Associations of Rap1 with Cell Wall Integrity, Biofilm Formation, and Virulence in Candida albicans.

Rap1 (repressor activator protein 1) is a multifunctional protein, playing important roles in telomeric and nontelomeric functions in many eukaryotes. Candida albicans Rap1 has been previously shown to be involved in telomeric regulation, but its other functions are still mostly unknown. In this study, we found that the deletion of the RAP1 gene altered cell wall properties, composition, and gene expression. In addition, deletion of RAP1 affected C. albicans biofilm formation and modulated phagocytosis and cytokine release by host immune cells. Finally, the RAP1 gene deletion mutant showed attenuation of C. albicans virulence in a Galleria mellonella infection model. Therefore, these findings provide new insights into Rap1 functions that are particularly relevant to pathogenesis and virulence of C. albicans. IMPORTANCE C. albicans is an important fungal pathogen of humans. The cell wall is the outermost layer of C. albicans and is important for commensalism and infection by this pathogen. Moreover, the cell wall is also an important target for antifungals. Studies of how C. albicans maintains its cell wall integrity are critical for a better understanding of fungal pathogenesis and virulence. This work focuses on exploring unknown functions of C. albicans Rap1 and reveals its contribution to cell wall integrity, biofilm formation, and virulence. Notably, these findings will also improve our general understanding of complex machinery to control pathogenesis and virulence of fungal pathogens.

The Effects of Freshwater Clam (Corbicula fluminea) Extract on Activated Hepatic Stellate Cells.

BACKGROUND: The extract of freshwater clams has been used to protect the body against liver diseases in traditional folk medicine. This study aims at investigating the effects of freshwater clam extract on activated hepatic stellate cells (aHSCs), which are critical contributors to liver fibrosis. METHODS: The aHSCs used in this study were derived from hepatic stellate cells that were isolated and purified from the livers of male Wistar rats and then transformed into the activated phenotype by culturing on uncoated plastic dishes. Freshwater clam extract (CE) was collected after the outflow from the live freshwater clams in a water bath at 100°C for 60 min. The effects of CE on aHSCs were analyzed by MTT assay, flow cytometry, Oil Red O (ORO) staining, western blot, and real-time RT-PCR. RESULTS: The results indicated that CE suppressed the proliferation of aHSCs through G0/G1 cell cycle arrest by downregulating cyclin D1 and upregulating p27. The expression levels of a-SMA, collagen I, TGF-ß, and TNF-α were inhibited in the CE-treated aHSCs. In addition, the CE treatment increased the lipid contents in aHSCs by promoting PPARγ expression. Furthermore, CE modulated the expression of ECM-related genes, i.e., by upregulating MMP-9 and downregulating TIMP-II. CONCLUSIONS: These data revealed that CE could induce the deactivation of aHSCs. We therefore suggest that CE has potential as an adjuvant therapeutic agent against hepatic fibrosis.

Ultrasonic treatment at different pH values affects the macromolecular, structural, and rheological characteristics of citrus pectin.

Ultrasonic degradation has become a promising strategy for producing modified pectin (MP). In this study, the impact of ultrasonic treatment at various pH values (4.0, 7.0, and 10.0) on the macromolecular, structural and rheological characteristics of citrus pectin was investigated. Results demonstrated that ultrasonic irradiation at the higher pH led to larger reductions in the intrinsic viscosity and weight-average molecular weight of pectin. The degradation kinetics of pectin at different pH values under ultrasound well fitted to a second-order reaction kinetics model. Acoustic cavitation, ß-elimination, and demethylation led to the breakage of glycosidic linkages of side chains and methoxyl groups of pectin, but did not have noticeable influences on the main chain of pectin. The ultrasonic treatment at a high pH led to an apparent change in the rheological characteristics of pectin. Therefore, ultrasonic treatment at various pH values can be developed as a viable means to prepare desirable MP.

Antioxidant and Neuroprotector Influence of Endo-Polyphenol Extract from Magnesium Acetate Multi-Stage Addition in the Oak Bracket Medicinal Mushroom, Phellinus baumii (Agaricomycetes).

The objective of this study was to explore the effect of magnesium acetate (MA) addition on the endo-polyphenol yield by Phellinus baumii and establish a feasible additive strategy. The optimal three-point MA addition strategy (0.05 g/L concentration of MA added at 0 h and 6 h, 0.9 g/L concentration of MA added at 12 h) was employed to obtain maximum endo-polyphenol yield. The maximum endo-polyphenol production was reached at 1.22 g/L, which was 1.39-fold higher than that of the control. Additionally, the endo-polyphenol showed stronger antioxidant activity in vitro compared with the control, including DPPH· scavenging capacity (78.76%) and Trolox equivalent antioxidant capacity (TEAC) (32.28 µmol Trolox/g sample). HPLC analysis showed that the endo-polyphenol production of the crude ethanol extracts was significantly higher than that of the control. Hispidin was isolated and identified from the ethanol extract of the culture mycelia from Ph. baumii with the three-point MA addition strategy. Hispidin showed a strong ability to scavenge DPPH free radicals and TEAC, equivalent to positive (vitamin C) value of 89.41% and 75.98%, respectively. Furthermore, hispidin protected H2O2-induced PC12 cells injured by decreased oxidative stress level. These results indicated that the MA multi-stage addition strategy was dependable, and could be used to develop new natural antioxidants for foods or medicines.

Three-phase partitioning system with dimethyl carbonate as organic phase for partitioning of exopolysaccharides from Phellinus baumii.

In this study, a three-phase partitioning (TPP) system with dimethyl carbonate (DMC) as organic phase and sodium citrate (SC) as salt phase was used for partitioning of exopolysaccharide (EPS), namely, EPS-D, from fermentation broth of Phellinus baumii. Results showed that the maximum extraction yield (EY) of EPS-D was 71.02% under the following modified optimal conditions: DMC to fermentation broth ratio 0.5:1.0 (v/v), SC concentration 19% (w/v), temperature 30 °C, and pH 4.0. EPS-D had higher EY, carbohydrate, and uronic acid contents compared with the EPS, designated as EPS-T, obtained from the TPP system with t-butanol and ammonium sulfate. EPS-D and EPS-T had different chemical compositions and molecular weights; however, their preliminary chemical structures basically remained unchanged. Moreover, EPS-D exhibited stronger free radical-scavenging capability and total antioxidant capacity than EPS-T. Therefore, the TPP system with DMC as an alternative solvent for t-butanol has great potential for efficient partitioning of natural biomolecules.

Employment of ARTP to Generate Phellinus baumii (Agaricomycetes) Strain with High Flavonoids Production and Validation by Liquid Fermentation.

To obtain Phellinus baumii strain with high flavonoids yield, ARTP was employed to generate mutants of a Ph. baumii strain, which were screened for higher flavonoids content. After five rounds of screening, four mutants were identified to produce more flavonoids than the wild type strain under optimal conditions, of which A67 was the mutant with the highest flavonoids productive capacity. When cultured in shake flasks, the maximum intracellular total flavonoids production of A67 reached 0.56 g/L, 86.67% higher than the total flavonoids in CK. Antagonistic testing, RAPD, and HPLC analysis suggested that ARTP caused changes of the genetic material and metabolites in Ph. baumii. In addition, the superiority of A67 to CK was proved by liquid fermentation using unstructured kinetic models, which was performed in a 50-L fermentor. The maximum intracellular total flavonoids production and dry mycelium weight of A67 reached 0.64 g/L and 15.24 g/L, which was an increase of 88.24% and 18.23% compared with CK, respectively. This work could provide an efficient and practical strategy to obtain high flavonoids production strains and the superiority of A67 could also provide a reference to further increase flavonoids production of Ph. baumii in large-scale production mode by submerged fermentation process.

Three-phase partitioning for the direct extraction and separation of bioactive exopolysaccharides from the cultured broth of Phellinus baumii.

In this study, three-phase partitioning (TPP) was used to directly extract and separate bioactive exopolysaccharides (EPSs) from a cultured broth of Phellinus baumii. The maximum extraction yield of EPS was 52.09% under the following optimal conditions: 20% (w/v) ammonium sulfate concentration, 1.0:1.5 (v/v) ratio of cultured broth to t­butanol, 30 min, and 35 °C. A multifrequency power ultrasound in a sequential mode coupled with TPP resulted in ~9.12% increment in extraction yield and ~80% reduction in extraction time compared with those of traditional TPP. The carbohydrate (88.21%) and uronic acid (3.37%) contents of partially purified EPS were higher than those of EPS-C obtained through conventional ethanol precipitation and separation methods. EPS and EPS-C exhibited similar preliminary structural characteristics and different monosaccharide compositions and molecular weights. The radical-scavenging abilities, antioxidant capacities, α­amylase and α­glycosidase inhibitory activities, and macrophage stimulation activities of EPS were also higher than those of EPS-C. Therefore, it could be concluded that TPP as a simple and green separation technique could be used to directly extract and separate bioactive EPS from the fermentation broths of mushrooms and other fungi.

Autophagy Inhibition Enhances SPCA-1 Cell Proliferation Inhibition Induced by By-1 from the Stout Camphor Medicinal Mushroom, Taiwanofungus camphoratus (Agaricomycetes).

Taiwanofungus camphoratus has been reported to have antitumor effects against various cancer cells. The aim of this study was to investigate the direct inhibitory effect of By-1 (3-isobutyl-l-methoxy-4-[4'-(3-methylbut-2-enyloxy)phenyl]-1H-pyrrole-2,5-dione), a compound from spent broth from submerged cultures of T. camphoratus, on human lung adenocarcinoma cells and to determine the molecular mechanism underlying this effect. The growth-inhibitory assay and colony formation assay showed that cell viability was significantly decreased. A By-1 concentration of 300 µmol/L caused 73.55% cell death and at a concentration of 240 µmol/L led to a 58% reduction in the number of colonies. The wound-healing assay showed that the distance of migration was 0.3 times shorter than that of untreated cells. Flow cytometry revealed that By-1 could suppress DNA synthesis, cause cell cycle arrest at the S phase, and induce apoptosis in a reactive oxygen species-dependent manner. Furthermore, the expression of caspase-3 and P53 was 4 times higher than that in untreated cells, and the antiapoptotic protein Bcl-2 was decreased 2 times compared with the protein in untreated cells. It is interesting to note that apoptosis and autophagy were both induced during treatment with By-1, and autophagy inhibition decreased cell proliferation. By-1 potently inhibited the growth of SPCA-1 cells by inducing cell cycle arrest and apoptosis. The combination of proapoptosis agents and antiautophagy agents could effectively enhance anticancer efficacy, which may be a new strategy in treating non-small cell lung cancer.

Structural elucidation of a polysaccharide from Flammulina velutipes and its immunomodulation activities on mouse B lymphocytes.

A novel polysaccharide FVPB2 was purified from fruiting bodies of Flammulina velutipes. Its structure was elucidated by monosaccharide composition and methylation analyses, UV-Visible and FTIR spectroscopy as well as NMR. FVPB2 was a homogeneous heteropolysaccharide (molecular weight ~ 1.50 × 104 Da) containing D-galactose, D-mannose, L-fucose, and D-glucose at molar ratio of 1.9:1.2:1:2.5. In vitro immunomodulatory studies showed FVPB2 induced proliferation of mouse spleen lymphocytes in a dose-dependent manner. The levels of IgM and IgG, secreted by B cells, increased after FVPB2 treatment. So FVPB2 has potential to be a new important immunomodulatory nutraceutical.

Fabrication and stabilization of biocompatible selenium nanoparticles by carboxylic curdlans with various molecular properties.

In this study, carboxylic curdlans (Cur-4, Cur-8, and Cur-24) with different molecular properties and chain conformations were used as stabilizer and capping agent to fabricate stable and water-dispersible selenium nanoparticles (SeNPs). Results showed that molecular properties and chain conformations of carboxylic curdlans remarkably influenced the size, morphology, structure, and stability of SeNPs and the carboxylic curdlan was ligated to SeNPs via OH⋯Se interaction. The as-prepared SeNPs was amorphous and showed homogeneous and monodisperse spherical structure with size of ∼50-90nm. The Cur-8-decorated SeNPs (SeNPs@Cur-8) exhibited smaller particle size (∼56nm) and greater stability than those of the others. The carboxylic curdlan-stabilized SeNPs exhibited excellent antioxidant capacities compared to the control SeNPs. Specifically, SeNPs@Cur-8 with smaller particle size possessed strong antioxidant efficacy. SeNPs@Cur-8 also exhibited low cytotoxic activity against SPCA-1 and HeLa cell lines in vitro.

Structural Characteristics of the Novel Polysaccharide FVPA1 from Winter Culinary-Medicinal Mushroom, Flammulina velutipes (Agaricomycetes), Capable of Enhancing Natural Killer Cell Activity against K562 Tumor Cells.

FVPA1, a novel polysaccharide, has been isolated from fruiting bodies of the culinary-medicinal mushroom Flammulina velutipes, a historically popular, widely cultivated and consumed functional food with an attractive taste, beneficial nutraceutical properties such as antitumor and immunomodulatory effects, and a number of essential biological activities. The average molecular weight was estimated to be ~1.8 × 104 Da based on high-performance size exclusion chromatography. Sugar analyses, methylation analyses, and 1H, 13C, and 2-dimensional nuclear magnetic resonance spectroscopy revealed the following structure of the repeating units of the FVPA1 polysaccharide Identification of this structure would conceivably lead to better understanding of the nutraceutical functions of this very important edible fungus. Bioactivity tests in vitro indicated that FVPA1 could significantly enhance natural killer cell activity against K562 tumor cells.

Antitumor Effect of By-1 from Spent Broth from Submerged Cultures of Stout Camphor Medicinal Mushroom, Taiwanofungus camphoratus (Higher Basidiomycetes), on A549 Adenocarcinoma Cells.

By-1 was obtained from spent broth from submerged cultures of Taiwanofungus camphoratus. This report evaluates the effects of By-1 on plate clone formation, wound healing, cell cycle, activated caspase-3 expression, and ROS release in A549 lung cancer cells. The result of plate clone formation assay revealed that By-1 could dramatically inhibit the viability of A549 cells in vitro. The inhibitory effect of By-1 on cell migration was tested using a wound healing assay. Proliferation rates of A549 cells were significantly inhibited following exposure to By-1 (12.5, 50, and 80 µg/mL). Flow cytometry revealed that the extracts increased, in a concentration-dependent manner, the number of cells in the G0/G1 phases of the cell cycle. The results of the caspase-3 experiment suggested that By-1 could induce A549 cells apoptosis, and this apoptosis was related to the release of reactive oxygen species by the A549 cells. All these results indicate that By-1 has potential in anti-lung cancer drug development.

Structural Characterization and Immunological Activities of a Novel Water-Soluble Polysaccharide from the Fruiting Bodies of Culinary-Medicinal Winter Mushroom, Flammulina velutipes (Agaricomycetes).

A water-soluble polysaccharide, designated FVPA2, was isolated from the fruiting bodies of Flammulina velutipes using DEAE Sepharose Fast Flow and gel-permeation chromatography. Its structure was elucidated by monosaccharide composition and methylation analysis, ultraviolet, Fourier transform infrared spectrometry, and nuclear magnetic resonance spectroscopy. Results showed that FVPA2 was a homogeneous heteropolysaccharide containing galactose, fucose, and mannose in a molar ratio of 5:1:1. High-performance liquid chromatography indicated its molecular weight as 3.4 × 104 Da. FVPA2 also has a repeating unit. In vitro immunomodulatory studies showed that Raw264.7 cells were stimulated to secret nitric oxide upon administration of 200-500 µg/mL FVPA2. FVPA2 also stimulated the proliferation of mouse spleen lymphocytes and B lymphocytes.

Clinicopathologic Factors and Thyroid Nodule Sonographic Features for Predicting Central Lymph Node Metastasis in Papillary Thyroid Microcarcinoma: A Retrospective Study of 1204 Patients.

OBJECTIVES: Preoperative prediction of lymph node metastasis is of clinical importance for the surgical treatment of thyroid tumor. The purpose of this study was to evaluate clinicopathologic factors and thyroid nodule sonographic features predictive of central lymph node metastasis in papillary thyroid microcarcinoma. METHODS: Clinicopathologic factors and thyroid nodule sonographic features of 1204 patients with papillary thyroid microcarcinoma were retrospectively reviewed from January 2014 to June 2015. Central lymph node dissection was performed on each patient. Univariate and multivariate analyses were performed to analyze the clinicopathologic factors and thyroid nodule sonographic features associated with central lymph node metastasis in papillary thyroid microcarcinoma. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the relevance of all potential predictive factors. RESULTS: Central lymph node metastasis was detected in 395 of the 1204 patients (32.81%). By univariate and multivariate analyses, younger age (≤43 years), male sex, larger tumor size (≥7 mm), multifocal papillary thyroid microcarcinoma and microcalcification were independently associated with central lymph node metastasis in papillary thyroid microcarcinoma (P < .05). The ORs were 1.920 (95% CI, 1.476-2.499), 1.665 (95% CI, 1.234-2.247), 1.534 (95% CI, 1.177-2.000), 2.120 (95% CI, 1.563-2.877), and 4.109 (95% CI, 3.118-5.414), respectively. CONCLUSIONS: Central lymph node metastasis is highly prevalent in papillary thyroid microcarcinoma. Younger age (≤43 years), male sex, larger tumor size (≥7 mm), multifocal papillary thyroid microcarcinoma, and microcalcification were independent predictors of central lymph node metastasis. Surgeons and radiologists need to pay more attention to patients with papillary thyroid microcarcinoma who have these risk predictors.

Evaluation of Thyroid Nodules by a Scoring and Categorizing Method Based on Sonographic Features.

OBJECTIVES: To assess sonographic features of thyroid nodules associated with malignancy and to establish a scoring and categorizing method based on sonographic features. METHODS: A total of 2445 patients with 2445 thyroid nodules were included and divided into 2 groups: benign (1493 cases) and malignant (952 cases). First, 10 sonographic features, including shape, border, margin, internal content, echogenicity, microcalcifications, posterior echo, halo, vascularization distribution, and vascularization degree, were defined, and all nodules were retrospectively evaluated. Second, the features associated with malignancy were selected by statistical analysis and were assigned weightings according to their odds ratios. Third, a total score for each nodule was obtained after the assigned weightings of the suspicious features were summed. Fourth, the malignancy rate of each total score was calculated. Then a modified version of the Thyroid Imaging Reporting and Data System (TI-RADS) was established with reference to the American College of Radiology's Breast Imaging Reporting and Data System. RESULTS: Seven independent features associated with malignancy were a taller-than-wide shape, an obscure border, an irregular margin, solid internal content, marked hypoechogenicity and hypoechogenicity, microcalcifications, and an internal vascularization distribution. The TI-RADS included 5 categories with different malignancy rates: category 3 (<2%), 4A (2%-5%), 4B (5%-50%), 4C (50%-90%), and 5 (≥ 90%). CONCLUSIONS: A modified version of TI-RADS was established on the basis of the sonographic features with different weightings according to the relative risk of malignancy. This system could be of great use in predicting the nature of thyroid nodules in a quantified and standardized way and also helping clinicians decide on the clinical management.

Hypoglycemic Effect of Ethanol and Ethyl Acetate Extract of Phellinus baumii Fruiting Body in Streptozotocin-Induced Diabetic Mice.

We investigated hypoglycemic effect of ethanol (EtOH) and ethyl acetate extract acetate (AcOEt) extracts in streptozotocin- (STZ-) induced diabetic mice. Our data showed the maximum inhibitory effect on the fasting plasma glucose (FPG) level was detected in STZ-induced diabetic mice administered with 400 mg/kg AcOEt extract of P. baumii. A lower glycated albumin (GA) level and a higher insulin level were observed in 400 mg/kg AcOEt and EtOH extract groups. Moreover, 400 mg/kg AcOEt and EtOH extract exhibited a stronger effect on increasing size and cell number of islets. The insulin expression level of ß-cells and integrated optical density (IOD) value were significantly increased by the administration of 400 mg/kg AcOEt and EtOH extracts. Taken together, AcOEt and EtOH extracts of P. baumii fruiting body exhibited considerable hypoglycemic effect on STZ-induced diabetic mice.

Sedum mexicanum Britt. Induces Apoptosis of Primary Rat Activated Hepatic Stellate Cells.

Background. Liver fibrosis is a significant liver disease in Asian countries. Sedum mexicanum Britt. (SM) has been claimed to have antihepatitis efficacy. In traditional folk medicine, a solution of boiling water-extracted SM (SME) is consumed to prevent and treat hepatitis. However, its efficacy has not yet been verified. The purpose of this study was to investigate the in vitro effect of SME on hepatoprotection. Methods. Hepatic stellate cells (HSCs) and hepatocytes (HCs) were isolated from the livers of the rats by enzymatic digestion and density gradient centrifugation. Results. Treating the HCs and aHSCs with SME caused a dose-dependent decrease in the viability of aHSCs but not that of HCs. In addition, treatment with SME resulted in apoptosis of aHSCs, as determined by DAPI analysis and flow cytometry. SME also increased the amount of cleaved caspase-3, cleaved caspase-9, and cleaved poly ADP-ribose polymerase (PARP) in aHSCs. Furthermore, SME treatment induced a dose-dependent reduction in Bcl-2 expression and increased the expression of Bax in aHSCs. Conclusions. SME did not cause cytotoxicity in HCs, but it induced apoptosis in aHSCs through the mitochondria-dependent caspase-3 pathway. Therefore, SME may possess therapeutic potential for liver fibrosis.

Intracellular trafficking of histone deacetylase 4 regulates long-term memory formation.

Histone acetylation is important for gene transcription, which is controlled by the balance between two kinds of opposing enzymes: histone acetyltransferases and histone deacetylases (HDACs). HDACs repress gene transcription by decreasing histone acetylation levels. Our hypothesis was that shuttling of Class II HDACs, such as HDAC4, between the nucleus and cytoplasm is critical for its function. We constructed mutants of mammalian HDAC4 that had different cellular locations and checked their function during memory formation using Caenorhabditis elegans as a model. The deletion of hda4, a homolog of HDAC4, was able to enhance learning and long-term memory (LTM) in a thermotaxis model. Transgenic experiments showed that mammalian wild-type HDAC4 rescued the phenotype of hda4-deleted worms but impaired LTM formation in wild-type worms. The cytosol-localized HDAC4 mutant was not able to alter the phenotype of knock-out worms but led to enhanced LTM formation in wild-type worms similar to hda4-deletion mutants. Constitutive nuclear localization of HDAC4 rescued the phenotype of deletion worms similar to wild-type HDAC4 but had no effect on wild-type worms. These results support our hypothesis that HDAC4's biological function is regulated by its intracellular distribution.