl-l-threose, (ii) the transformation of 1,2-O-isopropylidenyl sugar into suitable 1,2-di-O-acyl l-threose precursor, and (iii) the construction of target α-l-threose nucleoside phosphonate analogs by Vorbrüggen glycosidation reaction, deprotection of acyl group, and hydrolysis of diethyl group on phosphonate. The target nucleoside phosphonates were evaluated for their antitumour activities in cell culture-based assays. Compound 8g, 2-fluroadenosine phosphonate, showed remarkable activity against human breast cancer cell lines (MCF-7 and MDA-MB-231) with IC50 values of 0.476 and 0.391 µM, corresponding to 41- and 47-fold higher potency than the reference compound 5-FU, respectively. Subsequent investigations found that the compound 8g can inhibit the proliferation of breast cancer cells and cell cloning. The mechanistic studies indicated that compound 8g could cause DNA damage to breast cancer cells through the ATM-Chk1/Chk2-cdc25c pathway, leading to blockage of the G2/M phase cycle of breast cancer cells, which ultimately led to apoptosis. Moreover, 8g could inhibit the PI3K/AKT signaling pathway and induce apoptosis. These results indicate that compound 8g holds promising potential as an antitumour agent.

SOCS molecules: the growing players in macrophage polarization and function.

The concept of macrophage polarization is defined in terms of macrophage phenotypic heterogeneity and functional diversity. Cytokines signals are thought to be required for the polarization of macrophage populations toward different phenotypes at different stages in development, homeostasis and disease. The suppressors of cytokine signaling family of proteins contribute to the magnitude and duration of cytokines signaling, which ultimately control the subtle adjustment of the balance between divergent macrophage phenotypes. This review highlights the specific roles and mechanisms of various cytokines family and their negative regulators link to the macrophage polarization programs. Eventually, breakthrough in the identification of these molecules will provide the novel therapeutic approaches for a host of diseases by targeting macrophage phenotypic shift.

Astragaloside IV downregulates the expression of MDR1 in Bel­7402/FU human hepatic cancer cells by inhibiting the JNK/c­Jun/AP­1 signaling pathway.

Previous studies demonstrated that astragaloside IV (ASIV) is a potential P­glycoprotein (P­gp)­mediated multidrug resistance (MDR) reversal agent through mechanisms involving downregulation of the gene expression of mdr1. In order to investigate whether the c­Jun N­terminal kinase (JNK) signaling pathway is involved in the mechanism underlying ASIV­induced downregulated the expression of mdr1, the present study used 5­fluorouracil­resistant Bel­7402/FU human hepatic cancer cells as target cells. ASIV (0.1 mM) decreased the protein expression of phosphorylated (p)­JNK and p­c­Jun in the Bel­7402/FU cells, as determined using western blot analysis. Treatment with the JNK pathway inhibitor, SP600125, at a concentration of 11 µM, decreased the mRNA expression levels of mdr1 and P­gp, as determined using reverse transcription­quantitative polymerase chain reaction and western blot analyses, and similar effects were observed following exposure to ASIV. Furthermore, electrophoretic mobility shift assays demonstrated that the DNA­binding activity of activator protein­1 (AP­1) was decreased by 0.1 mM ASIV or 11 µM SP600125. Flow cytometric analysis revealed that 0.1 mM ASIV or 11 µM SP600125 increased the intracellular accumulation of fluorescent P­gp substrates, including rhodamine 123. Taken together, these results indicated that ASIV reversed the drug resistance of Bel­7402/FU cells by downregulating the expression of mdr1 via inhibition of the JNK/c­Jun/AP­1 signaling pathway.

Cryptotanshinone induces ROS-dependent autophagy in multidrug-resistant colon cancer cells.

The development of novel chemotherapeutic agents is highly desired for colon cancer treatment, in particular for the multidrug-resistant cancer types. Cryptotanshinone (CTS), an active quinoid diterpene isolated from Salvia miltiorrhiza Bunge, was previously reported to induce autophagy in various colon cancer cell lines. However, its mechanisms of action have not been fully understood. The current study aims to explore the mechanisms by which CTS induces autophagy in a multidrug-resistant human colon cancer cell line SW620 Ad300. Using MTT assay, CTS at 10 µM exhibited no significant cytotoxicity on human normal colon fibroblasts CCD-18Co, but induced 45.67% and 48.35% cell death in SW620 and SW620 Ad300 cells, respectively. Further studies revealed that CTS induced weak apoptosis (9.37%) and significant caspase-independent cell death in SW620 Ad300 cells. In the same cell line, CTS also induced significant autophagy, which was found to promote cell death and to mediate the cytotoxicity of CTS in these multidrug-resistant cells. Moreover, activation of ROS-p38 MAPK-NF-κB signaling pathway was involved in autophagic cell death induced by CTS in SW620 Ad300 cells. Interestingly, our results also demonstrated a complementary relationship between CTS-induced apoptosis and autophagic cell death in SW620 Ad300 cells. Taken together, CTS induces autophagic cell death in SW620 Ad300 cells via the ROS-p38 MAPK-NF-κB signaling pathway, and it might be a potential candidate as a chemotherapeutic agent for the treatment of multidrug-resistant colon cancer.

Development and in vivo evaluation of self-microemulsion as delivery system for α-mangostin.

α-Mangostin (MG) is a versatile bioactive compound isolated from mangosteen and possesses significant pharmacokinetic shortages. To augment the potential clinical efficacy, MG-loaded self-microemulsion (MG-SME) was designed and prepared in this study, and its potential as a drug loading system was evaluated based on the pharmacokinetic performance and tissue distribution feature. The formula of MG-SME was optimized by an orthogonal test under the guidance of ternary phase diagram, and the prepared MG-SME was characterized by encapsulation efficiency, size distribution, and morphology. Optimized high performance liquid chromatography method was employed to determine concentrations of MG and characterize the pharmacokinetic and tissue distribution features of MG in rodents. It was found that diluted MG-SME was characterized as spherical particles with a mean diameter of 24.6 nm and an encapsulation efficiency of 87.26%. The delivery system enhanced the area under the curve of MG by 4.75 times and increased the distribution in lymphatic organs. These findings suggested that SME as a nano-sized delivery system efficiently promoted the digestive tract absorption of MG and modified its distribution in tissues. The targeting feature and high oral bioavailability of MG-SME promised a good clinical efficacy, especially for immune diseases.

Thrombus aspirated from patients with ST-elevation myocardial infarction: Clinical and angiographic outcomes.

Objective To investigate differences in clinical and angiographic outcomes between patients with acute myocardial infarction with red and white thrombi. Methods A total of 137 patients with ST-segment elevation myocardial infarction undergoing primary percutaneous coronary interventions were included. Thrombus material was classified as white or red based on its pathology. Information on characteristics of thrombi was available for 97 (70.8%) patients. Results The total ischaemic time was significantly longer in the red thrombus group compared with the white thrombus group. The incidence of major adverse cardiovascular events in hospital was higher in the red thrombus group than in the white thrombus group (15.6% vs 0%). Multivariable logistic analysis showed that the total ischaemic time was the only predictor of thrombus composition (odds ratio 1.353; 95% confidence interval 1.003, 1.826). Conclusion Red thrombi were present in nearly two-thirds of cases, and were associated with a longer ischaemic time and higher incidence of major adverse cardiovascular events in hospital.

siRNA-mediated silencing of Wnt5a regulates inflammatory responses in atherosclerosis through the MAPK/NF-κB pathways.

Previous studies have demonstrated that the aberrant expression of Wnt5a occurs in atherosclerotic lesions. However, the precise role of Wnt5a in the pathogenesis of atherosclerosis remains largely unknown. The present study was undertaken to determine whether the RNA interference of Wnt5a in vivo by adenovirus (Ad)-mediated small interfering RNA (siRNA) transfection is capable of inhibiting the progression of atherosclerosis. Recombinant adenovirus carrying siRNA targeting Wnt5a (Ad-Wnt5a siRNA) was designed. Male apolipoprotein E-deficient (ApoE(-/-)) mice were fed a high-fat diet to induce the pathogenesis of atherosclerosis. Mice were randomly divided into 3 groups (n=15 in each group): the mock group, which received treatment with phosphate-buffered saline (PBS); the Ad-NC group, which received treatment with Ad-non-specific siRNA; and the Ad-Wnt5a siRNA group, which received treatment with Ad-Wnt5a siRNA. Treatment with Ad-Wnt5a siRNA markedly inhibited the mRNA and protein expression of Wnt5a in the aortic tissues. The knockdown of Wnt5a had no significant effect on blood lipid levels, but it suppressed atherosclerotic development and increased plaque stability, which was determined by hematoxylin and eosin staining, picrosirius red staining and Oil Red O staining. Furthermore, the mRNA and protein expression of inflammatory cytokines, including monocyte chemotactic protein-1 (MCP-1), cyclooxygenase-2 (COX-2), matrix metalloproteinase (MMP)-2 and MMP-9 was significantly downregulated in the Ad-Wnt5a siRNA group. In addition, the knockdown of Wnt5a inhibited the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. These results demonstrate that Ad-mediated Wnt5a silencing in vivo attenuates the development of atherosclerotic disease by reducing inflammatory mediators involved in the MAPK/NF-κB pathways.

Astragaloside â £ reduces the expression level of P-glycoprotein in multidrug-resistant human hepatic cancer cell lines.

Astragaloside is a saponin widely used in traditional Chinese medicine and has been reported to be a potent multidrug resistance (MDR) reversal agent. The present study investigated the role of astragaloside Ⅳ (ASIV) in the regulation of P-glycoprotein (P-gp, encoded by the mdr1 gene) and its effect on the reversal of MDR. The activity of ASIV was evaluated using human hepatic cancer cells Bel-7402 and the corresponding 5-fluorouracil (5-FU) resistant cells Bel-7402/FU. ASIV (0.08 mg/ml) potentiated the cytotoxicity of 5-FU which was demonstrated using the MTT assay on Bel-7402/FU cells. ASIV reduced the expression of P-gp as was revealed by immunocytochemistry. Accumulation and efflux studies with the P-gp substrate, rhodamine 123 (Rh123), demonstrated that ASIV inhibited P-gp-mediated drug efflux. Furthermore, it was demonstrated that ASⅣ enhanced the drug accumulation of 5-FU using a high performance liquid chromatography (HPLC) assay for drug resistant cells. Furthermore, ASIV may downregulate the expression of P-gp, which was examined using western blot analysis and polymerase chain reaction. In conclusion, the results of the present study indicated that ASIV reverses the drug resistance of Bel-7402/FU cells by downregulating the expression of mdr1. ASIV may represent a potent modulator of P-gp-mediated MDR in hepatic cancer therapy.

Simvastatin decreases stent-induced in-stent restenosis rate via downregulating the expression of PCNA and upregulating that of p27kip1.

BACKGROUND: The inhibition of the proliferation and migration of vascular smooth muscle cells (VSMC) is one cholesterol-independent effect of statins that could lower the rate of in-stent restenosis (ISR), even if the exact mechanism remains unclear. The aim of this study was to explore the exact inhibitory mechanisms of simvastatin on ISR in vivo. METHODS: Forty-five rabbits were randomized into 3 feeding groups of equal size (n=15): normal rabbit chow (normal group), a high-cholesterol diet (control group), and a simvastatin-enriched high-cholesterol diet (simvastatin group). Balloon de-endothelialization was first performed in the control and simvastatin groups after 3 days, followed by stent deployment at week 14. All rabbits were killed at week 18, and the histological changes of the ISR segments were observed. The expressions of cyclin-dependent kinase inhibitor p27 (p27kip1), proliferating cell nuclear antigen (PCNA), and α-smooth muscle (α-SM) actin were measured. RESULTS: In the simvastatin group compared to the control group, the neointimal thickness, neointimal area, and degree of stenosis decreased, while the residual lumen area increased significantly (P<0.05). Moreover, the expression of α-SM actin in the control group decreased by 55.4% compared to the normal group, while it increased by 29.7% with respect to the simvastatin group (P<0.05). Finally, the expression of p27kip1 increased, while that of PCNA decreased significantly in the simvastatin group compared to the control group (P<0.05). CONCLUSIONS: Simvastatin may inhibit VSMC phenotype modulation and proliferation by downregulating the expression of PCNA and upregulating that of p27kip1.