Improved production of germacrene A, a direct precursor of ß-elemene, in engineered Saccharomyces cerevisiae by expressing a cyanobacterial germacrene A synthase.

BACKGROUND: The sesquiterpene germacrene A is a direct precursor of ß-elemene that is a major component of the Chinese medicinal herb Curcuma wenyujin with prominent antitumor activity. The microbial platform for germacrene A production was previously established in Saccharomyces cerevisiae using the germacrene A synthase (LTC2) of Lactuca sativa. RESULTS: We evaluated the performance of LTC2 (LsGAS) as well as nine other identified or putative germacrene A synthases from different sources for the production of germacrene A. AvGAS, a synthase of Anabaena variabilis, was found to be the most efficient in germacrene A production in yeast. AvGAS expression alone in S. cerevisiae CEN.PK2-1D already resulted in a substantial production of germacrene A while LTC2 expression did not. Further metabolic engineering the yeast using known strategies including overexpression of tHMGR1 and repression of squalene synthesis pathway led to an 11-fold increase in germacrene A production. Site-directed mutagenesis of AvGAS revealed that while changes of several residues located within the active site cavity severely compromised germacrene A production, substitution of Phe23 located on the lateral surface with tryptophan or valine led to a 35.2% and 21.8% increase in germacrene A production, respectively. Finally, the highest production titer of germacrene A reached 309.8 mg/L in shake-flask batch culture. CONCLUSIONS: Our study highlights the potential of applying bacterial sesquiterpene synthases with improved performance by mutagenesis engineering in producing germacrene A.

Biotransformation of dietary phytoestrogens by gut microbes: A review on bidirectional interaction between phytoestrogen metabolism and gut microbiota.

Phytoestrogens are a class of plant produced polyphenolic compounds with diphenolic structure, which is similar to 17ß-estradiol. These phytoestrogens preferentially bind to estrogen receptors, however, with weak affinity. Recently, many studies have found that these phytoestrogens can be transformed by gut microbiota through novel enzymatic reactions into metabolites with altered bioactivity. Recent studies have also implied that these metabolites could possibly modulate the host gut ecosystem, gene expression, metabolism and the immune system. Thus, isolating gut microbes capable of biotransforming phytoestrogens and characterizing the novel enzymatic reactions involved are principal to understand the mechanisms of beneficial effects brought by gut microbiota and their metabolism on phytoestrogens, and to provide the theoretical knowledge for the development of functional probiotics. In the present review, we summarized works on gut microbial biotransformation of phytoestrogens, including daidzin (isoflavone), phenylnaringenin (prenylflavonoid), lignans, resveratrol (stilbene) and ellagitannins. We mainly focus on gut bacterial isolation, metabolic pathway characterization, and the bidirectional interaction of phytoestrogens with gut microbes to illustrate the novel metabolic capability of gut microbiota and the methods used in these studies.

[Effect of a chemical primer on the bond strength of a zirconia ceramic with self-adhesive resin cement].

OBJECTIVE: To evaluate the bond strength and durability of a self-adhesive resin cement with a zirconia ceramic pretreated by a zirconia primer. METHODS: Zirconia ceramic (Vita Inceram YZ) plates with a thickness of 2.5 mm were fired, polished, and then cleaned. Half of the polished ceramic plates were sandblasted with 50 µm alumina particles at 0.3 MPa for 20 s. The surface compound weight ratios were measured via X-ray fluorescence microscopy. The polished and sandblasted ceramic plates were directly bonded with self-adhesive resin cement (Biscem) or were pretreated by a zirconia primer (Z Primer Plus) before bonding with Biscem. The specimens of each test group were divided into two subgroups (n=10) and subjected to the shear test after 0 and 10,000 thermal cycles. The data were analyzed via three-way ANOVA. RESULTS: After air abrasion, 8.27% weight ratio of alumina attached to the zirconia surface. Compared with air abrasion, primer treatment more significantly improved the primary resin bond strength of the zirconia ceramic. The primary resin bond strength of the zirconia ceramic with no primer treatment was not affected by thermocycling (P>0.05). However, the primary resin bond strength of the zirconia ceramic with primer treatment was significantly decreased by thermocycling (P<0.05). CONCLUSION: Primer treatment can improve the primary resin bond strengths of zirconia ceramics. However, the bond interface of the primer is not stable and rapidly degraded during thermocycling.

[Effects of sandblasting on surface character and resin bond of zirconia ceramic].

OBJECTIVE: To evaluate the effect of Al2O3 particles sandblasting on the surface roughness, element composition and resin bond durability of zirconia ceramic. METHODS: Sixty 2.5 mm thick computer aided design and computer aided manufacture (CAD/CAM) zirconia ceramic (Vita Inceram YZ) plates were fired, polished and cleaned. Half of polished ceramic plates was sandblasted with 50 µm alumina particles at 0.3 MPa for 20 s. The surface roughness of polished and sandblasted ceramic surface were measured by 3D-laser scanning microscope, and the surface element weight and atom ratio of the ceramic surface were measured by energy disperse spectroscopy (EDS). Then polished and sandblasted ceramic plates were randomized into six groups. In Group 1 and 2 the polished and sandblasted ceramic plates were bonded irrespectively with conventional resin cement (DUOLINK). In Group 3 and 4 the ceramic plates were bonded with resin cement containing MDP (Panavia F), In Group 5 and 6 the specimens were pretreated with silane coupler acitivated by MDP (Clearfil Ceramic Primer), then bond with Panavia F. The specimens of each test group were then divided into two subgroups, and to received shear test after 0 and 10 000 time thermal cycle. The data was analyzed by one-way ANOVA and independent t test. RESULTS: Comparing with polishing, sandblasting reduced the oxygen atom and weight ratio of zirconia ceramic surface (P < 0.001), and increased the zirconium atom and weight ratio (P < 0.001), meanwhile increased the surface roughness (P < 0.001). The bond strength between ceramic plates and resin cement in all test groups decreased after thermocycling (P < 0.001). All specimen in test group 1 and 2 lost bond, and the bond strength of test group 3 and 5 [(0.59 ± 0.17), (0.89 ± 0.84) MPa] were significantly lower than that of test group 4 and 6 [(14.63 ± 3.03), (16.64 ± 1.90) MPa], and the bond strength of test group 6 were significanlty higher than that of test group 4. CONCLUSIONS: Sandblasting improves durability of bond between zirconia ceramic and resin cement containing MDP, not only by increasing the roughness and area of ceramic surface, but also by changing its surface element composition to obtain more chemical bond.

[Research on the resin bond durability of glass-infiltrated alumina ceramic].

OBJECTIVE: To analyze the effect of different silane coupling agents on the resin bond durability of glass-infiltrated alumina ceramic. Methods A glass-infiltrated alumina ceramic was silanized or not by three silane coupling agents. The treated ceramic surfaces were bonded with two resin cements. Their micro-bond strength were measured after 0, 30,000 thermal cycles. RESULTS: Before thermal cycling, resin cement A had lowest bond strength to ceramic, and ceramic treated by silane coupling agent A with two cements had lower bond strength than those treated by silane coupling agent B and C. After thermal cycling, cement A had no bond strength with no treated ceramic, only ceramic treated by silane coupling agent A with two cements had more than 5 MPa bond strength. CONCLUSION: The glass-infiltrated alumina cermaic treated by the silane coupling agent activated by 10-methacryloyloxydecyl-dihydrogen phosphate could obtain better bond durability with different type of resin cements.

Characterization of midgut trypsinogen-like cDNA and enzymatic activity in Plutella xylostella parasitized by Cotesia vestalis or Diadegma semiclausum.

Protein digestion in insects is a result of the action of a complex of proteinases present in the midgut. In this report we describe the cloning and sequencing of a trypsin cDNA from larvae of the lepidopteran herbivore Plutella xylostella. We investigated the expression of this gene and enzymatic activity of its translation product with N-a-benzoyl-l-arginine p-nitroanilide (BApNA) as substrate in P. xylostella larvae that were either unparasitized or parasitized by Cotesia vestalis or Diadegma semiclausum parasitoids. The full cDNA sequence consisted of an open reading frame (ORF) encoding 273 amino acid residues including 23 residues of a signal peptide, and the predicted mature trypsinogen-like enzyme had a molecular mass of 26.5 kDa. The amino acid sequence of this trypsinogen-like enzyme protein and phylogenetic relationship with other published trypsin enzyme proteins suggested that it may be a new proteinase in the trypsin protein family. Parasitism of D. semiclausum did not significantly change the mRNA transcript level or BApNAase activity in host larvae. By contrast, parasitization by C. vestalis induced higher transcript levels coupled with a higher level of BApNAase activity. The BApNAase activity in the midgut of nonparasitized or parasitized P. xylostella larvae increased to a maximum level at pH 12, and the parasitism by both C. vestalis and D. semiclausum increased sensitivity of the enzyme to pH values ranging from 2 to 9.5. These parasitoid-induced changes may represent host manipulation by the developing parasitoid larva.

Microshear bond strength of resin bonding systems to machinable ceramic with different surface treatments.

PURPOSE: The bond strength and bond durability of two high-viscosity dual-curing resin luting agents with different surface treatments of ceramic were investigated. MATERIALS AND METHODS: GN-I machinable ceramic surfaces were treated with 37% phosphoric acid for 30 s (PA), 5% hydrofluoric acid for 5 s (HF-5), 10 s (HF-10), or 30 s (HF-30), or blasting with 50-microm Al2O3 for 10 s (AB). The roughness of the ceramic surface was measured. Treated ceramic surfaces were bonded with three resin bonding systems (RBS): Ceramic Primer/LinkmaxHV (CP/LMHV), Monobond S/VariolinkIIHV (MBS/VLIIHV), or MBS/Heliobond/VLIIHV (MBS/HB/VLIIHV). A microshear test was used to measure the bond strength after 24 h (TC 0) and subsequent thermocycling (TC 10,000 at 4 degrees C and 60 degrees C). ANOVA was performed for statistical analysis, with significance set at p < 0.05. RESULTS: For three RBSs, bond strength at TC O was not affected, regardless of ceramic surface treatment (p > 0.05). All combined groups of ceramic surface treatment and resin bonding system decreased after 10,000 thermal cycles, especially groups treated with alumina blasting and bonded with each of three RBSs (p < 0.05). MBS/HB/VLIIHV with each surface treatment did not improve the bond strength and durability compared with MBS/VLIIHV. CONCLUSION: CP/LMHV and MBS/VLIIHV obtained sufficient bond strength and bond durability to GN-I machinable ceramic by only cleaning with PA; the application of HB may not be necessary.