Isolation and identification of belamcandaoids A-N from Belamcanda chinensis seeds and their inhibition on extracellular matrix in TGF-ß1 induced kidney proximal tubular cells.

Belamcandaoids A-N (1-14), fourteen new triterpenoids were isolated from the seeds of Belamcanda chinensis. Their structures including absolute configurations were assigned by using spectroscopic, computational, and crystallographic methods. All the compounds except 1 and 2 are 3,4-seco-triterpenoids belonging to fernane type. Biological evaluation results indicated that 3 and 13 could reduce fibronectin and collagen I expression respectively in TGF-ß1 induced kidney proximal tubular cells.

Phenolic Compounds from Belamcanda chinensis Seeds.

Two new sucrose derivatives, namely, belamcanosides A (1) and B (2), together with five other known compounds (3-7), were isolated from the seeds of Belamcanda chinensis (L.) DC. Their structures were identified based on spectroscopic data. Especially, the absolute configurations of fructose and glucose residues in 1 and 2 were assigned by acid hydrolysis, followed by derivatization and gas chromatography (GC) analysis. Among the known compounds, (-)-hopeaphenol (3), (+)-syringaresinol (4), and quercetin (5), were isolated from B. chinensis for the first time. In addition, biological evaluation of 1 and 2 against cholesterol synthesis and metabolism at the gene level was carried out. The results showed that compounds 1 and 2 could regulate the expression of cholesterol synthesis and metabolism-associated genes, including 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), squalene epoxidase (SQLE), low density lipoprotein receptor (LDLR), and sortilin (SORT1) genes in HepG2 cells.

First total synthesis of (+)-broussonetine W: glycosidase inhibition of natural product & analogs.

The first total synthesis of (+)-broussonetine W (4), a naturally-occurring pyrrolidine iminosugar isolated from the traditional Chinese medical plant Broussonetia kazinoki SIEB (Moraceae), has been completed through a concise synthetic route starting from the readily available d-arabinose derived cyclic nitrone 10 in 11 steps and 31% overall yield, with regioselective installation of the α,ß-unsaturated ketone functional group by the elimination of HBr from α-bromoketone as the key step. A number of analogs of (+)-broussonetine W (4) with variable side chain length, different polyhydroxylated pyrrolidine core configurations or saturated cyclohexanones have also been prepared to explore the glycosidase inhibition and the preliminary structure-activity relationship of this intriguing class of compounds. Glycosidase inhibition studies identified the natural product (+)-broussonetine W (4) as a selective and potent inhibitor of ß-galactosidase (IC50 = 0.03 µM), while its enantiomer was a selective and potent inhibitor of α-glucosidase (IC50 = 0.047 µM). It was found that the configuration of the polyhydroxylated pyrrolidine ring played a key role on their glycosidase inhibitory activities. The length of side chain and α,ß-unsaturated ketone functional group also exhibited some effect on their glycosidase inhibition.

Reference Genes for Expression Analyses by qRT-PCR in Enterobacter cancerogenus.

The Enterobacter cancerogenus strain EcHa1 was isolated from the dead larvae of Helicoverpa armigera, and has the potential for biocontrol of some Lepidoptera insects. In order to screen insecticidal-related genes by qRT-PCR, stable endogenous reference genes used for normalizing qRT-PCR data were selected and evaluated from 13 housekeeping genes (HKGs). The expression levels of the HKGs were determined using qRT-PCR under different experimental conditions, including two culture temperatures and three bacterial OD values. Five stability analysis methods (Ct, BestKeeper, NormFinder, geNorm, and RefFinder) were used to comprehensively rank the candidate genes. The results showed that the optimal reference genes varied under different experimental conditions. The combination of gyrA and gyrB was recommended as the best reference gene combination at 28 °C, while gyrA and rpoB was the best combination at 37 °C. When the OD values were 0.5, 1.0 and 2.0, the recommended reference gene combinations were ftsZ and gyrA, rpoB and gyrB, and gyrA and pyk, respectively. The most suitable reference genes were gyrA and gyrB under all experimental conditions. Using gyrA and gyrB as the reference genes for qRT-PCR, EcHa1 was found to invade all tissues of the H. armigera larvae, and expressed a candidate pathogenic factor Hcp at high levels in gut, Malpighian tubules, and epidermis tissues. This study not only establishes an accurate and reliable normalization for qRT-PCR in entomopathogenic bacteria but also lays a solid foundation for further study of functional genes in E. cancerogenus.

Design and synthesis of 6-C-alkyl-DMDP type nanomolar inhibitors of ß-galactosidase and ß-glucosidase based on broussonetine S and related derivatives.

Broussonetine S (9), its C-1' and C-10' stereoisomers, and their corresponding enantiomers have been synthesized from enantiomeric arabinose-derived cyclic nitrones, with cross metathesis (CM), epoxidation and Keck asymmetric allylation as key steps. Glycosidase inhibition assays showed that broussonetine S (9) and its C-10' epimer (10'-epi-9) were nanomolar inhibitors of bovine liver ß-galactosidase and ß-glucosidase; while their C-1' stereoisomers were 10-fold less potent towards these enzymes. The glycosidase inhibition results and molecular docking calculations revealed the importance of the configurations of pyrrolidine core and C-1' hydroxyl for inhibition potency and spectra. Together with the docking calculations we previously reported for α-1-C-alkyl-DAB derivatives, we designed and synthesized a series of 6-C-alkyl-DMDP derivatives with very simple alkyl chains. The inhibition potency of these derivatives was enhanced by increasing the length of the side chain, and maintained at nanomolar scale inhibitions of bovine liver ß-glucosidase and ß-galactosidase after the alkyl groups are longer than eight or ten carbons for the (6R)-C-alkyl-DMDP derivatives and their 6S epimers, respectively. Molecular docking calculations indicated that each series of 6-C-alkyl-DMDP derivatives resides in the same active site of ß-glucosidase or ß-galactosidase with basically similar binding conformations, and their C-6 long alkyl chains extend outwards along the hydrophobic groove with similar orientations. The increasing inhibitions of ß-glucosidase and ß-galactosidase with the number of carbon atoms in the side chains may be explained by improved adaptability of longer alkyl chains in the hydrophobic grooves. In addition, the lower ß-glucosidase and ß-galactosidase inhibitions of (6S)-C-alkyl-DMDP derivatives than their C-6 R stereoisomers can be attributed to the misfolding of their alkyl chains and resulted decreased adaptability in the hydrophobic groove. The work reported herein is valuable for design and development of more potent and selective inhibitors of ß-galactosidase and ß-glucosidase, which have potential in treatment of lysosomal storage diseases. Furthermore, part of the 6-C-alkyl-DMDP derivatives and their enantiomers were also tested as potential anti-cancer agents; all the compounds tested were found with moderate cytotoxic effects on MKN45 cells, which would indicate potential applications of these iminosugars in development of novel anticancer agents.

One new furanone analogue from the deep-sea fungus Purpureocillium sp. SCSIO 06693.

A new furanone analog, (E)-2-(8,9-dihydroxy-6,8-dimethyldec-4-en-2-yl)-met-hylfuran-3(2H)-one (1), together with six known compounds, including two diterpenoids (2 and 3), one butyrolactone (4) and three isocoumarins (5-7), were isolated from a deep-sea fungus, Purpureocillium sp. SCSIO 06693. Among them, compound 1 existed as two tautomeric forms (1a and 1b) differing in configuration of the furan ring. The chemical structures were elucidated by the basis of spectroscopic evidences, including HRESIMS, NMR and optical rotation. Isolated compounds were evaluated for their cytotoxic, antiviral, antibacterial, antioxidant, acetyl cholinesterase (AChE) and pancreatic lipase (PL) enzyme inhibitory activities. Biological evaluation results revealed that compound 4 showed modest antioxidant activity against DPPH with IC50 value of 72.03 µM. In addition, compounds 1-4 exhibited PL enzyme inhibitory activities.

Bisabolane-type sesquiterpenoids and bacillibactin from the marine-derived fungus Aspergillus sydowii SCSIO 41041 and their enzyme inhibiting activity.

Twelve compounds, including eleven bisabolane-type sesquiterpenoids (1 - 11), and one bacillibactin (12) were identified from marine-derived fungus Aspergillus sydowii SCSIO 41041 isolated from Creseis acicula. The chemical structures were elucidated by the basis of spectroscopic evidences, including HRESIMS, NMR and optical rotation. Biologically, all compounds were evaluated for their acetyl cholin-esterase (AChE) enzyme, pancreatic lipase (PL) enzyme, neuraminidase (NA) and phosphodiesterase 4 (PDE4) inhibitory activities. Compound 12 displayed significant inhibitory activity against neuraminidase (NA) with an IC50 value of 24.0 µM, which was equivalent to the positive drug oseltamivir phosphate (IC50 value of 20.0 µM). And the NA inhibitory activity was confirmed by molecular docking analysis.

Nanomolar ß-glucosidase and ß-galactosidase inhibition by enantiomeric α-1-C-alkyl-1,4-dideoxy-1,4-imino-arabinitol derivatives.

A series of α-1-C-alkyl DAB (1,4-dideoxy-1,4-imino-d-arabinitol) and LAB (1,4-dideoxy-1,4-imino-l-arabinitol) derivatives with aryl substituents have been designed as analogues of broussonetine W (12), and assayed as glycosidase inhibitors. While the inhibition spectrum of α-1-C-alkyl DAB derivative 16 showed a good correlation to that of broussonetine W (12), introduction of substituents on the terminal aryl (17a-f) or hydroxyl groups at C-1' position of the alkyl chains (18a-e) decreased their α-glucosidase inhibitions but greatly improved their inhibitions of bovine liver ß-glucosidase and ß-galactosidase. Furthermore, epimerization of C-1' configurations of compounds 18a-e clearly lowered their inhibition potency of bovine liver ß-glucosidase and ß-galactosidase. Notably, some of the α-1-C-alkyl DAB derivatives were also found to have potent human lysosome ß-glucosidase inhibitions. In contrast, enantiomers of compounds 18a-e and 1'-epi-18a-e generally showed increased α-glucosidase inhibitions, but sharply decreased bovine liver ß-glucosidase and ß-galactosidase inhibitions. Molecular docking calculations unveiled the novel two set of binding modes for each series of compounds; introduction of C-1' hydroxyl altered the conformations of the pyrrolidine rings and orientation of their long chains, resulting in improved accommodation in the hydrophobic grooves. The compounds reported herein are very potent ß-glucosidase and ß-galactosidase inhibitions with novel binding mode; and the structure-activity relationship provides guidance for design and development of more pyrrolidine pharmacological chaperones for lysosomal storage diseases.

Sidt2 is a key protein in the autophagy-lysosomal degradation pathway and is essential for the maintenance of kidney structure and filtration function.

The regulation and homeostasis of autophagy are essential for maintaining organ morphology and function. As a lysosomal membrane protein, the effect of Sidt2 on kidney structure and renal autophagy is still unknown. In this study, we found that the kidneys of Sidt2-/- mice showed changes in basement membrane thickening, foot process fusion, and mitochondrial swelling, suggesting that the structure of the kidney was damaged. Increased urine protein at 24 h indicated that the kidney function was also damaged. At the same time, the absence of Sidt2 caused a decrease in the number of acidic lysosomes, a decrease in acid hydrolase activity and expression in the lysosome, and an increase of pH in the lysosome, suggesting that lysosomal function was impaired after Sidt2 deletion. The accumulation of autophagolysosomes, increased LC3-II and P62 protein levels, and decreased P62 mRNA levels indicated that the absence of the Sidt2 gene caused abnormal autophagy pathway flow. Chloroquine experiment, immunofluorescence autophagosome, and lysosome fusion assay, and Ad-mcherry-GFP-LC3B further indicated that, after Sidt2 deletion, the production of autophagosomes did not increase, but the fusion of autophagosomes and lysosomes and the degradation of autophagolysosomes were impaired. When incubating Sidt2-/- cells with the autophagy activator rapamycin, we found that it could activate autophagy, which manifested as an increase in autophagosomes, but it could not improve autophagolysosome degradation. Meanwhile, it further illustrated that the Sidt2 gene plays an important role in the smooth progress of autophagolysosome processes. In summary, the absence of the Sidt2 gene caused impaired lysosome function and a decreased number of acidic lysosomes, leading to formation and degradation disorders of the autophagolysosomes, which eventually manifested as abnormal kidney structure and function. Sidt2 is essential in maintaining the normal function of the lysosomes and the physiological stability of the kidneys.

Dysregulation of FOXO transcription factors in Epstein-Barr virus-associated gastric carcinoma.

Epstein-Barr virus (EBV) infection is associated with the development of gastric cancer (GC). Forkhead box class O (FOXO) transcription factors play important roles in tumor suppression. This study aims to investigate the interplay between EBV and FOXOs in EBV-associated GC (EBVaGC). The results showed that EBV infection of GC cells led to the downregulation of FOXO1 by the inhibition of its mRNA and protein expression. FOXO3 protein is repressed by EBV infection. FOXO4 mRNA is upregulated in EBV-positive cell lines, while its protein expression is downregulated. FOXO1, FOXO3 and FOXO4 proteins are upregulated following PI3K inhibition in GT39 cells, confirming that they are partially suppressed by the PI3K/AKT pathway. However, the upregulation of FOXO1 and FOXO3 by single transfection with LMP1 or LMP2A implies that the dysregulation of FOXOs in EBVaGC is affected by various EBV latent genes and that PI3K/AKT signaling is not the only mechanism of FOXO regulation.

Evaluating the effects of transgenic Bt rice cultivation on soil stability.

Insecticidal crystal (Cry) proteins produced by genetically modified rice that enter the soil via pollen dispersal, plant residues, and root exudation may disturb soil health. In the present study, we assessed the influences of transgenic Bt rice (i.e., HH1 with Cry1Ab/Cry1Ac) cultivation on the dynamics of soil carbon and nutrients under field conditions during 2013-2016. Transgenic treatments (transgenic Bt rice vs. its parental line (i.e., MH63) of non-Bt rice) have no consistently significant effects on soil property, including available nitrogen, available phosphorus, available potassium, total nitrogen, and total phosphorus, while apparent seasonal changes were observed. Besides, the variations of soil nutrients in the paddy field of transgenic Bt rice did not exceed their resistance capacities, except total organic carbon (TOC; RS (resistance) = 1.51) and total potassium (TK; RS = 2.62) in 2013 and TK (RS = 1.94) in 2014. However, the TOC and soil nutrient of TK in the paddy field of transgenic Bt rice have recovered to the pre-perturbation status after harvest (RL (resilience) = 1.01, F = 0.01, P = 0.91; RL = 0.98, F = 0.34, P = 0.58; RL = 0.99, F = 1.26, P = 0.29). Moreover, the paddy yield of transgenic Bt rice was consistently higher than that of its parental line of non-Bt rice. These results suggested that the cultivation of transgenic Bt rice has no adverse impact on soil stability in terms of soil carbon and nutrients and paddy yield.

Belamchinanes A-D from Belamcanda chinensis: Triterpenoids with an Unprecedented Carbon Skeleton and Their Activity against Age-Related Renal Fibrosis.

Belamchinanes A-D (1-4), four triterpenoids with an unprecedented skeleton, were isolated from the seeds of Belamcanda chinensis and fully characterized. The structures of belamchinanes feature a 4/6/6/6/5 polycyclic system in which a four-membered carbocyclic ring bridges the C-1 and C-11 positions of a classical triterpenoid framework. A plausible pathway for the biosynthesis of 1-4 is proposed. Biological studies reveal that 1-4 dose-dependently protect age-related renal fibrosis in vitro .

Clinical Significance of Dynamics of Programmed Death Ligand-1 Expression on Circulating CD14+ Monocytes and CD19+ B Cells with the Progression of Hepatitis B Virus Infection.

Programmed death-1 (PD-1) expression has been revealed to be upregulated on T cells and contributes to T cell exhaustion in patients with hepatitis B virus (HBV) infection. In this study, we investigated the dynamic expression of programmed death ligand-1 (PD-L1), the ligand of PD-1, on circulating CD14+ monocytes and CD19+ B cells of HBV-infected patients at the stages of chronic HBV (CHB) infection, liver cirrhosis (LC), and hepatocellular carcinoma (HCC), respectively. The results showed that compared with healthy controls, the levels of PD-L1 expression on CD14+ and CD19+ populations were both upregulated in CHB, LC, and HCC groups. Although there was no significant difference of PD-L1 expression on CD14+ population among three disease groups, further analysis demonstrated that the frequency of CD14+PD-L1+ population was negatively correlated with HBV DNA load, the levels of alanine aminotransaminase (ALT), and the levels of aspartate aminotransferase (AST), respectively, at CHB stage, while it did not present significant correlation with such parameters at LC stage and was only positively correlated with HBV DNA load at HCC stage. Similarly, the levels of PD-L1 expression on CD19+ population also did not present much difference among three disease groups. Intriguingly, the frequencies of CD19+PD-L1+ population at CHB and LCC stages were both positively correlated with the levels of ALT and AST, but they were not significantly correlated with HBV DNA load. Thereby, the current study elucidated the dynamics of PD-L1 expression on monocytes and B cells, along with the dynamic regulation of PD-1 on T cells, which had a close relationship during the progression of HBV infection. Collectively, our findings demonstrated that in the course of HBV infection development, PD-L1 expression on CD14+ monocytes and CD19+ B cells varied and significantly correlated with clinical parameters, which could be utilized as a potential clinical indicator.

[Effects of nitrogen and carbon addition and arbuscular mycorrhiza on alien invasive plant Ambrosia artemisiifolia].

A greenhouse control experiment was conducted to explore the effects of nitrogen and carbon addition and arbuscular mycorrhiza (AM) on the growth of alien invasive plant Ambrosia artemisiifolia (common ragweed). Nitrogen addition had no significant effects on the morphological indices, biomass and its allocation, and absolute growth rate of A. artemisiifolia, but increased the nitrogen content in the aboveground and underground parts of the plant significantly. Carbon addition increased the content of soil available nitrogen. In this case, the biomass allocation in root system for nutrient (nitrogen) absorption promoted, resulting in a remarkable decrease of branch number, total leaf area, specific leaf area (SLA), and leaf mass ratio. As a result, the total biomass decreased significantly. The symbiosis of A. artemisiifolia and AM fungi had great influence on the common ragweed's soil nitrogen acclimation, which enhanced its resource-capture by the increase of SLA, and this effect was more significant when the soil nitrogen content was low. AM fungi played an important role in the growth of A. artemisiifolia in low-nitrogen environment.