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Ba0.6Sr0.4TiO3/Mg1−xZnxO (MZO, x = 0, 0.05, 0.10, 0.15 and 0.20) ceramic composites were prepared by traditional ceramic processing. The crystal structure, fracture surface morphology, and dielectric properties were investigated. The samples with x = 0, 0.05 and 0.10 exhibited favorable sintering behavior, and homogeneous diphase microstructure was obtained. Nevertheless, the microstructure of the samples with x = 0.15 and 0.20 was inhomogeneous and abnormal grain growth could be observed, and the abnormal grain growth induced the degradation of dielectric strength. The sample with x = 0.10 has relatively low dielectric loss (1.26×10−3) and the optimal FOM value (about 174).

A combined promoter expression vector pBV–PAL for high-level expression of phenylalanine ammonia lyase gene of Rhodosporidium toruloides was constructed. Pal gene was cloned and inserted into the region between SalI and PstI restriction sites of expression vector pBV220 (containing PLPR promoter) to obtain recombinant expression vector pBV220–PAL. The tac promoter obtained from the plasmid pKtac was inserted into the expression vector pBV220–PAL to construct expression vector pBV–PAL. The recombinant plasmid pBV220–PAL and pBV–PAL were introduced into Escherichia coli JM109 by transformation. The result showed that the transformant E. coli JM109 (pBV–PAL) gave a much higher PAL activity than that transformant E. coli JM109 (pBV220–PAL). Recombinant PAL expression level of the transformant JM109 (pBV–PAL) was about 9.6% of total cellular protein, specific enzyme activity was 2.3-fold higher than that of the transformant JM109 (pBV220–PAL), reached 35 U/g (dry cells weight, DCW). PAL specific activity of 123 U/g (DCW) could be achieved in a 5-l fermentor. 80.5% conversion rate of trans-cinnamic acid to l -phenylalanine and 5.12 g/l l -phenylalanine were obtained after 3 h bioconversion using the transformant JM109 (pBV–PAL). The recombinant strain JM109 containing the combined promoter expression vector pBV–PAL was shown to be effective and practical to product l -phenylalanine.

Ba0.6Sr0.4TiO3/Mg0.9Zn0.1O (BST/MZO) ceramic composites with different MZO contents were prepared by traditional ceramic process. The crystal structure, fracture surface morphology, and dielectric properties were systematically investigated. The results show that the BST/MZO ceramic composites possess diphase structure, dense, and uniform morphology. The composites have relatively low dielectric loss (in the order of 10−3) at microwave frequency. The ceramics all retain substantial tunability (more than 20% at 8 kV/mm DC field) and excellent dielectric strength (more than 19.5 kV/mm). The BST-50 wt% MZO sample has the optimal FOM value (about 256) and should be a better candidate for tunable microwave applications.

Aim: To improve phenylalanine ammonia lyase (E.C.4·3·1·5-PAL) activity in recombinant Escherichia coli. Some methods for enrichment of PAL activity in recombinant E. coli JM109 were described. In an effort to create a rich enzyme source these methods would lead to improvements in the production of l-phenylalanine. Methods and Results: The possibilities of enriching PAL activity in recombinant E. coli was investigated by using individual and combinations of amino acids, organic solvents and surfactants. PAL activity was induced by adding combination of l-phenylalanine and l-tyrosine, activities as high as 64·3 U g−1of cells were obtained and enzyme activity was enriched by over 3·5-fold in comparison with the control. Permeabilization with cetyl trimethyl ammonium bromide or the acetone significantly enriched cellular PAL activity, which improved over 8·2- and 9·0-fold compared with the control, as high as 148·5 and 164·5 U g−1of cells respectively. Conclusion: These efforts may provide some effective methods for enhancing l-phenylalanine ammonia lyase activity. Significance and Impact of the Study: These approaches for manipulating recombinant E. coli in an effort to create a rich enzyme source would serve as a biotechnologically important protocol for production of l-phenylalanine.

A novel enzyme immobilization approach was used to generate mesoporous enzymes-silica composite microparticles by co-entrapping gelatinized starch and cross-linked phenylalanine ammonia lyase (PAL) aggregates (CLEAs) containing gelatinized starch into biomemitic silica and subsequently removing the starch by α-amylase treatment. During the preparation process, the gelatinzed starch served as a pore-forming agent to create pores in CLEAs and biomimetic silica. The resulting mesoporous CLEAs-silica composite microparticles exhibited higher activity and stability than native PAL, conventional CLEAs and PAL encapsulated in biomimetic silica. Furthermore, the mesoporous CLEAs-silica composite microparticles displayed good reusability due to its suitable size and mechanical properties and had excellent stability for storage. The superior catalytic performances were attributed to the combinational unique structure from the intra-cross-linking among enzyme aggregates and hard mesoporous silica shell, which not only decreased the enzyme-support negative interaction and mass-transfer limitations, but also improved the mechanical properties and monodispersity. This approach will be highly beneficial for preparing various bioactive mesoporous composites with excellent catalytic performance.

To verify c-Myc can regulate the expression of lncRNA H19 directly in non-small cell lung cancer (NSCLC) and clarify the molecular mechanism on how lncRNA H19 promote the cell cycle progression of NSCLC. The mRNA levels of lncRNA H19 in NSCLC tissues and cells, the adjacent tissues and normal cells were determined by RT-PCR. The expression change of lncRNA H19 in NSCLC cells after transfection with pcDNA3.1-c-Myc or c-Myc-siRNA was determined by RT-PCR, respectively. Targeted role of c-Myc on the promoter of H19 was studied by luciferase reporter assay. Chromosome immune coprecipitation (ChIP) was used to confirm the relationship between c-Myc and H19. MiRNAs that have base-pairing with H19 was predicted by online software. The relationship between H19 and miR-107 was determined by disturbing and overexpressing the expression of H19. The influence of the changes of H19 and miR-107 on cell cycle progression was determined by flow cytometry. The mRNA levels of lncRNA H19 in NSCLC tissues and cells were significantly higher than the adjacent tissues and normal cells, respectively. The expression of H19 increased or decreased accordingly with the overexpression and knockdown of c-Myc. The activity of the promoter of H19 was strengthened by c-Myc. While the expression of miR-107 increased or decreased with the overexpression and knockdown of H19, respectively. The number of cells in G2/M stage decreased significantly with the knockdown of H19 and miR-107 compared with the control group. Our study demonstrates that lncRNA H19, which is induced by c-Myc, is up-regulated in NSCLC. H19 influences the mitotic progression of NSCLC cell lines.

Objective: The present study observed the clinical outcomes of the sodium cantharidinate/vitamin B6/GP regimen in the treatment of middle-late stage Non-small-cell lung carcinoma (NSCLC). Materials and Methods: Eighty-six cases of NSCLC were selected and randomized into two groups. Forty-five cases in the treatment group were subject to 30 ml cantharidinate/vitamin B6+ GP regimen, for four 21-day cycles. Forty-one cases in the control group were subject to regular GP regimen, for four 21-day cycles. Results: The effectiveness rate was 57.8% in the treatment group and 36.6% in the control group, suggesting statistical difference (P < 0.05); life quality in the treatment group was significantly better than that in the control group (P < 0.05); the occurrence rate of the toxic/adverse effects were significantly lower in the treatment group compared with that in the control group (P < 0.05). Conclusion: Sodium cantharidinate/vitamin B6/GP regimen had fair effectiveness and synergistically improved the clinical outcomes. It lowered the toxic/adverse effects and its application is worth further investigation and promotion.

Phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis was encapsulated within polyethyleneimine-mediated biomimetic silica. The main factors in the preparation of biomimetic silica were optimized by response surface methodology (RSM). Compared to free PAL (about 2 U), the encapsulated PAL retained more than 43 % of their initial activity after 1 h of incubation time at 60 degrees C, whereas free PAL lost most of activity in the same conditions. It was clearly indicated that the thermal stability of PAL was improved by encapsulation. Moreover, the encapsulated PAL exhibited the excellent stability of the enzyme against denaturants and storage stability, and pH stability was improved by encapsulation. Operational stability of 7 reaction cycles showed that the encapsulated PAL was stable. Nevertheless, the K-m value of encapsulated PAL in biomimetic silica was higher than that of the free PAL due to lower total surface area and increased mass transfer resistance.

The industrial use of phenylalanine ammonia lyase (PAL), an interesting biocatalyst for manufacture of L-phenylalanine (L-Phe) by reversing the enzyme reaction with high concentration of trans-cinnamic acids (t-CA) and ammonia, has been hampered by a lack of long-term stability and low activity toward substrates. In this study, it is shown that the PAL activity of such a CLEA can be improved by molecular imprinting with a suitable substrate. PAL was imprinted with t-CA and subsequently cross-linked with glutaraldehyde (iCLEAs). Compared to free PAL, PAL stability in the iCLEAs against substrate inhibition was significantly improved, furthermore, the iCLEAs exhibited good reusability. These results indicated that the procedure might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application.

In order to specifically support the technology trend of increased miniaturization of Micro Electro Mechanical Systems (MEMS) devices, highly porous ZrCoCe non-evaporable getter (NEG) film has been produced by DC magnetron sputtering from a preformed ZrCoCe alloy target. The porous film is composed of columnar crystals, which is further built up with assembled ZrCoCe amorphous or nanocrystalline grains. Gas sorption investigation shows that this film can be activated at low temperature and exhibits excellent stable sorption characteristics. Sorption properties can be further improved with elevating the activation temperatures due to the microstructure modification. The capability of ZrCoCe films withstanding wafer physical or chemical cleaning processes is investigated, indicating their compatibility with MEMS vacuum packaging and the appropriate way to store them.

Cordyceps militaris is a potential harborer of biometabolites for herbal drugs. For a long time, C. militaris has gained considerable significance in several clinical and biotechnological applications. Much knowledge has been gathered with regard to the C. militaris's importance in the genetic resources, nutritional and environmental requirements, mating behavior and biochemical pharmacological properties. The complete genome of C. militaris has recently been sequenced. This fungus has been the subject of many reviews, but few have focused on its biotechnological production of bioactive constituents. This mini-review focuses on the recent advances in the biotechnological production of bioactive compositions of C. militaris and the latest advances on novel applications from this laboratory and many others.

Novel hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase (HM-PAL-CLEAs) were developed by co-aggregation of enzyme aggregates with magnetite nanoparticles and subsequent crosslinking with glutaraldehyde. The HM-PAL-CLEAs can be easily separated from the reaction mixture by using an external magnetic field. Analysis by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) indicated that PAL-CLEAs were inlayed in nanoparticle aggregates. The HM-PAL-CLEAs revealed a broader limit in optimal pH compared to free enzyme and PAL-CLEAs. Although there is no big difference in Km of enzyme in CLEAs and HM-PAL-CLEAs, Vmax of HM-PAL-CLEAs is about 1.75 times higher than that of CLEAs. Compared with free enzyme and PAL-CLEAs, the HM-PAL-CLEAs also exhibited the highest thermal stability, denaturant stability and storage stability. The HM-PAL-CLEAs retained 30% initial activity even after 11 cycles of reuse, whereas PAL-CLEAs retained 35% of its initial activity only after 7 cycles. These results indicated that hybrid magnetic CLEAs technology might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application.

Cross-linked enzyme aggregates of glucoamylase (GAs-CLEAs) were prepared. The effect and concentration of aggregating agent as well as those of cross-linking agent were studied. It was effective for GAs-CLEAs production to use ammonium sulfate (80 %-saturation), and subsequently cross-link for 2 h with 4.5 % (v/v) glutaraldehyde. Some changes in optimum pH and temperature values of the enzyme activity were recorded after immobilization. Moreover, the thermal stability of the resulting GAs-CLEAs was also investigated. Compared to the free enzyme, the GAs-CLEAs exhibited the expected increasing thermal stability. These CLEAs are suitable for all starch-based industries. To the best of our knowledge, this is the first report of immobilization of GAs as cross-linked enzyme aggregates.

Cross-linked enzyme aggregate (CLEA) technology has been regarded as an effective carrier-free immobilization method. This method is very attractive due to its simplicity and robustness, as well as for the possibility of using the crude enzyme extract and the opportunity to co-immobilize multiple different enzymes. The resulting CLEAs generally exhibit high catalyst productivities, improved storage and operational stability and are easy to recycle. Nowadays, although the technology has been applied to various enzymes, some undesirable properties have limited its further application. To overcome these limitations, novel strategies have been developing in recent years. This mini-review focuses on process optimization, new improved strategies and the latest advances on CLEAs technology.

Cross-linked enzyme aggregates (CLEAs) have been recently proposed as an alternative to conventional immobilization methods on solid carriers. However, the low cross-linking efficiency causes the major activity loss and instability in the conventional protocol for CLEA preparation. Herein, the effects of bovine serum albumin and starch addition on the cross-linking efficiency of CLEAs of phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis were evaluated. A co-aggregation strategy was developed to improve cross-linking efficiency by adding starch and bovine serum albumin (BSA). CLEAs of PAL prepared in the presence of BSA and starch (PSB-CLEAs) retained 36 % activity, whereas CLEAs prepared without BSA and starch (PAL-CLEAs) retained only 8 % activity of the starting enzyme preparation. Compared with PAL-CLEAs, the thermal stability of PSB-CLEAs has improved considerably, maintaining 30 % residual activity after 4 h of incubation at 70 °C, whereas the PAL-CLEAs have only 13 % residual activity. PSB-CLEAs also exhibited the expected increased stability of PAL against hydrophilic organic solvents, superior operability, and higher storage stability. The proposed technique of preparing CLEAs using co-aggregation with starch and BSA would rank among the potential strategies for efficiently preparing robust and highly stable enzyme aggregates.

During the two-stage submerged fermentation of medicinal mushroom Cordyceps militaris, it was found that K(+), Ca(2+), Mg(2+), and Mn(2+) were favorable to the mycelial growth. The EPS production reached the highest levels in the media containing Mg(2+) and Mn(2+). However, Ca(2+) and K(+) almost failed to increase significantly exopolysaccharides (EPS) production. Sodium dodecyl sulfate (SDS) significantly enhanced EPS production compared with that of without adding SDS when SDS was added on static culture stage of two-stage cultivation process. The presence of Tween 80 in the medium not only simulated mycelial growth but also increased EPS production. By response surface methods (RSM), EPS production reached its peak value of 3.28 g/L under optimal combination of 27.6 mM Mg(2+), 11.1 mM Mn(2+), and 0.05 mM SDS, which was 3.76-fold compared with that of without metal ion and surfactant. The results obtained were useful in better understanding the regulation for efficient production of EPS of C. militaris in the two-stage submerged culture.

Curdlan is a microbial polysaccharide composed exclusively of β-(1,3)-linked glucose residues. Until now only bacteria belonging to the Alcaligenes and Agrobacterium species have been reported to produce Curdlan. In this study, a bacterium capable of producing extracellular Curdlan, identified as Pseudomonas sp. on the basis of 16S rDNA gene sequencing, was isolated from soil samples. From the HPLC, permethylation linkage analysis, (13)C NMR, and FT-IR analytical data, the polysaccharide consisted exclusively of glucose; the most prominent sugar was 1,3-linked glucose, and most glycosidic bonds joining these sugar residues were of the β-type. This also supported that the exopolysaccharide produced by Pseudomonas sp. was actually Curdlan. In addition, the Pseudomonas sp. was studied for the production of Curdlan by conventional "one-factor-at-a-time technique" and response surface methodology (RSM). It was observed that glucose and yeast extract were the most suitable carbon source and nitrogen source for Curdlan production, respectively. By using RSM, Curdlan production was increased significantly by 188%, from 1.25 to 2.35 g/L, when the strain was cultivated in the optimal condition developed by RSM, and the highest Curdlan production rate of 0.81 g/(L h) was obtained. To the best of the authors' knowledge, this is the first report on Curdlan production by Pseudomonas sp.

Cross-linked enzyme aggregates of phenylalanine ammonia lyase (PAL-CLEAs) from Rhodotorula glutinis were prepared. The effects of the type of aggregating agent, its concentration, and that of cross-linking agent were studied. PAL-CLEAs production was most effective using ammonium sulfate (40 % saturation), followed by cross-linking for 1 h with 0.2 % (v/v) glutaraldehyde. Moreover, the storage and operational stability of the resulting PAL-CLEAs were also investigated. Compared to the free enzyme, the PAL-CLEAs exhibited the expected increased stability of the enzyme against various deactivating conditions such as pH, temperature, denaturants, and organic solvents and showed higher storage stability than its soluble counterpart. Additionally, the reusability of PAL-CLEAs with respect to the biotransformation of L-phenylalanine was evaluated. PAL-CLEAs could be recycled at least for 12 consecutive batch reactions without dramatic activity loss, which should dramatically increase the commercial potential of PAL for synthesis of L: -phenylalanine. To the best of our knowledge, this is the first report of immobilization of PAL as cross-linked enzyme aggregates.

Resource-based cities occupy a prominent position in the process of industrialization and urbanization in China. After the long-term development, most resource-based cities are faced with the situation that their resources are decreasing and exhausted. In the period of post-resource economy, the value of natural resources must be compensated reasonably to achieve sustainable development. This paper analyzes the prominent characteristics of resource-based cities in the period of post-resource economy and expounds the necessity of compensation. On this basis, the paper, from the perspectives of government, society, enterprise and legal system, puts forward the relevant suggestions on improving the compensation system in the period of post-resource economy.

A separable and highly-stable enzyme system was developed by adsorption of phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis in amino-functionalized macroporous silica gel and subsequent enzyme crosslinking. This resulted in the formation of cross-linked enzyme aggregates (PAL-CLEAs) into macroporous silica gel (MSG-CLEAs). The effect of adsorptive conditions, type of aggregating agent, its concentration as well as that of cross-linking agent was studied. MSG-CLEAs production was most effective using ammonium sulfate (40%-saturation), followed by cross-linking for 1 h with 1.5% (v/v) glutaraldehyde. The resulting MSG-CLEAs extended the optimal temperature and pH range compared to free PAL and PAL-CLEAs. Moreover, MSG-CLEAs exhibited the excellent stability of the enzyme against various deactivating conditions such as temperature and denaturants, and showed higher storage stability compared to the free PAL and the conventional PAL-CLEAs. Such as, after 6 h incubation at 60°C, the MSG-CLEAs still retained more than 47% of the initial activity whereas PAL-CLEAs only retained 7% of the initial activity. Especially, the MSG-CLEAs exhibited good reusability due to its suitable size and active properties. These results indicated that PAL-CLEAs on MSG might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application.