Recent insights into glucans biosynthesis and engineering strategies in edible fungi.

Fungal α/ß-glucans have significant importance in cellular functions including cell wall structure, host-pathogen interactions and energy storage, and wide application in high-profile fields, including food, nutrition, and pharmaceuticals. Fungal species and their growth/developmental stages result in a diversity of glucan contents, structures and bioactivities. Substantial progresses have been made to elucidate the fine structures and functions, and reveal the potential molecular synthesis pathway of fungal α/ß-glucans. Herein, we review the current knowledge about the biosynthetic machineries, including: precursor UDP-glucose synthesis, initiation, elongation/termination and remodeling of α/ß-glucan chains, and molecular regulation to maximally produce glucans in edible fungi. This review would provide future perspectives to biosynthesize the targeted glucans and reveal the catalytic mechanism of enzymes associated with glucan synthesis, including: UDP-glucose pyrophosphate phosphorylases (UGP), glucan synthases, and glucanosyltransferases in edible fungi.

Functional Characterization and Structural Basis of the ß-1,3-Glucan Synthase CMGLS from Mushroom Cordyceps militaris.

ß-1,3-Glucan synthases play key roles in glucan synthesis, cell wall assembly, and growth of fungi. However, their multi-transmembrane domains (over 14 TMHs) and large molecular masses (over 100 kDa) significantly hamper understanding of their catalytic characteristics and mechanisms. In the present study, the 5841-bp gene CMGLS encoding the 221.7 kDa membrane-bound ß-1,3-glucan synthase CMGLS in Cordyceps militaris was cloned, identified, and structurally analyzed. CMGLS was partially purified with a specific activity of 87.72 pmol/min/µg, a purification fold of 121, and a yield of 10.16% using a product-entrapment purification method. CMGLS showed a strict specificity to UDP-glucose with a Km value of 84.28 µM at pH 7.0 and synthesized ß-1,3-glucan with a maximum degree of polymerization (DP) of 70. With the assistance of AlphaFold and molecular docking, the 3D structure of CMGLS and its binding features with substrate UDP-glucose were proposed for the first time to our knowledge. UDP-glucose potentially bound to at least 11 residues via hydrogen bonds, π-stacking ,and salt bridges, and Arg 1436 was predicted as a key residue directly interacting with the moieties of glucose, phosphate, and the ribose ring on UDP-glucose. These findings would open an avenue to recognize and understand the glucan synthesis process and catalytic mechanism of ß-1,3-glucan synthases in mushrooms.

Preparation, characterization, and bioactivity evaluation of oligosaccharides from Atractylodes lancea (Thunb.) DC.

Sixteen oligosaccharide monomers with the degree of polymerization 3 to 18 (DP 3 to DP 18) and three active fractions (DP 3-9, DP 8-11, and DP 11-17) were separated from Atractylodes lancea (Thunb.) DC. by optimized fast protein liquid chromatography coupled with refractive index detector (FPLC-RID) and preparation hydrophilic interaction chromatography (Pre-HILIC). Gas chromatography-mass spectrometer (GC-MS), liquid chromatography tandem mass spectrometry (LC-MS/MS), nuclear magnetic resonance (NMR) spectroscopy, and methylation analysis showed that the oligosaccharide in A. lancea was 1-kestose [ß-D-fructofuranosyl-(2 â†’ 1)-ß-D-fructofuranosyl-(2 â†’ 1)-α-D-glucopyranoside] (inulin-type fructooligosaccharides, FOS). Particularly, DP 3-9 showed the best capacity in stimulating phagocytic, NO, and cytokines production on RAW264.7 cells than any other purified oligosaccharide monomers and active fractions. It could also activate T-cells in Peyer's patch cells and enhance the production of colony stimulation factors. Besides, FPLC-RID showed a good capacity for large-scale preparation of DP 3-9 with the recovery of more than 93%. The bioactivity of sixteen FOS monomers (DP 3 to DP 18) and three FOS fractions (DP 3-9, DP 8-11, and DP 11-17) investigated in this study are beneficial for the utilization of FOS as a functional ingredient in novel product development.

The role of thymidylate synthase in non-small cell lung cancer treated with pemetrexed continuation maintenance therapy.

Pemetrexed continuation maintenance therapy has been proven to be beneficial for patients with advanced non-squamous non-small cell lung cancer (NSCLC). However, the eligibility criteria for maintenance treatment are too simple. This study sought to evaluate thymidylate synthase (TS) as a predicting biomarker for pemetrexed continuation maintenance treatment in NSCLC. Specimens were collected from 87 patients treated with pemetrexed continuation maintenance therapy before and after four-cycle induction chemotherapy. Real-time quantitative PCR was used to detect TS expression in tissues. The TS expression level was correlated with characteristic clinical data, radiographic response, progression-free time (PFS) and overall survival (OS). Low total TS expression (<8.47) was associated with improved PFS (median: 4.7 months vs. 3.5 months, p = 0.034) and improved OS (time from random assignment: 16.4 months vs. 11.7 months, p = 0.026; time from induction: 19.7 months vs. 14.8 months, p = 0.022). Our results indicate that in NSCLC patients treated with pemetrexed continuation maintenance therapy, low TS expression is associated with improved PFS and OS.

Significance of thymidylate synthase expression for resistance to pemetrexed in pulmonary adenocarcinoma.

Pemetrexed (PEM), a multi-targeted antifolate, has promising clinical activity in non-squamous non-small cell lung cancer. However, the majority of patients eventually acquire resistance to PEM. To evaluate the resistant mechanisms, the A549 lung adenocarcinoma cell line was exposed to stepwise increasing PEM concentrations of 1.6, 6.4 and 16 µM to establish three PEM-resistant lung cancer cell lines, A549/PEM-1.6, -6.4 and -16. Growth inhibition was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Expression of the genes encoding thymidylate synthase (TS), reduced folate carrier (RFC) and folypoly-γ-glutamate synthetase (FPGS) were analyzed by quantitative real-time reverse transcription polymerase chain reaction. The three A549 cell lines showed more resistance to PEM (3.7-, 17.3- and 38.0-fold, respectively) compared with that of the parental cell line, which also showed cross-resistance to cisplatin, but not to docetaxel, vinorelbine and 5-Fluorouracil (5-FU). TS gene expression was significantly increased in three PEM-resistant cells, relative to that of the parental cells, in a PEM dose-dependent manner. Knockdown of TS expression with siRNA enhanced the cytotoxicity of PEM in A549/PEM-16 cells. By contrast, the levels of RFC and FPGS gene expression in A549/PEM-1.6 and -6.4 cells were significantly decreased, whereas the levels of the two genes were restored in A549/PEM-16 cells. In summary, PEM-resistant A549 cells remained sensitive to docetaxel, vinorelbine and 5-FU. TS expression appeared to be associated with resistance to PEM, which may be a predictive marker for PEM sensitivity in lung adenocarcinoma.

[Correlation between the methylation of SULF2 and WRN promoter and chemosensitivity to irinotecan in gastric cancer].

OBJECTIVE: To explore the relationship between SULF2 and WRN promoter methylation and chemosensitivity to irinotecan, and also the clinicopathological features in patients with gastric cancer. METHODS: The chemosensitivity to irinotecan was tested by MTT assay. The methylation of SULF2 and WRN promoter in the fresh gastric cancer tissues was detected by methylation specific PCR. The differences of chemosensitivity and clinicopathological features of the methylation group were compared with that of the non-methylation group. The tumor growth in nude mice bearing human gastric cancer xenografts treated with CPT-11was also observed. RESULTS: The methylation rates of SULF2 and WRN were 28.4% (29/102) and 23.5% (24/102), respectively. There were no significant association between promoter methylation and clinicopathological features of patients including age, gender, histologic type, lymphatic invasion, and TNM Stage. In all the 102 cases, there were 30 cases of irrinotecan-sensitive group, and 72 cases of the irrinotecan-resistant group. The SULF2 methylation rate was 46.7% (14/30)in the sensitive group, and 20.8% (15/72) in the resistant group (P = 0.008),The WRN methylation rate was 33.3% (10/30) in the sensitive group, and 19.4% (14/72) in the resistant group (P = 0.214). Gastric cancer tissues were more sensitive to irrinotecan when both the genes were methylated. The nude mice bearing human gastric cancer xenografts with SULF2 methylation were more sensitive to irrinotecan. CONCLUSIONS: The detection of SULF2 and WRN promoter methylation may provide evidence for screening and targeting the most sensitive gastric cancer subpopulation suitable for personalized irrinotecan chemotherapy.

The role of cyclin D1 expression and patient's survival in non-small-cell lung cancer: a systematic review with meta-analysis.

BACKGROUND: The role of cell-cycle protein cyclin D1 in lung cancer remains controversial. To clarify its impact on survival in non-small-cell lung cancer (NSCLC), we performed a meta-analysis on the currently available medial literature to quantitatively assess its role on survival of NSCLC according to cyclin D1 status. METHOD: Published studies that investigated the association between cyclin D1 expression and NSCLC survival were identified. Meta-analysis was performed by using a DerSimonian-Laird model. Funnel plot was used to investigate publication bias and sources of heterogeneity were identified by using meta-regression analysis. RESULT: A total of 24 studies with 2731 patients were evaluable for this meta-analysis. No statistical significance was found that cyclin D1 expression was associated with poor prognosis in NSCLC (hazard ratio 1.08 [95% CI, 0.80-1.45]) without publication bias found. But there was significant heterogeneity present; meta-regression analysis was used to explore the sources of heterogeneity and revealed that the outcome of analysis was influenced by cutoff values and ethnicity. No difference between positive and negative studies on study quality assessment was present. CONCLUSION: The cyclin D1 expression is unlikely to be useful as a prognostic marker for NSCLC in clinical practice from current evidence. The conclusion should be confirmed by a large well-designed prospective study.

Evaluation of oral chemotherapy with capecitabine and cyclophosphamide plus thalidomide and prednisone in prostate cancer patients.

PURPOSE: The aim of this study was to evaluate clinical outcomes of second-line chemotherapy with capecitabine and cyclophosphamide (CTX) plus thalidomide and prednisone in refractory advanced castrate-resistant prostate cancer (CRPC) patients. METHODS: We retrospectively reviewed patients with advanced CRPC who had previously progressed to first-line docetaxel-based chemotherapy. Patients were given second-line chemotherapy with capecitabine and CTX plus thalidomide and prednisone throughout the course. Patients were evaluated for response and toxicity, and treatment was continued until the disease progression or excessive toxicity was noted. RESULTS: From April 2007 to February 2010, a total of 28 patients (median age, 72.8 ± 2.9 years) received second-line chemotherapy. The median cycle and duration of metronomic chemotherapy were six (range: 1-12) cycles and 6.3 (range 1.5-20.5) months, respectively. Prostatic-specific antigen was decreased by more than 50% in 10 (35.7%) of the 28 patients. All patients had bone metastases, and 8 patients (28.6%) had measurable soft tissue lesions. Among the 8 patients, 1 patient achieved partial response, and 3 patients had stabilized disease. With a median follow-up time of 29.5 (95% CI, 26.4-33.4) months, median composite progression-free survival and overall survival were 4.7 (95% CI, 3.4-5.7) months and 19.5 (95% CI, 18.9-25.5) months, respectively. No grade 3-4 toxicity was observed, and none of the patients experienced grade 3-4 hematological and nonhematological toxicities. CONCLUSIONS: These data suggested that oral combination second-line chemotherapy with capecitabine and CTX plus thalidomide and prednisone offers promising activity with an excellent safety profile for patients with advanced CRPC.

[Study on optimization of formulation of Danggui Liuhuang effervescent granules].

OBJECTIVE: To optimize the formulation of Danggui Liuhuang effervescent granules. METHODS: By means of quadratic regression rotation-orthogonal combination design, the effect of the proper proportion between citric acid and sodium bicarbonate, as well as the proper quantity of polyethylene glycol 6000 and sodium cyclamate on the dissolubility and pH of effervescent granules was studied. RESULTS: The best formulation was as follows: citric acid: sodium bicarbonate = 0.75: 1, the percentage of polyethylene glycol 6000 and cyclamate was 3.25% and 0.89%, respectively. CONCLUSION: The dissolubility and pH of the effervescent granules are better and the taste is satisfactory.