•We show high-yield production of diacetyl by Bacillus sp.•We improved diacetyl production by blocking ALDC in Bacillus sp.•Diacetyl biosynthetic pathway was amplified by overexpressing ALS in Bacillus sp.•Fe3+ enhanced the NOD of α-acetolactate to diacetyl in the engineered Bacillus sp.Diacetyl, a highly valuable product that is extensively used as an ingredient of food, tobacco, and daily chemicals such as perfumes, can be produced from the nonenzymatic oxidative decarboxylation of α-acetolactate during bacterial fermentation and converted to acetoin and 2,3-butanediol by 2,3-butanediol dehydrogenase. In the present study, Bacillus sp. DL01, which gives high acetoin production, was metabolically engineered to improve diacetyl production. After the deletion of α-acetolactate decarboxylase (ALDC)-encoding gene (alsD) by homologous recombination, the engineered strain, named Bacillus sp. DL01-ΔalsD, lost ALDC activity and produced 1.53 g/L diacetyl without acetoin and 2,3-butanediol accumulation. The channeling of carbon flux into diacetyl biosynthetic pathway was amplified by an overexpressed α-acetolactate synthase (ALS)-encoding gene (alsS) in Bacillus sp. DL01-ΔalsD-alsS, which produced 4.02 g/L α-acetolactate and 1.94 g/L diacetyl, and the conversion from α-acetolactate to diacetyl was increased by 1-fold after 20 mM Fe3+ was added to the fermentation medium. A titer of 8.69 g/L diacetyl, the highest reported diacetyl production, was achieved by fed-batch fermentation in optimal conditions using the metabolically engineered strain of Bacillus sp. DL01-ΔalsD-alsS. These results are of great importance as a new method for the efficient production of diacetyl by food-safe bacteria.Download high-res image (82KB)Download full-size image

Quercetin (Qu) is known as a dietary antioxidant with numerous bioactivities, but its function in anti-cancer has not been fully investigated. Here, we show that Qu at low doses (≤ 10 μM) significantly enhances the inhibition of 10-hydroxy camptothecin (HCPT) on the proliferation of MCF7, BGC823 and HepG2 cells. A plasmid DNA relaxation assay indicates that the inhibition of HCPT on the catalytic activity of topoisomerase I (Topo I) is increased by Qu at 10 μM. Compared to the treatment by Qu or HCPT alone, phosphorylation at Ser139 of γH2A.X in MCF7 cells starts to increase significantly (P < 0.05) at 6 h when treated by the combination of 10 μM Qu and 0.62 μM HCPT. Moreover, the combinational group successively arrests MCF7 cells at G1, S and G2/M phases from 12 h to 48 h via up-regulation of p21 and induces apoptosis at 24 h by triggering intrinsic cell death pathways. In addition, the inhibition effects of the combinational group on the proliferation of MCF7 cells are eliminated by pretreatment with 100 μM Z-VAD-FMK (a caspase inhibitor). Finally, by using nude mice xenografting assay of MCF7 cells, we demonstrate that tumor inhibition rates of combinational group are significantly higher than single-drug group. In summary, the synergic anti-cancer mechanism of Qu and HCPT in MCF7 cells is through the combined inhibitory effects of Qu and HCPT on Topo I, which synergistically induce cell cycle arrest and apoptosis by triggering DNA damage.Download high-res image (92KB)Download full-size image

Quercetin (Qu) is known as a dietary antioxidant with numerous bioactivities, but its function in anti-cancer has not been fully investigated. Here, we show that Qu at low doses (≤ 10 μM) significantly enhances the inhibition of 10-hydroxy camptothecin (HCPT) on the proliferation of MCF7, BGC823 and HepG2 cells. A plasmid DNA relaxation assay indicates that the inhibition of HCPT on the catalytic activity of topoisomerase I (Topo I) is increased by Qu at 10 μM. Compared to the treatment by Qu or HCPT alone, phosphorylation at Ser139 of γH2A.X in MCF7 cells starts to increase significantly (P < 0.05) at 6 h when treated by the combination of 10 μM Qu and 0.62 μM HCPT. Moreover, the combinational group successively arrests MCF7 cells at G1, S and G2/M phases from 12 h to 48 h via up-regulation of p21 and induces apoptosis at 24 h by triggering intrinsic cell death pathways. In addition, the inhibition effects of the combinational group on the proliferation of MCF7 cells are eliminated by pretreatment with 100 μM Z-VAD-FMK (a caspase inhibitor). Finally, by using nude mice xenografting assay of MCF7 cells, we demonstrate that tumor inhibition rates of combinational group are significantly higher than single-drug group. In summary, the synergic anti-cancer mechanism of Qu and HCPT in MCF7 cells is through the combined inhibitory effects of Qu and HCPT on Topo I, which synergistically induce cell cycle arrest and apoptosis by triggering DNA damage.Download high-res image (92KB)Download full-size image

Quercetin (Qu) is known as a dietary antioxidant with numerous bioactivities, but its function in anti-cancer has not been fully investigated. Here, we show that Qu at low doses (≤ 10 μM) significantly enhances the inhibition of 10-hydroxy camptothecin (HCPT) on the proliferation of MCF7, BGC823 and HepG2 cells. A plasmid DNA relaxation assay indicates that the inhibition of HCPT on the catalytic activity of topoisomerase I (Topo I) is increased by Qu at 10 μM. Compared to the treatment by Qu or HCPT alone, phosphorylation at Ser139 of γH2A.X in MCF7 cells starts to increase significantly (P < 0.05) at 6 h when treated by the combination of 10 μM Qu and 0.62 μM HCPT. Moreover, the combinational group successively arrests MCF7 cells at G1, S and G2/M phases from 12 h to 48 h via up-regulation of p21 and induces apoptosis at 24 h by triggering intrinsic cell death pathways. In addition, the inhibition effects of the combinational group on the proliferation of MCF7 cells are eliminated by pretreatment with 100 μM Z-VAD-FMK (a caspase inhibitor). Finally, by using nude mice xenografting assay of MCF7 cells, we demonstrate that tumor inhibition rates of combinational group are significantly higher than single-drug group. In summary, the synergic anti-cancer mechanism of Qu and HCPT in MCF7 cells is through the combined inhibitory effects of Qu and HCPT on Topo I, which synergistically induce cell cycle arrest and apoptosis by triggering DNA damage.Download high-res image (92KB)Download full-size image

Quercetin (Qu) is known as a dietary antioxidant with numerous bioactivities, but its function in anti-cancer has not been fully investigated. Here, we show that Qu at low doses (≤ 10 μM) significantly enhances the inhibition of 10-hydroxy camptothecin (HCPT) on the proliferation of MCF7, BGC823 and HepG2 cells. A plasmid DNA relaxation assay indicates that the inhibition of HCPT on the catalytic activity of topoisomerase I (Topo I) is increased by Qu at 10 μM. Compared to the treatment by Qu or HCPT alone, phosphorylation at Ser139 of γH2A.X in MCF7 cells starts to increase significantly (P < 0.05) at 6 h when treated by the combination of 10 μM Qu and 0.62 μM HCPT. Moreover, the combinational group successively arrests MCF7 cells at G1, S and G2/M phases from 12 h to 48 h via up-regulation of p21 and induces apoptosis at 24 h by triggering intrinsic cell death pathways. In addition, the inhibition effects of the combinational group on the proliferation of MCF7 cells are eliminated by pretreatment with 100 μM Z-VAD-FMK (a caspase inhibitor). Finally, by using nude mice xenografting assay of MCF7 cells, we demonstrate that tumor inhibition rates of combinational group are significantly higher than single-drug group. In summary, the synergic anti-cancer mechanism of Qu and HCPT in MCF7 cells is through the combined inhibitory effects of Qu and HCPT on Topo I, which synergistically induce cell cycle arrest and apoptosis by triggering DNA damage.Download high-res image (92KB)Download full-size image

Seafood is sometimes wasted due to the growth of psychrotolerant microbes which secrete proteases and break down proteins. Stenotrophomonas maltophilia FF11, isolated from frozen Antarctic krill, grows at a wide range of temperatures and secretes more proteases at low temperatures. According to zymogram analysis, two kinds of proteases were produced from this strain. A major protease was produced largely at 15 °C, but not at 37 °C. The temperature-dependent secreted protease was purified to homogeneity. Its molecular mass was determined at 37.4 kDa and its amino acid sequence was also obtained. This protease is a member of the subtilase group according to the NCBI blast analysis. The enzyme was highly stable at high salt concentration (4 M). Interestingly, its activity increased about 1.6-fold under high salt condition. The enzyme remains active and stable in different organic solvents (50 %, v/v) such as dimethylsulfoxide, dimethyl formamide, dioxane and acetone. These properties may provide potential applications in quality control for sea foods, in protein degradation at high salt concentration, in biocatalysis and biotransformation within non-aqueous media, such as detergent and transesterification.

•Markedly improved fructose mono-acylation catalyzed by CALB in ILs/2M2B co-solvent system.•Selectivity of mono-acylation in ILs/2M2B co-solvent governed by the change of CALB kinetic parameters.•Kinetic parameters variance determined by conformational changes of CALB binding substrates.Fructose monoesters are eco-friendly nonionic surfactants in various applications. Selective preparation of mono-acylated fructose is challenging due to the multiple hydroxyl sites available for acylation both chemically and enzymatically. Ionic liquids (ILs) have profound impacts not only on the reaction media but also on the catalytic properties of enzymes in the acylation process. In this study, utilizing an IL co-solvent system, selective synthesis of mono-acylated fructose with lauric acid catalyzed by immobilized Candida antarctica lipase B (CALB) was investigated. The imidazolium-based ILs selected as co-solvents with 2-methyl-2-butanol (2M2B) markedly improved the ratios of monolauroyl fructose in the presence of 60% [BMIM][TfO] (v/v) and 20% [BMIM][BF4] (v/v), in which the mono-acylated fructose was 85% and 78% respectively. Based on a Ping-Pong Bi−Bi model, a kinetic equation was fitted, by which the kinetic parameters revealed that the affinity between fructose and acyl-enzyme intermediate was enhanced. The inhibition effect of fructose on free enzyme was weakened in the presence of IL co-solvents. The conformation of CALB binding substrates also changed in the co-solvent system as demonstrated by Fourier transform infrared spectra. These results demonstrated that the variation of CALB kinetic characteristics was a crucial factor for the selectivity of mono-acylation in ILs/2M2B co-solvents.

•New gene delivery vector, folate–PEI–modified pullulan (P–PEI–FA), was synthesized.•It can efficiently wrap pDNA/siRNA at optimal N/P, help pDNA against degradation.•P–PEI–FA/pDNA showed lower cytotoxicity, displayed high gene transfection efficiency.•P–PEI–FA/siRNA at N/P ratio of 12.5 demonstrated enhanced gene silencing effect.•P–PEI–FA can assist DNA or siRNA targeting to FR-overexpressing cells.Folate receptor (FR)-mediated gene/short interfering RNA (siRNA) targeting shows advantage for the delivery of gene/siRNA into specific FR-overexpressing cancer cells. In this study, the non-targeted gene vector P–PEI was synthesized by grafting low-molecular-weight (1 kDa) branched polyethyleneimine (PEI) to succinylated pullulan, and the targeted gene vector P–PEI–FA was synthesized by coupling the carboxyl of folate (FA) to the amino of PEI. Gel electrophoresis retardation assay demonstrated that both P–PEI and P–PEI–FA can efficiently wrap pDNA and siRNA with electrostatic interaction at N/P ratios higher than 1.56 and can protect pDNA from degradation by DNase I and serum. Compared with PEI/pDNA, P–PEI/pDNA and P–PEI–FA/pDNA showed lower cytotoxicity against different cells. Under serum-containing conditions, compared with Lipofamine 2000/DNA and Lipofamine2000/siRNA, P–PEI–FA/DNA at N/P ratio of 6.25 displayed higher gene transfection efficiency, whereas P–PEI–FA/siRNA at N/P ratio of 12.5 demonstrated better enhanced gene silencing effect. P–PEI–FA/siRNA can also deliver FAM-labeled siRNA to endosomes and escape. Moreover, the gene transfection and silencing effects of P–PEI–FA were higher than those of P–PEI, and were dependent on the dose of FA in FR+ HeLa cells. Thus, P–PEI-FA can assist DNA or siRNA targeting to FR-overexpressing cells, and the uptake pathway of P–PEI–FA/siRNA was FR-mediated endocytosis. These results indicate that P–PEI–FA is a potential candidate for safe and targeted gene delivery applications.

•Tocopheryl pullulan-based (PUTC) self-assembling nanomicelles were fabricated.•These micelles showed low CMC and dispersed uniformly with regular spherical shape.•High entrapment efficiency and in vitro sustained release of HCPT in PUTC micelles•HCPT–PUTC micelles accumulated in cell nuclei and showed higher anticancer activity.•Blank PUTC micelles were biocompatible and showed low cytotoxicity to normal cells.Amphiphilic α-tocopherol pullulan polymers (PUTC1, PUTC2, and PUTC3) with different degrees of substitution were synthesized as new carriers for anticancer drugs. The polymers easily self-assembled into nanomicelles through dialysis method. The critical micelle concentrations (CMCs) were 38.0, 8.0, and 4.3 mg/L for PUTC1, PUTC2, and PUTC3, respectively. 10-Hydroxycamptothecin (HCPT) used as a model drug was successfully loaded into the PUTC nanomicelles. Transmission electron microscopy images demonstrated that HCPT-loaded PUTC nanomicelles were almost spherical and had sizes ranging within 171.5–257.8 nm that increased with increased HCPT-loading content, as determined by dynamic laser scattering. The highest encapsulation efficiency of HCPT in PUTC nanomicelles reached 98.3%. The in vitro release of HCPT from PUTC micelles demonstrated sustained release for over 80 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays showed that blank PUTC micelles were nontoxic to normal cells and that the HCPT-loaded PUTC2 nanomicelles showed higher cytotoxicity than the free drug, which was attributed to the enhanced cellular uptake of drug-loaded nanomicelles. Biodistribution experiments showed that PUTC micelles provided an excellent approach to rapid drug transport into cell nuclei. Moreover, the cellular uptake of micelles was found to be an energy-dependent and actin polymerization-associated endocytic process by endocytosis inhibition experiments. These results suggested that PUTC nanomicelles had considerable potential as a drug carrier for drug intracellular delivery in cancer therapy.

Gln49-PLA2 is a phospholipase A2 isolated from the Gloydius ussurensis snake venom. In this paper, we studied its effect on the function of neural conduction. Electrophysiological studies demonstrated that Gln49-PLA2 reduced the amplitude of the action potential and the velocity of nerve conduction on isolated mouse sciatic nerve. Patch clamp recordings confirmed that Gln49-PLA2 significantly decreased neural excitability by the potentiation of sodium channels and the blockade of potassium channels in nerve terminal. In freshly isolated hippocampal pyramidal neurons, 54.25% of potassium current was inhibited by 20 μg/ml Gln49-PLA2. However, sodium current was potentiated by 158.99% under the same condition. These findings demonstrate that the effect of Gln49-PLA2 on ion channels is the main mechanism of analgesic action.

A block copolymer of 2-dimethylaminoethyl methacrylate (DMAEMA) and glycidyl methacrylate (GMA) was grafted onto the surface of magnetic nanoparticles (Fe3O4) via atom transfer radical polymerization. The resultant PGMA-b-PDMAEMA-grafted-Fe3O4 magnetic nanoparticles with amino and epoxy groups were characterized by Fourier transform infrared spectroscopy, powder X-ray diffraction, thermo-gravimetric analysis, and scanning electron microscopy. Lipase from Burkholderia cepacia was successfully immobilized onto the magnetic nanoparticles by physical adsorption and covalent bonding. The immobilization capacity of the magnetic particles is 0.5 mg lipase per mg support, with an activity recovery of up to 43.1% under the optimum immobilization condition. Biochemical characterization shows that the immobilized lipase exhibits improved thermal stability, good tolerance to organic solvents with high lg P, and higher pH stability than the free lipase at pH 9.0. After six consecutive cycles, the residual activity of the immobilized lipase is still over 55% of its initial activity.A block copolymer of 2-dimethylaminoethyl methacrylate (DMAEMA) and glycidyl methacrylate (GMA) was grafted onto the surface of magnetic nanoparticles (Fe3O4) via atom transfer radical polymerization. The resultant PGMA-b-PDMAEMA-grafted-Fe3O4 magnetic nanoparticles with amino and epoxy groups were employed to immobilize the lipase from Burkholderia cepacia successfully by physical adsorption and covalent bonding. Biochemical characterization shows that the immobilized lipase exhibits improved thermal stability, good tolerance to organic solvents with high log P, and higher pH stability than the free lipase at pH 9.0. After six consecutive cycles, the residual activity of the immobilized lipase is still over 55% of its initial activity.Download full-size image

•Lipase (CALB) shows selectivity for esterification degree (DE) of fructose laurate.•High DE product synthesized more effective in methyl ethyl ketone by CALB enzyme.•Conformational changes of CALB controlling high DE selectivity in methyl ethyl ketone.Sugar esters of fatty acids have many applications as biocompatible and biodegradable emulsifiers, which are determined by their degrees of esterification (DE). Direct esterification of fructose with lauric acid in organic media used commercial immobilized Candida antarctica lipase B (CALB) was investigated for DE. Significant difference of DE was observed between 2-methyl-2-butanol (2M2B) and methyl ethyl ketone (MEK), as di-ester/mono-ester molar ratio of 1.05:1 in 2M2B and 2.79:1 in MEK. Fourier transform infrared (FTIR) spectra showed that the secondary structure of the enzyme binding mono-ester presented distinct difference in 2M2B and MEK. Contents of β-turn and antiparallel β-sheet of CALB in 2M2B were 26.9% and 16.2%, respectively, but 19.1% and 13.2% in MEK. To understand the relationship between the conformational changes and differences of DE, mono-ester and fatty acid were directly employed for synthesis of di-ester. The maximum initial velocity of di-ester synthesis in MEK was 0.59 mmol g (enzyme)−1 h−1, which was 2.19-fold as greater as that in 2M2B, indicating that CALB conformation in MEK was preferred for the synthesis of di-ester. These results demonstrated that the conformation of CALB binding mono-ester affected by organic solvents essentially determined DE.Download full-size image

Background10-Hydroxy camptothecin (HCPT), a naturally occurring alkaloid, is a clinical drug for cancer chemotherapy. Baicalein (BA) is a flavonoid extracted from the root of Scutellaria baicalensis. The synergistic anti-cancer effect of BA and HCPT has not been reported.PurposeTo explore whether and how BA enhances the anti-cancer effect of HCPT in BGC823 cells.MethodsCell viability was measured by MTT assay. Apoptosis and cell cycle were analyzed through flow cytometry and western blotting analysis. DNA damage was determined by a comet assay. The activity of topoisomerase I (Topo I) was detected by the plasmid DNA relaxation assay. The synergistic anti-cancer effect of BA and HCPT in vivo was tested by BGC823 xenografted tumor model.ResultsBA at non-toxic doses prominently enhanced the anti-cancer activities of HCPT in BGC823, MCF7 and SMMC7721 cells. Combination treatment of BA and HCPT induced BGC823 cells apoptosis mainly via intrinsic rather than extrinsic pathways, and preferentially arresting cell cycle in G1 and G2 phases with the aid of p21. Of note, p53, the upstream regulator of cell apoptosis and cycle, was increased by 5 folds in combination group. It helped to further trigger DNA damage and inhibit Topo I catalytic activity after combination treatment of BA and HCPT. Moreover, the BGC823 xenografted tumor growth rate in nude mice was repressed in a greater degree (P < 0.01) in the combinational group than the single-drug group.ConclusionHCPT and BA, a new and effective combination therapy, synergistically target Topo I and up-regulate p53 to induce cell apoptosis and cell cycle arrest.Download high-res image (91KB)Download full-size image