Improvement of laccase activity by silencing PacC in Ganoderma lucidum.

Ganoderma lucidum is a representative white-rot fungus that has great potential to degrade lignocellulose biomass. Laccase is recognized as a class of the most important lignin-degrading enzymes in G. lucidum. However, the comprehensive regulatory mechanisms of laccase are still lacking. Based on the genome sequence of G. lucidum, 15 laccase genes were identified and their encoding proteins were analyzed in this study. All of the laccase proteins are predicted to be multicopper oxidases with conserved copper-binding domains. Most laccase proteins were secreted enzymes in addition to Lac14 in which the signal peptide could not be predicted. The activity of all laccases showed the highest level at pH 3.0 or pH 7.0, with total laccase activity of approximately 200 U/mg protein. Silencing PacC resulted in a 5.2 fold increase in laccase activity compared with WT. Five laccase genes (lac1, lac6, lac9, lac10 and lac14) showed an increased transcription levels (approximately 1.5-5.6 fold) in the PacC-silenced strains versus that in WT, while other laccase genes were downregulated or unchanged. The extracellular pH value was about 3.1, which was more acidic in the PacC-silenced strains than in the WT (pH 3.5). Moreover, maintaining the fermentation pH resulted in a downregulation of laccase activity which is induced by silencing PacC. Our findings indicate that in addition to its function in acidification of environmental pH, PacC plays an important role in regulating laccase activity in fungi.

Regulation of glutamine synthetase activity by transcriptional and posttranslational modifications negatively influences ganoderic acid biosynthesis in Ganoderma lucidum.

Glutamine synthetase (GS), a central nitrogen metabolic enzyme, plays important roles in the nitrogen regulation network and secondary metabolism in fungi. However, the mechanisms by which external nitrogen sources regulate fungal GS activity have not been determined. Here, we found that GS activity was inhibited under nitrate conditions in Ganoderma lucidum. By constructing gs-silenced strains and adding 1 mM GS inhibitor to inhibit GS activity, we found that a decrease in GS activity led to a decrease in ganoderic acid biosynthesis. The transcription of gs increased approximately five fold under nitrate conditions compared with that under ammonia. Electrophoretic mobility shift and yeast one-hybrid assay showed that gs was transcriptionally regulated by AreA. Although both gs expression and GS protein content increased under nitrate conditions, the GS activity still decreased. Treatment of recombinant GS with SIN-1 (protein nitration donor) resulted in a strengthened nitration accompanied by a 71% decrease in recombinant GS activity. Furthermore, intracellular GS could be nitrated from mycelia cultivated under nitrate conditions. These results indicated that GS activity could be inhibited by NO-mediated protein nitration. Our findings provide the first insight into the role of transcriptional and posttranslational regulation of GS activity in regulating secondary metabolism in fungi.

GCN4 Regulates Secondary Metabolism through Activation of Antioxidant Gene Expression under Nitrogen Limitation Conditions in Ganoderma lucidum.

Nitrogen limitation has been widely reported to affect the growth and development of fungi, and the transcription factor GCN4 (general control nonderepressible 4) is involved in nitrogen restriction. Here, we found that nitrogen limitation highly induced the expression of GCN4 and promoted the synthesis of ganoderic acid (GA), an important secondary metabolite in Ganoderma lucidum. The activated GCN4 is involved in regulating GA biosynthesis. In addition, the accumulation of reactive oxygen species (ROS) also affects the synthesis of GA under nitrogen restrictions. The silencing of the gcn4 gene led to further accumulation of ROS and increased the content of GA. Further studies found that GCN4 activated the transcription of antioxidant enzyme biosynthesis genes gr, gst2, and cat3 (encoding glutathione reductase, glutathione S-transferase, and catalase, respectively) through direct binding to the promoter of these genes to reduce the ROS accumulation. In conclusion, our study found that GCN4 directly interacts with the ROS signaling pathway to negatively regulate GA biosynthesis under nitrogen-limiting conditions. This provides an essential insight into the understanding of GCN4 transcriptional regulation of the ROS signaling pathway and enriches the knowledge of nitrogen regulation mechanisms in fungal secondary metabolism of G. lucidum.IMPORTANCE Nitrogen has been widely reported to regulate secondary metabolism in fungi. Our study assessed the specific nitrogen regulatory mechanisms in Ganoderma lucidum. We found that GCN4 directly interacts with the ROS signaling pathway to negatively regulate GA biosynthesis under nitrogen-limiting conditions. Our research highlights a novel insight that GCN4, the nitrogen utilization regulator, participates in secondary metabolism through ROS signal regulation. In addition, this also provides a theoretical foundation for exploring the regulation of other physiological processes by GCN4 through ROS in fungi.

Dual functions of AreA, a GATA transcription factor, on influencing ganoderic acid biosynthesis in Ganoderma lucidum.

Nitrogen metabolism repression (NMR) has been well studied in filamentous fungi, but the molecular mechanism of its effects on fungal secondary metabolism has been generally unexplored. Ganoderic acid (GA) biosynthesis in Ganoderma lucidum differs between ammonia and nitrate nitrogen sources. To explain the functions of NMR in secondary metabolism, AreA, which is a core transcription factor of NMR, was characterized in G. lucidum. The transcription level of AreA was dramatically increased (approximately 4.5-folds), with the nitrate as the sole nitrogen source, compared with that with ammonia as the source. In addition, the expression of related genes involved in NMR was changed (upregulated of MeaB and downregulated of Nmr and GlnA) when AreA was knockdown. Yeast one-hybrid and electrophoretic mobility shift assay results showed that AreA could directly bind to the promoter of fps (encoding farnesyl-diphosphate synthase) to activate its expression. However, GA biosynthesis was increased (27% in the ammonia source and 77% in the nitrate source) in AreAi mutant strains versus that in control strains. These results showed that another important factor must participate in regulating GA biosynthesis other than the direct activation of AreA. Furthermore, we found that the content of nitric oxide (NO) was increased approximately 2.7-folds in the nitrate source compared with that in the ammonia. By adding the NO donor (SNP) or scavenger (cPTIO) and using NR-silenced or NR-overexpressed strains, we found that there was a negative correlation between the NO contents and GA biosynthesis. NO generated by nitrate reductase (NR) during the nitrogen utilization burst and could negatively influence GA biosynthesis. As a global transcription factor, AreA could also regulate the expression of NR. Our studies provide novel insight into the dual functions of AreA in GA biosynthesis during nitrogen assimilation.

Mechanism of Huayu Jianpi Fangshi decoction in urolithiasis prevention: a randomized trial.

BACKGROUND: This study aims to explore the mechanism of the Huayu Jianpi Fangshi decoction in urolithiasis prevention. METHODS: The present study was designed as a randomized, double-blinded, placebo-controlled clinical trial. Sixty patients with the qi stagnation and blood stasis, spleen deficiency, and dampness obstruction types of urolithiasis were randomly divided into two groups: the treatment group and the control group (n=30 in both groups). Patients in the treatment group were treated with the Huayu Jianpi Fangshi decoction, while patients in the control group were treated with the Huayu Fangshi placebo decoction. Both treatments were taken orally two times per day. All patients received treatment over the course of four weeks. The main outcome indicators included the Tamm-Horsfall protein (THP) expression levels, osteopontin, and inter-α-trypsin inhibitor heavy chain 3 (ITIH3) in the patients' urine as well as changes in 24-h urinary citric acid, urinary magnesium levels, and Traditional Chinese Medicine (TCM) syndrome scores. RESULTS: The results of the present study revealed a significant increase in the total citric acid excretion level (244.75±59.62 vs. 297.48±57.91 mmol/L, P<0.01), significant decrease in the total urinary THP level (10.83±7.73 vs. 6.37±6.10 mg/L, P<0.05), significant decrease in the total ITIH3 level (9.51±6.32 vs. 6.14±4.46 mg/L, P<0.05) in the patients' 24-h urine, and a significant elevation of the total TCM syndrome score (5% vs. 23%, P<0.01) in the treatment group when compared with the control group. CONCLUSIONS: The Huayu Jianpi Fangshi decoction can increase the excretion of urinary citric acid, decrease the expression levels of urinary THP and ITIH3 in patients with cured urolithiasis, and reduce the TCM syndrome score.

Mouse Model to Explore the Therapeutic Effect of Nano-Doxorubicin Drug Delivery System on Bladder Cancer.

To study the therapeutic effect of nano-dosin-loaded drug system in mouse bladder cancer, a luciferase-labeled mouse bladder cancer cell line and a mouse bladder cancer model were constructed. In vivo imaging monitors tumor growth and uses a combination of photothermal, immune, and chemotherapy to treat the mouse model. With doxorubicin as an antitumor drug carrier, the drug loading, in vitro drug release, cytotoxicity and behavior in cells of mesoporous nano particle-targeted drug delivery system were studied. The cells were injected into the bladder through the urethra, and the mouse bladder cancer subcutaneous model was treated with gelatin-coated single-walled carbon nanotube-encapsulated mouse granulocytes-macrophage colony-stimulating factor and doxorubicin. In the process of using, the use of near-infrared light for irradiation, thereby achieving the combined effect of photothermal, immune and chemotherapy. The experimental results show that the prepared doxorubicin prodrug delivery system can enhance the targeted therapeutic effect and reduce the toxicity and side effects of the drug. Especially for those cancer cells or tissues with overexpression of folate receptors, it has a better therapeutic effect and provides reference for the treatment of subsequent bladder cancer.

[A clinical study on psycho-behavior therapy for premature ejaculation].

OBJECTIVE: To investigate the clinical efficacy of psycho-behavior therapy for premature ejaculation (PE). METHODS: A total of 58 PE patients that met the study criteria were treated by psycho-behavior therapy, 2-3 times a week, for a 6-time course. After the treatment, the therapeutic effect was assessed by observation of the changes in the patients' CIPE-5 scores. RESULTS: The rates of cure, effectiveness, ineffectiveness and overall effectiveness were 46.55% (27/58), 32.76% (19/58), 20.69% (12/58) and 79.31%, respectively. The CIPE-5 scores of the patients were elevated from 7.97 +/- 2.30 before the treatment to 22.50 +/- 6.64 after it, and the differences were statistically significant. The psycho-behavior therapy obviously prolonged the ejaculation latency of the patients, increased the sexual satisfaction of both the patients and their spouses, lessened the patients' sexual anxiety and nervousness, and decreased the difficulty in retarding ejaculation, with statistically significant differences from pretreatment. CONCLUSION: Psychobehavior therapy has remarkable therapeutic effect on premature ejaculation.

[Recognition and thoughts for diagnosis and treatment of chronic prostatitis in integrated traditional Chinese and Western medicine].

The etiology, pathogenesis, and diagnostic criteria of chronic prostatitis were reviewed in this article. Based on clinical practice, the authors systematically discussed the thoughts and methods for the treatment of chronic prostatitis by integrated traditional Chinese and Western medicine. Meanwhile, advice on disputed problems in clinical study of prostatits were given, such as curative effect estimation value of the number of leukocytes in expressed prostatic secretion (EPS) and bacterial culture in EPS, the opportunity and treatment course of antibiotics, National Institutes of Health chronic prostatitis symptom index, classification of syndromes of traditional Chinese medicine (TCM), TCM symptom score, and clinical study period.

Functions of reactive oxygen species in apoptosis and ganoderic acid biosynthesis in Ganoderma lucidum.

Ganoderma lucidum is a medicinal fungus that is widely used in traditional medicine. Fungal PacC is recognized as an important transcription factor that functions during adaptation to environmental pH, fungal development and secondary metabolism. Previous studies have revealed that GlPacC plays important roles in mycelial growth, fruiting body development and ganoderic acid (GA) biosynthesis. In this study, using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay, we found that the apoptosis level was increased when PacC was silenced. The transcript and activity levels of caspase-like proteins were significantly increased in the PacC-silenced (PacCi) strains compared with the control strains. Silencing PacC also resulted in an increased reactive oxygen species (ROS) levels (∼2-fold) and decreased activity levels of enzymes involved in the antioxidant system. Further, we found that the intracellular ROS levels contributed to apoptosis and GA biosynthesis. Adding N-acetyl-cysteine and vitamin C decreased intracellular ROS and resulted in the inhibition of apoptosis in the PacCi strains. Additionally, the GA biosynthesis was different between the control strains and the PacCi strains after intracellular ROS was eliminated. Taken together, the findings showed that silencing PacC can result in an intracellular ROS burst, which increases cell apoptosis and GA biosynthesis levels. Our study provides novel insight into the functions of PacC in filamentous fungi.