In this study, we designed and synthesized 12 novel aloperine derivatives with different core structures. Among them, compound 3 with a ten-membered ring core was obtained through a special ring expansion reaction after γ-H Huffman elimination of quaternary ammonium salt, and the structure was verified by X-single crystal diffraction. Furthermore, their antiviral activity against human β-coronavirus HCoV-OC43 was evaluated by CCK-8 assay. Quaternary ammonium salt 2a and 3 had a good inhibitory effect against HCoV-OC43, and 2a had the highest anti-HCoV-OC43 activity with an EC₅₀ values of 3.77 μmol·L^-1 and a SI value of over 53.1. Schrӧdinger molecular docking results showed that both 2a and 3 might display their anti-HCoV-OC43 activity by directly acting on host TMPRSS2 and SR-B1. The results expanded the structural types of endocyclic aloperine and the function against coronavirus, and provided useful scientific data for the development of pharmaceutical applications of these compounds.

Dihydroflavonol 4-reductase (DFR) plays an essential role in the biosynthesis of anthocyanin and regulation of plant flower color. Based on the transcriptome data of Cistanche tubulosa (Schenk) Wight, a full-length cDNA sequence of CtDFR gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR). CtDFR contains an open reading frame (ORF) of 1 263 bp which encodes 420 amino acids with a predicted molecular weight of 47.5 kDa. The sequence analysis showed that CtDFR contains a nicotinamide adenine dinucleotide phosphate (NADPH) binding domain and a specific substrate binding domain. The expression analysis indicated that CtDFR was highly expressed in red and purple flowers, and the relative expression levels were 4.04 and 19.37 times higher than those of white flowers, respectively. The recombinant CtDFR protein was expressed in E.coli BL21 (DE3) using vector pET-28a-CtDFR and was purified. In vitro enzyme activity analysis, CtDFR could reduce three types of dihydroflavonols including dihydrokaempferol, dihydroquercetin, and dihydromyricetin to leucopelargonidin, leucocyanidin and leucodelphinidin. Subcellular localization analysis showed that CtDFR was mainly localized in the cytoplasm. These results demonstrate that CtDFR plays an important role in regulation of flower color in C. tubulosa and make a valuable contribution for the further investigation on the regulation mechanism of C. tubulosa (Schenk) Wight flower color.

Background Perfluoroalkyl substances (PFASs) are classified as persistent organic pollutants and have been widely detected in human. Studies investigating the associations between PFASs exposure and estimated glomerular filtration rate (eGFR) yielded inconsistent results, and little is known about the effects of PFASs on eGFR in population without kidney disease. Objective To explore the associations of exposure to PFASs with eGFR and renal dysfunction in population without kidney disease. Methods A total of 609 participants with an eGFR > 60 mL·min⁻¹·1.73 m⁻² and without renal impairment matched for sex and age (1∶1) were recruited from endocrinology department and medical examination center of two hospitals in Tianjin, China, from April 2021 to March 2022. Each subject was interviewed using a structured questionnaire to collect information about sex, age, height, weight, disease history, smoking, alcohol intake, etc. Clinical parameters were obtained from medical record, such as fasting blood glucose (FBG), creatinine (Cre), total cholesterol (TC), and triglyceride (TG). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured by professionals using standard methods. The serum concentrations of PFASs were determined by liquid chromatography/mass spectrometry. Multivariable linear and logistic regression models were performed to evaluate the associations of PFASs exposure with eGFR and renal dysfunction, respectively. Subgroup analyses stratified by age and sex were also performed to assess the modified effects of covariates on the associations of PFASs exposure with eGFR. Results There were 283 males, accounting for 46.5% of the total population. The mean age of the participants was (56.86±12.47) years, and the average body mass index (BMI) was (25.59±3.84) kg·m⁻². Perfluoro-n-octanoic acid (PFOA), perfluorohexane sulfonic acid (PFHxS), perfluorooctane sulfonic acid (PFOS), perfluoro-n-nonanoic acid (PFNA), perfluoro-n-decanoic acid (PFDA), perfluoro-n-undecanoic acid (PFUnDA), sodium 1H, 1H, 2H, 2H-perfluoro-1-octanesulfonate (6:2 FTS), and perfluoropentane sulfonic acid (PFPeS) were positive in more than 75% of serum samples, and the corresponding median concentrations were 9.50, 1.67, 17.22, 1.86, 1.41, 0.78, 0.42, and 0.43 μg·L⁻¹, respectively. After full adjustments of sex, age, BMI, hypertension, diabetes, TC, TG, smoking, and drinking, the linear regression models showed that log₂-transformed PFHxS concentration was negatively associated with eGFR (b=−1.160, 95%CI: −2.280, −0.410). Compared with the lowest exposure tertile, the estimated change of eGFR in the highest tertile for PFHxS was significantly decreased (b=−2.471, 95%CI: −4.574, −0.368). Furthermore, compared with males, the negative association of PFHxS with eGFR was strengthened among females (female: b=−1.281, 95%CI: −2.388, −0.174; male: b=−0.781, 95%CI: −2.823, 1.261, P_interaction=0.043). Conclusion A significant negative association between serum PFHxS and eGFR is observed in the sampled population without kidney disease, and females are more susceptible to PFASs exposure than the males.

Qualitative and quantitative analysis of 2-(2-phenylethyl) chromones in sodium chloride(NaCl)-treated suspension cells of Aquilaria sinensis was conducted by UPLC-Q-Exactive-MS and UPLC-QQQ-MS/MS. Both analyses were performed on a Waters T3 column(2.1 mm×50 mm, 1.8 μm) with 0.1% formic acid aqueous solution(A)-acetonitrile(B) as mobile phases at gradient elution. MS data were collected by electrospray ionization in positive ion mode. Forty-seven phenylethylchromones was identified from NaCl-treated suspension cell samples of A. sinensis using UPLC-Q-Exactive-MS, including 22 flindersia-type 2-(2-phenylethyl) chromones and their glycosides, 10 5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones and 15 mono-epoxy or diepoxy-5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones. Additionally, 25 phenylethylchromones were quantitated by UPLC-QQQ-MS/MS. Overall, the rapid and efficient qualitative and quantitative analysis of phenylethylchromones in NaCl-treated suspension cells of A. sinensis by two LC-MS techniques, provides an important reference for the yield of phenylethylchromones in Aquilariae Lignum Resinatum using in vitro culture and other biotechnologies.
Chromones ; Sodium Chloride ; Chromatography, Liquid ; Flavonoids ; Tandem Mass Spectrometry ; Thymelaeaceae

Objectives: To investigated the safety and efficacy of treating patients with acute non-ST-segment elevation myocardial infarction (NSTEMI) and elevated levels of N-terminal pro-hormone B-type natriuretic peptide (NT-proBNP) with levosimendan within 24 hours of first medical contact (FMC). Methods: This multicenter, open-label, block-randomized controlled trial (NCT03189901) investigated the safety and efficacy of levosimendan as an early management strategy of acute heart failure (EMS-AHF) for patients with NSTEMI and high NT-proBNP levels. This study included 255 patients with NSTEMI and elevated NT-proBNP levels, including 142 males and 113 females with a median age of 65 (58-70) years, and were admitted in the emergency or outpatient departments at 14 medical centers in China between October 2017 and October 2021. The patients were randomly divided into a levosimendan group (n=129) and a control group (n=126). The primary outcome measure was NT-proBNP levels on day 3 of treatment and changes in the NT-proBNP levels from baseline on day 5 after randomization. The secondary outcome measures included the proportion of patients with more than 30% reduction in NT-proBNP levels from baseline, major adverse cardiovascular events (MACE) during hospitalization and at 6 months after hospitalization, safety during the treatment, and health economics indices. The measurement data parameters between groups were compared using the t-test or the non-parametric test. The count data parameters were compared between groups using the χ² test. Results: On day 3, the NT-proBNP levels in the levosimendan group were lower than the control group but were statistically insignificant [866 (455, 1 960) vs. 1 118 (459, 2 417) ng/L, Z=-1.25,P=0.21]. However, on day 5, changes in the NT-proBNP levels from baseline in the levosimendan group were significantly higher than the control group [67.6% (33.8%,82.5%)vs.54.8% (7.3%,77.9%), Z=-2.14, P=0.03]. There were no significant differences in the proportion of patients with more than 30% reduction in the NT-proBNP levels on day 5 between the levosimendan and the control groups [77.5% (100/129) vs. 69.0% (87/126), χ²=2.34, P=0.13]. Furthermore, incidences of MACE did not show any significant differences between the two groups during hospitalization [4.7% (6/129) vs. 7.1% (9/126), χ²=0.72, P=0.40] and at 6 months [14.7% (19/129) vs. 12.7% (16/126), χ²=0.22, P=0.64]. Four cardiac deaths were reported in the control group during hospitalization [0 (0/129) vs. 3.2% (4/126), P=0.06]. However, 6-month survival rates were comparable between the two groups (log-rank test, P=0.18). Moreover, adverse events or serious adverse events such as shock, ventricular fibrillation, and ventricular tachycardia were not reported in both the groups during levosimendan treatment (days 0-1). The total cost of hospitalization [34 591.00(15 527.46,59 324.80) vs. 37 144.65(16 066.90,63 919.00)yuan, Z=-0.26, P=0.80] and the total length of hospitalization [9 (8, 12) vs. 10 (7, 13) days, Z=0.72, P=0.72] were lower for patients in the levosimendan group compared to those in the control group, but did not show statistically significant differences. Conclusions: Early administration of levosimendan reduced NT-proBNP levels in NSTEMI patients with elevated NT-proBNP and did not increase the total cost and length of hospitalization, but did not significantly improve MACE during hospitalization or at 6 months.
Male ; Female ; Humans ; Aged ; Natriuretic Peptide, Brain ; Simendan/therapeutic use* ; Non-ST Elevated Myocardial Infarction ; Heart Failure/drug therapy* ; Peptide Fragments ; Arrhythmias, Cardiac ; Biomarkers ; Prognosis

OBJECTIVE: To investigate the effects and mechanisms of equol and its enantiomers on urethane-induced lung cancer in mice. METHODS: A total of 120 5-week-old male C57BL/6 mice were randomly divided into 8 groups: lung cancer tumor control group (CG), genistein control group (GCG), low dose racemic equol group (LEG), high dose racemic equol group (HEG), low dose R-equol group (LRE), high dose R-equol group (HRE), low dose S-equol group (LSE) and high dose S-equol group (HSE). Urethane was injected subcutaneously twice a week for 4 weeks to induce lung cancer and then the mice were fed for 4 months. The body weight and food intake of each group were measured and recorded weekly. After the mice were sacrificed, the blood, livers and lungs of the mice were collected. The incidence of lung cancer in each group was recorded. The concentration of serum superoxide dismutase (SOD), malondialdehyde (MDA) and 8-hydroxydeoxygunosine (8-OHdG) were detected by the corresponding kits. Western blotting was used to detect the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the livers. Between-group differences in body weight and food intake of the mice were compared using repeated measures ANOVA, and ANOVA for the differences between non-repeated measurements, with post hoc analysis using Tukey's method if there were between-group differences. Comparisons of categorical data were performed by chi-square test, and if there were differences between the groups, the Bonferroni method was used for pairwise comparison. RESULTS: A total of 49 in the 120 mice developed lung cancer. The overall incidence of lung cancer was 40.8%. Compared with the control group, the incidence of lung cancers in each experimental group was lower, and the difference was statistically significant. The incidence of lung cancer in the high-dose experimental group was significantly lower than that in the low-dose experimental group. However, the incidence of lung cancer was similar in the three equol groups and the genistein group at the same dose. Compared with the control group, the high-dose experimental group had higher serum SOD concentration, lower MDA and 8-OHdG concentrations, and the differences were statistically significant. Western blotting analysis showed that the expression levels of Nrf2 protein in the experimental groups were higher than those in the control group except the low-dose racemic equol group, and the Nrf2 protein expression level in the high-dose equol groups was higher than that in the low-dose equol groups. CONCLUSION Racemic equol and its enantiomers mayinhibit lung carcinogenesis through antioxidant effects.
Animals ; Body Weight ; Equol ; Genistein ; Lung Neoplasms/prevention & control* ; Male ; Mice ; Mice, Inbred C57BL ; NF-E2-Related Factor 2 ; Superoxide Dismutase ; Urethane/toxicity*

Lysine decarboxylase is a key enzyme involved in the upstream biosynthesis of lycopodium alkaloids (LAs) such as huperzine A, contributing to the decarboxylation of lysine to 1,5-pentanediamine (cadaverine). Three lysine decarboxylase genes (HsLDC-L1, HsLDC-L2, HsLDC-L3) were successfully cloned from Huperzia serrata using transcriptomic sequence data mining strategy combined with reverse transcription PCR. The physicochemical properties, secondary and tertiary structures, amino acid identities, and evolutionary relationship of the three LDCs were analyzed by online bioinformatics analysis platforms and DNAMAN, MEGA 7.0 software, revealing that all of these proteins had the conserved PLP binding domain and active site residues were completely conserved in LDCs. Phylogenetic analysis showed that these LDCs were located in the same branch as other known LDCs from LA-producing plants. Accordingly, the ORFs of these three HsLDCs were inserted into different expression plasmids for further expression in E. coli. However, only HsLDC-L1 was successfully expressed in E. coli BL21 (DE3) by inserting into a pCold TF vector. The recombinant protein was purified by Ni²⁺ affinity chromatography purification. HsLDC-L1 contains 469 amino acid residues, with a calculated molecular weight of 50.50 kDa. HsLDC-L1 expectedly catalyzed the decarboxylation of lysine to produce cadaverine. In addition, HsLDC-L1 can also catalyze the generation of putrescine from ornithine. However, it cannot catalyze the decarboxylation of tyrosine, phenylalanine, tryptophan and histidine. The results not only provide insight into the biosynthesis of LAs including huperzine A, but also provide a critical genetic element for the overproduction of Δ¹-piperideine and pelletierine, the essential biosynthetic precursors of LAs, using synthetic biology strategies.

Objective: To analyze the efficacy of sinonasal adenoid cystic carcinoma (ACC) with perineural invasion (PNI), and explore the prognostic value of PNI on sinonasal adenoid cystic carcinoma. Methods: The clinical data of 105 patients with sinonasal ACC admitted to Cancer Hospital, Chinese Academy of Medical Sciences from January 2000 to December 2016 were retrospectively reviewed. All patients were restaged according to American Joint Committee on Cancer 8th edition. Follow-up visits were conducted to obtain information of treatment failure and survival outcome. The Log rank test was used for univariate analysis of prognostic factors, and Cox regression model was used for multivariate prognostic analysis. Results: The maxillary sinus (n=59) was the most common primary site, followed by the nasal cavity (n=38). There were 93 patients with stage Ⅲ-Ⅳ. The treatment modalities included surgery alone (n=14), radiotherapy alone (n=13), preoperative radiotherapy plus surgery (n=10), and surgery plus postoperative radiotherapy (n=68). The median follow-up time was 91.8 months, the 5-year local control (LC), distant metastasis-free survival (DMFS), progression-free survival (PFS), and overall survival (OS) rates were 72.6%, 73.0%, 52.9% and 78.0%, respectively. There were 33 patients (31.4%) with PNI-positive. The 5-year DMFS, PFS, and OS rates of PNI-positive group were 53.7%, 29.4% and 56.5%, respectively, which were significantly inferior to those of PNI-negative group (80.8%, 63.0% and 86.8%, respectively, P<0.05), while there was no significant difference in the 5-year LC rate between both groups (64.5% vs 76.5%, P=0.273). The multivariate Cox regression analysis showed PNI was one of the poor prognostic factors of DMFS (HR=3.514, 95%CI: 1.557-7.932), PFS (HR=2.562, 95%CI: 1.349-4.866) and OS (HR=2.605, 95%CI: 1.169-5.806). Among patients with PNI-positive, the 5-year LC, PFS and OS rates of patients received surgery combined with radiotherapy were 84.9%, 41.3% and 72.7%, respectively, which were significantly higher than 23.3%, 10.0% and 26.7% of patients receiving surgery or radiotherapy alone (P<0.05). Conclusion: The presence of PNI increases the risk of distant metastasis in patients with sinonasal ACC. Compared with patients with PNI-negative, the prognosis of patients with PNI-positive is relatively poor, and surgery combined with radiotherapy for PNI-positive sinonasal ACC results in good clinical outcomes.
Carcinoma, Adenoid Cystic/pathology* ; Humans ; Paranasal Sinus Neoplasms/therapy* ; Prognosis ; Proportional Hazards Models ; Retrospective Studies

Cytochrome P450 reductase (CPR) is essential for the electron transport chain of cytochrome P450s, playing an indispensable role in electron transfer in vivo. In this study, one cDNA encoding cytochrome P450 reductase (Ascpr1) was identified from the callus of Aquilaria sinensis. Ascpr1 contains an open reading frame of 2 124 bp. The deduced protein is composed of 707 amino acids, with a predicted molecular weight of 78.82 kD. Phylogenetic analysis revealed that AsCPR1 is a type Ⅱ CPR protein closely related to the CPR from Theobroma cacao. Transmembrane prediction using TMHMM 2.0 indicated that the amino acids 52-71 of AsCPR1 comprise a transmembrane region. After truncating of 67 amino acid residues from N-terminal, the truncated AsCPR1 was successfully expressed in E. coli Transetta (DE3). Further purification of the recombinant AsCPR1 by affinity chromatography and determination of the enzymatic activity allowed the reducing ability of AsCPR1 to cytochrome C in vitro. The results pave the way for further study on the synthesis of defensive chemicals involved in P450s and the functions of CPR in self-defense of A. sinensis.

Adult ; Body Mass Index ; Cross-Sectional Studies ; Diabetes Mellitus, Type 2/epidemiology* ; Humans ; Obesity/epidemiology* ; Vitamin D ; Vitamin D Deficiency ; Vitamins ; Waist Circumference