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Substrate concentrations greatly influence bacterial growth and metabolism. However, optimal nitrogen concentrations for anammox bacteria in nitrogen-limited environments remain unclear. Here, we observed enhanced nitrogen metabolism and anabolism of anammox bacteria at low nitrogen conditions. Efficient nitrogen removal was achieved at ammonium and nitrite influent concentration of 30 mg/L under HRT of 1 hr, with an average nitrogen removal rate (NRR) of 0.73 kg N/(m3 ·day) in I-ABR composed of four compartments. The highest anammox activity of 6.25 mmol N/ (gVSS·hr) was observed in the fourth compartment (C4) with the lowest substrate levels (ammonium and nitrite of 11.6 mg/L and 7 mg/L). This could be resulted from the highest expression level of genes involved in nitrogen metabolism in C4, which was 1.49-1.67 times higher than that in other compartments. Besides, the second compartment (C2) exhibited the most active anabolism at ammonium and nitrite of 17 mg/L and 13 mg/L, respectively, which contributed to the most active amino acid synthesis and thus the highest EPS (1.35 times higher) in C2. This enhanced amino acid auxotrophy between anammox bacteria with heterotrophs, and consequently, heterotrophs thrived and competed for nitrite. These results hint at the potential application of anammox process in micro-polluted water. PRACTITIONER POINTS: High nitrogen removal and efficient biomass retention at low nitrogen concentrations under short HRT was achieved in I-ABR. Optimal concentrations for anammox nitrogen removal and anabolism were discussed under low nitrogen concentrations. More active anabolism contributed to enhanced amino acid synthesis and thus higher EPS contents. Low substrate levels led to enhanced expression of genes involved in nitrogen metabolism and thus high anammox activity.

Although the niche differentiation of anammox bacteria has been extensively observed in a lab-scale reactor, the inherent mechanism for this ecological phenomenon is still elusive. Here, we combined the long-term reactor operation, genome-centered metagenome, and metatranscriptome analyses to gain insight into the substrate competition and niche differentiation of Candidatus Jettenia and Candidatus Brocadia. After 146 days of operation, we found the anammox bacterial population shifted from Ca. Jettenia to Ca. Brocadia in the immobilization-anaerobic baffled reactor (I-ABR) with the ammonium and nitrite concentrations of 30 mg/L. Importantly, the genome and transcript comparisons of Ca. Jettenia and Ca. Brocadia showed that Ca. Brocadia harbored more complete function in cell chemotaxis, flagellar assembly, and two-component system and more redundant function in nitrite reduction, in which the genes were also highly expressed. Ca. Brocadia out-competed Ca. Jettenia at the mainstream condition. Meanwhile, though the highest biomass concentration led to the highest nitrogen removal rate (NRR) in the first compartment (C1), the competition of Ca. Jettenia and Ca. Brocadia could also affect the NRR of different compartments through affecting the bacterial activity. Substrate competition of anammox bacteria led to higher transcript activity of Ca. Jettenia and Ca. Brocadia in the second (C2) and fourth (C4) compartments, respectively. Further, high transcript activity of Ca. Brocadia led to the higher NRR in C4. A comparison of metabolic potential based on the metagenome-assembled genome adds a different dimension for understanding the discrepantly physiological characteristics and competition of anammox bacteria for wastewater treatment.

Background Breast cancer is the most frequently diagnosed malignancy among women and the second leading cause of cancer death worldwide. Among which nuclear estrogen receptor (nER) negative breast cancer is always with much poor prognosis. Recently, membrane G protein coupled estrogen receptor (GPER), a newly recognized estrogen receptor has been documented to take essential part in the development and treatment of breast cancer. The present study was designed to investigate the anti nER negative breast cancer effect of cryptotanshinone (CPT), an important active compound of traditional Chinese medicine Danshen and its possible molecular pathway. Methods The following in vitro tests were performed in nER negative but GPER positive breast cancer SKBR-3 cells. The effect of CPT on cell proliferation rate and cell cycle distribution was evaluated by MTT cell viability test and flow cytometry assay respectively. The role of PI3K/AKT pathway and the mediated function of GPER were tested by western blot and immunofluorescence. Technique of gene silence and the specific GPER agonist G-1 and antagonist G-15 were employed in the experiments to further verify the function of GPER in mediating the anticancer role of CPT. Results The results showed that proliferation of SKBR-3 cells could be blocked by CPT in a time and dose dependent manner. CPT could also exert antiproliferative activities by arresting cell cycle progression in G1 phase and down regulating the expression level of cyclin A, cyclin B, cyclin D and cyclin-dependent kinase 2 (CDK2). The antiproliferative effect of CPT was further enhanced by G-1 and attenuated by G-15. Results of western blot and immunofluorescence showed that expression of PI3K and p-AKT could be downregulated by CPT and such effects were mediated by GPER which were further demonstrated by gene silence test. Conclusion The current study showed that the antiproliferative action of CPT on SKBR-3 cells was realized by inhibition of GPER mediated PI3K/AKT pathway. These findings provide further validation of GPER serving as useful therapeutic target.

Vidarabine (ARA) was one of the earliest marine-related compounds to be used clinically for antiviral therapy, however, its fast metabolism is the main defect of this drug. To overcome this, we designed and synthesized a group of phosphamide-modified ARA compounds using ProTide technology. With a phosphamide modification, these compounds could become the substrate of specific phospholipase enzymes expressed in the liver. Among all 16 synthesized compounds, most showed stronger activity against herpes simplex virus type 1 (HSV-1) than ARA (EC50 of approximately 10 μM). The top three compounds were compound 2 (EC50 = 0.52 ± 0.04 μM), compound 6 (EC50 = 1.05 ± 0.09 μM) and compound 15 (EC50 = 1.18 ± 0.08 μM) (about 2 times higher than Sp type compound 2). This study provides evidence for use of the phosphamide modification, which could give ARA higher activity and liver cell targeting.

Substrate concentrations generally fluctuate in wastewaters. However, how anammox biomass behaves to overcome the stress of alternating feed loadings remains unclear. Here, we combined long-term reactor operation, batch tests, 16S rRNA transcript sequencing, and metabolomics analysis to investigate the aggregation of anammox biomass under the regulation of c-di-GMP, a key second messenger, in response to alternating feed loadings. We demonstrated that the aggregation process was significantly faster under alternating loadings and was significantly correlated with higher levels of c-di-GMP and extracellular polymeric substances (EPS) production. The increase in c-di-GMP was positively correlated with a higher relative transcript expression level in the c-di-GMP pathway-dependent community. The targeted metabolomics results indicated that the increased production of fructose 6-phosphate and UDP-N-acetyl-d-glucosamine, the precursor substances for the synthesis of exopolysaccharides, was induced by higher levels of c-di-GMP. Consequently, the granulation process was accelerated via EPS production. Higher levels of intracellular hydrophobic amino acids were also positively correlated with increased extracellular protein levels, considering the significant increase in peptides under alternating loadings. On the basis of our findings, we believe that c-di-GMP regulation and EPS production of the anammox biomass are important mechanisms to enhance its tolerance against unfavorable feed stress. These results highlight the role of c-di-GMP in anammox biomass as it works to survive in unfavorable niches.

Antioxidant action is critical for maintaining the normal cardiovascular function and vascular endothelial cell is an important target of estrogen action through estrogen receptor (ER) pathway. This study is carried out to explore the antioxidant effect of carnosol in bovine aortic endothelial cells (BAECs) via ER pathway. The ER subtype specific estrogenic effect of carnosol was further demonstrated by luciferase reporter gene assay in human embryonic kidney (HEK) 293 cells. Carnosol was extracted from Chinese medicine Rosmarinus officinalis. ER positive BAECs were employed in cell proliferation assay and cell apoptosis tests. Oxidative stress by intracellular reactive oxygen species (ROS) were measured via 2'7'-dichlorofluorescein (DCF) production. ERα and ERβ specific antagonists 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole (MPP) and 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-yl]phenol (PHTPP) were employed as tools in the experiment. ER negative HEK 293 cells were employed in luciferase reporter gene assay. The results indicate that carnosol can effectively attenuate H(2)O(2) induced slowing down of cell growth and increasing of cell apoptosis. At the meantime, carnosol pretreating can also effectively reduce the H(2)O(2) induced intracellular ROS elevation in BAECs. ERα and ERβ antagonist, especially ERα antagonist, can effectively decrease the above antioxidant effects of carnosol. The reporter gene analysis further demonstrates that the action of carnosol on inducing ERE dependent luciferase expression is realized via ER pathway. The conclusion is that carnosol can exert antioxidant effects towards oxidative stress induced by H(2)O(2) in BAECs. And such effects are realized via ER, especially ERα pathway. The results contribute to explain the mechanism of cardiovascular protective function of carnosol in postmenopausal women.

A new triterpenoid saponin, tenuifoside A, was isolated together with three known triterpenoid saponins 2, 3, and 4 from the roots of Polygala tenuifolia Willd. With the help of chemical and spectral analyses (IR, MS, 1D-NMR, and 2D-NMR), the structure of the new saponin was elucidated as 3-O-beta-d-glucopyranosyl presenegenin 28-O-beta-d-xylopyranosyl-(1 --3)-beta-d-xylopyranosyl-(1 --4)-[beta-D-apiofuranosyl-(1 --3)]-alpha-L-rhamnopyranosyl-(1 --2)-[4-O-p-methoxycinnamoyl]-[alpha-l-rhamnopyranosyl-(1 --3)]-beta-d-fucopyranosyl ester (1). Three known triterpenoid saponins (2-4) were identified on the basis of spectroscopic data.

Two new triterpenoid saponins, named cernuaside C and D, have been isolated from Pulsatilla cernua (Thunb.) Bercht. et Opiz. The structures of the two new triterpenoid saponins were elucidated as 3-O-beta-D-xylopyranosyl(1 --2)-[alpha-L-rhamnopyranosyl(1 --3)]-alpha-L-arabinopyranosyl oleanolic acid 28-alpha-L-rhamnopyranosyl(1 --4)-beta-D-glucopyranosyl(1 --6)-beta-D-glucopyranoside. (1) and 3-O-alpha-L-arabinopyranosyl (1 --3)-alpha-L-rhamnopyranosyl(1 --2)-alpha-L-arabinopyranosyl oleanolic acid 28-O-beta-D-glucopyranoside (2) by 1D, 2D-NMR techniques, ESI-MS analysis as well as chemical methods.

A new triterpenoid, saponin hehuanoside A, was isolated together with the known triterpenoid saponins 2, 3, and 4 from the stem bark of Albizia julibrissin. With the help of chemical and spectral analyzes (IR, MS, 1D-NMR, and 2D-NMR), the structure of the new triterpenoid saponin was elucidated as 21-O-[(6S)-2-trans-2,6-dimethyl-6-O-beta-d-quinovopyranosyl-2,7-octadienoyl]-3-O-beta-d-xylopyranosyl-(1 --2)-beta-d-fucopyranosyl-(1 --6)-beta-d-2-deoxy-2-acetamidoglucopyrasyl acacic acid 28-O-alpha-l-arabinofuranosyl-(1 --4)-[-beta-d-glucopyranosyl-(1 --3)]-alpha-l-rhamnopyranosyl-(1 --2)-beta-d-glucopyranosyl ester (1). Three known triterpenoid saponins 2-4 were identified on the basis of spectroscopic data.

A new furospirostanol saponin, ophiofurospiside A (1), was isolated together with the known steroidal glycosides 2, 3, and 4 from the tubers of Ophiopogon japonicus (Thunb.) Ker-Gawl. Using chemical and spectral analyses (IR, MS, 1D NMR, and 2D NMR), the structure of 1 was established as 26-O-beta-d-glucopyranosyl-(22S, 25R)-furospirost-5-ene-3beta, 17alpha, 26-triol-3-O-[alpha-l-rhamnopyranosyl-(1 --> 2)]-[beta-d-xylopyranosyl-(1 --> 4)]-glucopyranoside (1). Three known steroidal saponins 2-4 were identified on the basis of spectroscopic data.

AIM: To study the role of hepatic sinusoidal capillarization and perisinusoidal fibrosis in rats with alcohol-induced portal hypertension and to discuss the pathological mechanisms of alcohol-induced hepatic portal hypertension. METHODS: Fifty SD rats were divided into control group (n=20) and model group (n=30). Alcoholic liver fibrosis rat model was induced by intragastric infusion of a mixture containing alcohol, corn oil and pyrazole (1 000:250:3). Fifteen rats in each group were killed at wk 16. The diameter and pressure of portal vein were measured. Plasma hyaluronic acid (HA), type IV collagen (CoIV) and laminin (LN) were determined by radioimmunoassay. Liver tissue was fixed in formalin (10%) and 6-μm thick sections were routinely stained with Mallory and Sirius Red. Liver tissue was treated with rabbit polyclonal antibody against LN and ColIV. Hepatic non-parenchymal cells were isolated, total protein was extracted and separated by SDS-PAGE. MMP-2 and TIMP-1 protein expression was estimated by Western blotting. RESULTS: The diameter (2.207 ± 0.096 vs 1.528 ± 0.054 mm, P

AIM: To study the effects of Radix Puerariae flavones (RPF) on liver lipid metabolism in ovariectomized (OVX) rats. METHODS: Forty adult female Wistar rats were randomly divided into four groups: OVX group; sham-OVX group; OVX + estrogen group and OVX + RPF group. One week after operation rats of the first two groups were treated with physiological saline, rats of OVX + estrogen group with estrogen (1 mg/kg·b.w.) and rats of OVX + RPF group with RPF (100 mg/kg·b.w.), respectively for 5 weeks. After the rats were killed, their body weight, the weight of the abdominal fat and uterus were measured, and the levels of total cholesterol (TC) and triglyceride (TG) in liver homogenate were determined. RESULTS: Compared with the sham-OVX group, the body mass of the rats in OVX group was found increased significantly; more abdominal fat in store; TC and TG in liver increased and uterine became further atrophy. As a result, the RPF was found to have an inhibitive action on those changes of various degrees. CONCLUSION: RPF has estrogen-like effect on lipid metabolism in liver and adipose tissue.

AIM: To observe the effect of compound Biejiaruangan decoction (CBJRGC) (composite prescription of Carapax trionycis for softening the liver) on proliferation, activation, excretion of collagen and cytokine of hepatic stellate cells (HSCs) and to find the mechanism of prevention and treatment of hepatic fibrosis by CBJRGC. METHODS: Using MTT, immunohistochemistry and image analysis technology, the related indexes for proliferation, activation, excretion of collagen and cytokine of hepatic stellate cells were detected in 24 h, 48 h, and 72 h after adminstration of different dosages of CBJRGC. RESULTS: Statistical analysis showed that serum collected from rat perfused with CBJRGC could restrain the proliferation of HSC in 48 h and 72 h especially in high and medium dosage groups, markedly decrease the expression of desmin, synapsin and platelet derived growth factor (PDGF) in HSC in 24 h, 48 h and 72 h, as well as the expression of α-SMA, collagen III, TIMP and TGFβ1 in 48 h and 72 h, decrease the excretion of collagen I in 72 h. CBJRGC serum had no significant effect on collagens I, III and TIMP in 24 h. CONCLUSION: CBJRGC serum has a good curative effect on hepatic fibrosis. Its main mechanism may be related to the following factors. The drug serum can restrain the proliferation and activation of HSC, decrease the number of activated HSC and the total number of HSC, the excretion of collagens I, III, enhance the degradation of collagen and restore the balance of synthesis and degradation of collagen, inhibit the expression of transforming growth factor β1 (TGFβ1) and platelet derived growth factor (PDGF) in HSC, block and delay the process of hepatic fibrosis. Synapsin is a new marker of activation of HSC, which provides a theoretical and testing basis for neural regulation in the developing process of hepatic fibrosis.