GoldMag nanocomposite-functionalized graphene sensing platform for one-step electrochemical immunoassay of alpha-fetoprotein

A new flow-through electrochemical immunosensor was designed for sensitive detection of alpha-fetoprotein (AFP) in human serum by using nanogold-functionalized magnetic graphene nanosheets as immunosensing probes. Initially, amino functionalized magnetic beads were covalently immobilized on the surface of graphene oxide nanosheets (MGPs), then nanogold particles were adsorbed on the amino groups of the MGPs to construct GoldMag nanocomposites functionalized graphene nanosheets (GMGPs), and then horseradish peroxidase-anti-AFP conjugates (HRP-anti-AFP) were assembled onto the surface of nanogold particles (bio-GMGP). With the aid of an external magnet, the formed bio-GMGPs were attached onto the base electrode in the flow system. With a non-competitive immunoassay format, the injected sample containing AFP antigens was produced transparent immunoaffinity reaction with the immobilized HRP-anti-AFP on the bio-GMGPs. The formed immunocomplex inhibited partly the active center of HRP, and decreased the labeled HRP toward the reduction of H(2)O(2). The performance and factors influencing the performance of the immunosensor were investigated in detail. Under optimal conditions, the electrochemical immunosensor displayed a wide working range of 0.01-200 ng mL(-1) with a low detection limit (LOD) of 1.0 pg mL(-1) AFP (at 3s(B)). Intra- and inter-assay coefficients of variation (CV) were below 10%. In addition, the methodology was validated with real serum samples, receiving a good correlation with the results obtained from commercially available electrochemiluminescence automated analyzer.     

Production of glutamine synthetase in <Emphasis Type="Italic">Escherichia coli</Emphasis> using SUMO fusion partner and application to <Emphasis Type="SmallCaps">l</Emphasis>-glutamine synthesis

l-glutamine (Gln) is an important conditionally necessary amino acid in human body and potential demand in food or medicine industry is expected. High efficiency of l-Gln production by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system has been developed. We report here first the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of recombinant Bacillus subtilis GS. In order to obtain GS with high Gln-forming activity, safety and low cost for food and pharmaceutics industry, 0.1% (w/v) lactose was selected as inducer. The fusion protein was expressed in totally soluble form in E. coli, and expression was verified by SDS–PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel nitrilo-triacetic acid (Ni–NTA) resin chromatography with a yield of 625 mg per liter fermentation culture. After the SUMO/GS fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni–NTA column. Finally, about 121 mg recombinant GS was obtained from 1 l fermentation culture with no less than 96% purity. The recombinant purified GS showed great transferase activity (23 U/mg), with 25 U recombinant GS in a 50 ml reaction system, a biosynthesis yield of 27.5 g/l l-Gln was detected by high pressure liquid chromatography (HPLC) or thin-layer chromatography. Thus, the application of SUMO technology to the expression and purification of GS potentially could be employed for the industrial production of l-Gln.     

Comparison of covalent immobilization of amylase on polystyrene pellets with pentaethylenehexamine and pentaethylene glycol spacers

α-Amylase from Aspergillus oryzae was covalently immobilized onto polystyrene pellets with pentaethylenehexamine (PS-PEHA-Ald) and pentaethylene glycol (PS-PG-Ald) carrying a terminal aldehyde group. Optimum immobilization occured at pH 8.0 and 25 °C, and at pH 7.0 and 35 °C for PS-PEHA-Ald and PS-PG-Ald, respectively. PS-PEHA-Ald immobilized enzyme retained approximately 75% of the initial activity over 45 days of storage, 70% of the initial activity after nine runs of recycling and displayed the better resistance to detrimental metal ions. PS-PG-Ald immobilized enzyme retained approximately 50% of the initial activity in 8h at 70 °C. The catalytic efficiencies of PS-PEHA-Ald immobilized and PS-PG-Ald immobilized amylase were 1.42 and 1.29 times higher than that of native enzyme. The activation energy of the reaction mediated by the amylase was reduced by 58.1% and 57.3% when PS-PEHA-Ald and PS-PG-Ald used as support respectively.     

Enhancement effect of hematite nanoparticles on fermentative hydrogen production

The effects of hematite nanoparticles concentration (0-1600 mg/L) and initial pH (4.0-10.0) on hydrogen production were investigated in batch assays using sucrose-fed anaerobic mixed bacteria at 35 °C. The optimum hematite nanoparticles concentration with an initial pH 8.48 was 200 mg/L, with the maximum hydrogen yield of 3.21 mol H₂/mol sucrose which was 32.64% higher than the blank test. At 200 mg/L hematite nanoparticles concentration, further initial pH optimization experiments indicated that at pH 6.0 the maximum hydrogen yield reached to 3.57 mol H₂/mol sucrose and hydrogen content was 66.1%. The slow release of hematite nanoparticles had been recorded by transmission electron microscopy (TEM). In addition, TEM analysis indicated that the hematite nanoparticles can affect the shape of bacteria, namely, its length increased from ca. 2.0-3.6 μm to ca. 2.6-5.6 μm, and width became narrower.     

Oxalate production at different initial Pb²+ concentrations and the influence of oxalate during solid-state fermentation of straw with Phanerochaete chrysosporium

The production of oxalate at different initial Pb²⁺ concentrations during solid-state fermentation of straw with Phanerochaete chrysosporium was investigated. It was found that the maximal peak value of oxalate concentration (22.84 mM) was detected at the initial Pb²⁺ concentration of 200 mg kg⁻¹ dry straw, while the minimum (15.89 mM) at the concentration of 600 mg Pb²⁺ kg⁻¹ dry straw, and at moderate concentration of Pb²⁺ the capability of oxalic acid secretion was enhanced. In addition, it was also found that more oxalic acid accumulation went together with better Pb²⁺ passivation effect and higher manganese peroxidase (MnP) activity. The present findings will improve the understandings of the interactions of heavy metals with white-rot fungi and the role of oxalate in lignin degradation system, which could provide useful references for more efficient treatment of Pb-contaminated lignocellulosic waste.     

Introduction of glycine and proline residues onto protein surface increases the thermostability of endoglucanase CelA from Clostridium thermocellum

A saturation mutagenesis library was constructed at the position 329 of the endoglucanase CelA from Clostridium thermocellum based on previous results (Yi and Wu, 2010), and one mutation, S329G, was identified to contribute to the enhanced thermostability. The result inspired a rational design approach focusing on the introduction of Gly or Pro residue onto the protein surface, which led to the identification of two additional beneficial mutations, H194G and S269P. Combination of these three mutations resulted in a mutant with a 10-fold increase in half-life of inactivation (60 min) at 86°C without compromising activity compared with the wild-type. Its reaction temperature for maximum activity increased from 75 to 85°C. The results provide valuable thermostability-related structural information on this thermophilic enzyme.     

Estimation of nitrogen dynamics in a vertical-flow constructed wetland

The vertical-flow constructed wetland (VFCW) is a promising engineering technique for removal of excess nutrients and certain pollutants from wastewater and stormwater. The aim of this study was to develop a model using the STELLA software for estimating nitrogen (N) dynamics in an artificial VFCW (i.e., a substrate column with six zones) associated with a growing Cyperus alternifolius species under a wetting (wastewater) -to-drying ratio of 1:3. The model was calibrated by our experimental data with a reasonable agreement prior to its applications. Simulations showed that rates of NH₄⁺-N and NO₃⁻-N leaching decreased with increasing zone number (or column depth), although such a decrease was much more profound for NH₄⁺-N. Our simulations further revealed that rate of NH₄⁺-N leaching decreased with time within each zone, whereas rate of NO₃⁻-N leaching increased with time within each zone. Additionally, both the rates of NH₄⁺-N and NO₃⁻-N leaching through zones followed the water flow pattern: breakthrough during wetting period and cessation during drying period. In general, the cumulative amounts of total nitrogen (TN) were in the following order: leaching > denitrification > uptake > settlement. About 54% of the TN from the wastewater flowed out of the VFCW system, 18% of TN lost due to denitrification, 6% of TN was taken up by roots of a single plant (one hill), and the rest of 22% TN from the wastewater was removed from other mechanisms, such as volatilization, adsorption, and deposition. This study suggested that to improve the overall performance of a VFCW for N removal, prevention of N leaching loss was one of the major issues.     

99mTc- and Re-labeled 6-dialkylamino-2-naphthylethylidene derivatives as imaging probes for β-amyloid plaques

Based on the conjugate strategy, two neutral ^99mTc labeled 2-(1-(6-(dialkylamino)naphthalen-2-yl)ethylidene)malononitrile (DDNP) and 1-(6-(dialkylamino)naphthalen-2-yl)ethanone (ENE) derivatives, and their corresponding rhenium complexes were synthesized. In vitro fluorescent staining indicated that the corresponding rhenium derivatives selectively stained the β-amyloid (Aβ) plaques in the brain sections of AD model mice with low background. Compared with FDDNP and FENE, the affinities of the corresponding rhenium derivatives to Aβ aggregates decreased about 10-14-fold. In vivo biodistribution experiments in normal mice showed that ^99mTc-MAMA-ENE displayed medium initial brain uptake (0.65 %ID/g at 2 min) with a reasonable washout from the brain (0.19 %ID/g at 2 h) while ^99mTc-MAMA-DDNP showed a low brain uptake (0.28 %ID/g at 2 min). Further optimize these ^99mTc-labeled tracers in order to improve their binding affinities to Aβ plaques and diffusion through the blood brain barrier may generate useful imaging agents for SPECT.     

Efficient synthesis of trisaccharide saponins and their tumor cell killing effects through oncotic necrosis

To investigate the relationship of cytotoxicity and saponins with varied aglycones, based on the structure of indioside E 1, a natural derived anti-tumor active ingredient from Chinese medicinal plant Solanum indicum L., five novel saponins 2-6 bearing the same trisaccharide moiety together with 1 were efficiently synthesized via a transglycosylation strategy. MTT assay revealed the killing effects to tumor cells of the synthesized saponins are varied with the change of aglycones. Furthermore, time-lapse microscopy, LDH release, PI staining, and immunocytochemical investigations demonstrated that the cell death caused by neosaponin 2 was through oncotic necrosis involving plasma membrane perturbation and destruction of cytoskeleton.     

Synthesis and structure-activity relationships of novel benzoxaboroles as a new class of antimalarial agents

A series of boron-containing benzoxaborole compounds was designed and synthesized for a structure-activity relationship investigation surrounding 7-(HOOCCH₂CH₂)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (1) with the goal of discovering a new antimalarial treatment. Compound 1 demonstrates the best potency (IC₅₀ = 26 nM) against Plasmodium falciparum and has good drug-like properties, with low molecular weight (206.00), low ClogP (0.86) and high water solubility (750 μg/mL at pH 7).