Further improvement in ganoderic acid production in static liquid culture of Ganoderma lucidum by integrating nitrogen limitation and calcium ion addition.

To further improve the ganoderic acid (GA) production, a novel integrated strategy by combining nitrogen limitation and calcium ion addition was developed. The effects of the integrated combination on the content of GA-T (one powerful anticancer compound), their intermediates (squalene and lanosterol) and on the transcription levels of GA biosynthetic genes in G. lucidum fermentation were investigated. The maximum GA-T content with the integrated strategy were 1.87 mg/ 100 mg dry cell weight, which was 2.1-4.2 fold higher than that obtained with either calcium ion addition or nitrogen limitation alone, and it is also the highest record as ever reported in submerged fermentation of G. lucidum. The squalene content was increased by 3.9- and 2.2-fold in this case compared with either individual strategy alone. Moreover, the transcription levels of the GA biosynthetic genes encoding 3-hydroxy-3-methyglutaryl coenzyme A reductase and lanosterol synthase were also up-regulated by 3.3-7.5 and 1.3-2.3 fold, respectively.

Increased polysaccharide production and biosynthetic gene expressions in a submerged culture of Ganoderma lucidum by the overexpression of the homologous α-phosphoglucomutase gene.

This study aimed to improve the production of polysaccharide by engineering the biosynthetic pathway in Ganoderma lucidum through the overexpression of α-phosphoglucomutase (PGM) gene. PGM is responsible for the linkage between sugar catabolism and sugar anabolism. The effects of PGM gene overexpression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production and transcription levels of three genes encoding the enzymes involved in polysaccharide biosynthesis, including PGM, UDP-glucose pyrophosphorylase (UGP), and ß-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in G. lucidum overexpressing the PGM gene were 23.67 mg/100 mg dry weight and 1.76 g/L, respectively, which were higher by 40.5 and 44.3% than those of the wild-type strain. The transcription levels of PGM, UGP and GLS were upregulated by 4.77-, 1.51- and 1.53-fold, respectively, in the engineered strain, suggesting that increased polysaccharide biosynthesis may result from a higher expression of those genes.

Microphase separation in nanocomposite gels.

Microphase separation in poly(N-isopropylacrylamide)(PNIPA)-clay nanocomposite hydrogels (NC gels) is investigated by means of contrast-variation small-angle neutron scattering (CV-SANS) and dynamic light scattering (DLS). By using CV-SANS, it is revealed that microphase separation occurs in NC gels above the lower-critical solution temperature (LCST) of PNIPA aqueous solutions. The observed partial scattering functions show that only the spatial distribution of PNIPA chains is highly distorted by microphase separation and PNIPA chains are preferentially adsorbed on the clay surfaces, where the PNIPA-rich phase forms nanoscaled bicontinuous structure mediated by the clay particles. Additional DLS measurements for dilute solutions with PNIPA and/or the clay nanoparticles confirm that aggregation of PNIPA above the LCST is dramatically suppressed by addition of clay particles. Based on these observations, we conclude that strong affinity between the polymer and clay has a significant effect on the phase separation in NC gels and allows one to tune the length scale of the phase separation phenomenon by clay concentration.

Proposal of a modified American Joint Committee on Cancer staging scheme for resectable pancreatic ductal adenocarcinoma with a lymph node ratio-based N classification: A retrospective cohort study.

The recently launched 8th edition of the American Joint Committee on Cancer (AJCC) staging scheme for pancreatic ductal adenocarcinoma (PDAC) did not account for the impact of the total examined lymph node count on prognostic accuracy. In this population-based cohort study, we proposed a modified AJCC staging scheme by incorporating a lymph node ratio (LNR)-based N classification for patients with resectable PDAC.We analyzed 8615 patients with resectable PDAC from the Surveillance, Epidemiology, and End Results database between 2004 and 2013. The optimal cut-off points for LNR were identified by recursive partitioning, and an LNR-based N classification was designed accordingly.The LNR-based N classification could further stratify patients with the 8th AJCC N1 and N2 disease into subgroups with significantly different overall survival (P < .001 for both). By replacing the 8th AJCC N classification with the corresponding LNR-based N classification, we further proposed a modified AJCC staging scheme. The modified AJCC staging outperformed the 8th AJCC staging in terms of the discriminatory capacity measured by the concordance index and Akaike information criterion, and the prognostic homogeneity assessed by using the likelihood ratio chi-squared test and stratified survival analysis.Replacing the 8th AJCC N classification with the LNR-based N classification can improve the prognostic performance of the 8th AJCC staging scheme for PDAC.

Enhancement of ganoderic acid production by constitutively expressing Vitreoscilla hemoglobin gene in Ganoderma lucidum.

The Vitreoscilla hemoglobin (VHb) gene was expressed in Ganoderma lucidum to enhance antitumor ganoderic acid (GA) production. The effects of VHb expression on the accumulation of GAs and lanosterol (intermediate) and the transcription of GA biosynthesis genes were also investigated. In VHb-expressing G. lucidum, the maximum concentrations of four individual GAs (GA-S, GA-T, GA-Mk and GA-Me) were 19.1±1.8, 34.6±2.1, 191.5±13.1 and 45.2±2.8µg/100mg dry weight, respectively, which were 1.4-, 2.2, 1.9- and 2.0-fold higher than those obtained in the wild-type strain. Moreover, the maximum lanosterol concentration in the strain expressing VHb was 1.28-fold lower than that in the wild-type strain. The transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase genes were up-regulated by 1.6-, 1.5-, and 1.6-fold, respectively, in the strain expressing VHb. This work is beneficial in developing an efficient fermentation process for the hyperproduction of GAs.

Improved polysaccharide production in a submerged culture of Ganoderma lucidum by the heterologous expression of Vitreoscilla hemoglobin gene.

Expression of Vitreoscilla hemoglobin (VHb) gene was used to improve polysaccharide production in Ganoderma lucidum. The VHb gene, vgb, under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase gene promoter was introduced into G. lucidum. The activity of expressed VHb was confirmed by the observation of VHb specific CO-difference spectrum with a maximal absorption at 419 nm for the transformant. The effects of VHb expression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production and transcription levels of three genes encoding the enzymes involved in polysaccharide biosynthesis, including phosphoglucomutase (PGM), uridine diphosphate glucose pyrophosphorylase (UGP), and ß-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in the vgb-bearing G. lucidum were 26.4 mg/100mg dry weight and 0.83 g/L, respectively, which were higher by 30.5% and 88.2% than those of the wild-type strain. The transcription levels of PGM, UGP and GLS were up-regulated by 1.51-, 1.55- and 3.83-fold, respectively, in the vgb-bearing G. lucidum. This work highlights the potential of VHb to enhance G. lucidum polysaccharide production by large scale fermentation.

Enhanced Production of Polysaccharide Through the Overexpression of Homologous Uridine Diphosphate Glucose Pyrophosphorylase Gene in a Submerged Culture of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Higher Basidiomycetes).

This study aimed to improve polysaccharide production by engineering the biosynthetic pathway in Ganoderma lucidum through the overexpression of the homologous UDP glucose pyrophosphorylase (UGP) gene. The effects of UGP gene overexpression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production, and transcription levels of 3 genes encoding the enzymes involved in polysaccharide biosynthesis, including phosphoglucomutase (PGM), UGP, and α-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in G. lucidum overexpressing the UGP gene were 24.32 mg/100 mg dry weight and 1.66 g/L, respectively, which were higher by 42% and 36% than those of the wild-type strain. The transcription levels of PGM, UGP, and GLS were up-regulated by 1.6, 2.6, and 2.4-fold, respectively, in the engineered strain, suggesting that increased polysaccharide biosynthesis may result from a higher expression of those genes.

Deformation mechanism of nanocomposite gels studied by contrast variation small-angle neutron scattering.

Contrast-variation small-angle neutron scattering (CV-SANS) was applied to investigate the deformation mechanism of high-performance nanocomposite polymer hydrogels (NC gels) consisting of polymer chains and inorganic clay platelets. Anisotropic SANS functions were obtained at various stretching ratios, lambda 's up to lambda=9 and were decomposed to three partial structure factors, S(ij)(Q parallel,Q perpendicular). Here, the subscripts i and j denote the polymer (P) or clay (C) and Q parallel and Q perpendicular are the magnitude of the scattering vectors along and perpendicular to the stretching directions, respectively. SCC(Q parallel,Q perpendicular) and S_{PP}(Q parallel,Q perpendicular) suggested that the orientation of clay platelets saturated by lambda approximately 3 , while the polymer chain stretching continued by further stretching. On the other hand, SCP(Q parallel,Q perpendicular) , only available by CV-SANS, indicated the presence of a polymer-enriched layer adsorbed to clay surface, which are responsible for large extensibility of NC gels over 1000% strain and large toughness exceeding 780 kPa.

Unusually high hydrophobicity and its changes observed on the newly-created surfaces of PNIPA/clay nanocomposite hydrogels.

The surface wettability of cross-sections of polymeric hydrogels was studied, focusing particularly on poly(N-isopropylacrylamide) (PNIPA) hydrogels below their lower critical solution temperature (LCST). It was found that nanocomposite hydrogels (N-NC gels) with organic (PNIPA)/inorganic (clay) network structures exhibit extraordinarily high contact angles for water (theta(w)) on newly-created, cross-sectional surfaces produced by cutting prior to measurement. Values of theta(w) for N-NC gels were observed in the range of 100 degrees-131 degrees and changed depending on the composition, the environment and the measuring time. It was shown that hydrophobic surfaces (high theta(w)) are formed most effectively in N-NC gels with specific clay and water contents. Also, during long-term measurements, high values of theta(w) showed unique changes which strongly depended on the clay concentration (i.e. network density). Further, the hydrophobic surface of N-NC gels changed to hydrophilic in contact with surface water and rapidly reverted to hydrophobic on subsequent drying. Also, contrary to the conventional hydrophobic surfaces of solids, a water droplet on the hydrophobic surface of an N-NC gel did not fall, even on a vertical surface, because of the strong interaction between the droplet and the gel surface. The mechanism for creating high values of theta(w) was attributed to the amphiphilicity of PNIPA chain in PNIPA/clay networks below the LCST and, more specifically, to the spontaneous alignment of N-isopropyl groups of PNIPA chains at the gel-air interface.