Diversity and virulence of soil-dwelling fungi Isaria spp. and Paecilomyces spp. against Solenopsis invicta (Hymenoptera: Formicidae)

To comprehend the diversity and potential control of soil-dwelling fungi, Isaria and Paecilomyces, against the red imported fire ant Solenopsis invicta (Hymenoptera, Formicidae), an investigation was carried out between 2004 and 2008. From 258 soil samples collected from 16 central and southern provinces and cities in China, a total of 171 isolates of the genra Isaria and Paecilomyce were isolated, and the species I. javanicus, P. marquandii and I. fumosoroseus were found more abundant than I. cateniobliquus, P. carneus, P. inflatus and P. lilacinus. Geographic differences of isolating rates were observed as well. Samples from the southern areas had higher fungal isolating rates than those from the central areas. Subsequently, 47 isolates were further tested for pathogencity against the red imported fire ant. All isolates except P115 showed certain pathogenic potential (the mean is 52.3% at 4000 conidiospores/mL) to the ant. I. javanicus was the most effective species with a mean pathogenicity of 80.6%, while pathogenicities of P. marquandii, P. gunni and I. fumosoroseus were 44, 21 and 49%, respectively. Furthermore, the more effective isolates P028 of I. javanicus and P003 of I. fumosoroseus were tested in a virulence experiment. The LD₅₀ values of P028 and P003 against major and miner workers were determined as 412,280 and 854,451 conidiospores/cm², respectively. Meanwhile, the LT₅₀ values at 1000 conidiospores/cm² were 7.1 and 6.6 d in isolate P003 and 6.8 and 6.6 d in isolate P028.     

Biomolecule-stabilized Au nanoclusters as a fluorescence probe for sensitive detection of glucose

In this work, biomolecule-stabilized Au nanoclusters were demonstrated as a novel fluorescence probe for sensitive and selective detection of glucose. The fluorescence of Au nanoclusters was found to be quenched effectively by the enzymatically generated hydrogen peroxide (H(2)O(2)). By virtue of the specific response, the present assay allowed for the selective determination of glucose in the range of 1.0×10(-5) M to 0.5×10(-3) M with a detection limit of 5.0×10(-6) M. The absorption spectroscopy, X-ray photoelectron spectroscopy (XPS) and fluorescence decay studies were then performed to discuss the quenching mechanism. In addition, we demonstrated the application of the present approach in real serum samples, which suggested its great potential for diagnostic purposes.     

Amperometric sensors based on Ni/Al and Co/Al layered double hydroxides modified electrode and their application for hydrogen peroxide detection

Two different hydrogen peroxide sensors were constructed with Ni/Al and Co/Al layered double hydroxides (LDHs) modified glassy carbon electrodes (GCE). Ni (Co)/Al-LDHs were synthesized by electrochemical method and were characterized by scanning electron microscopy (SEM) and energy dispersive spectrometry (EDS). The advantages and shortcoming of the two hydrogen peroxide sensors were described in detail. Compared to Co/Al-LDHs modified electrode, sensors fabricated by Ni/Al-LDHs showed quicker heterogeneous electron transfer rate constants (k(s)), lower detection and better reproducibility. But Co/Al-LDHs modified electrode held the advantages of wider linear range and higher sensitivity. Further more, the different catalytic redox mechanisms of hydrogen peroxide on the Ni/Al/GCE and Co/Al/GCE were firstly comparatively explored.     

Electrochemical aptasensor using the tripropylamine oxidation to probe intramolecular displacement between target and complementary nucleotide for protein array

Tripropylamine (TPA) has different oxidation efficiency at double stranded (ds)-and single stranded (ss)-DNA-modified electrodes. Using this property, a simple but sensitive biosensor using TPA oxidation to probe the intramolecular displacement was constructed with the analysis of lysozyme as model for the first time. After the complementary ss-DNA strand of anti-lysozyme aptamer was immobilized onto gold electrode via gold-thiol bond, the incubation with the aptamer resulted in the formation of ds-DNA. Lysozyme (in 10 μL sample) binding with aptamer displaced the complementary strand because of the high affinity of lysozyme and its aptamer, corresponding to the dissociation of the ds-DNA. The modified electrode was swept in 20mM TPA solution from 0.2 to 0.95 V. The difference in oxidation current was used to quantify the content of lysozyme with a linear range from 1.0 pM to 1.1 nM. That means 10 amol or 6.0 × 10(6) lysozyme molecules can be detected. Because the signal is produced from the preconcentrated TPA at the electrode surface, the high sensitivity is achieved over the single site labelling strategy. The proposed method is simple, stable, specific, and time-saving while the complicated sample pre-treatment and the labelling to the DNA strand are avoided. The biosensor was validated by the analysis of the diluted egg white sample directly. The recovery and reproducibility were 93.3-100% and 1.4-4.2%, respectively.     

A label-free electrochemical DNA sensor based on exonuclease III-aided target recycling strategy for sequence-specific detection of femtomolar DNA

The present work demonstrates a rapid, single-step and ultrasensitive label-free and signal-off electrochemical sensor for specific DNA detection with excellent discrimination ability for single-nucleotide polymorphisms, taking advantage of Exonuclease III (Exo III)-aided target recycling strategy to achieve signal amplification. Exo III has a specifical exo-deoxyribonuclease activity for duplex DNAs in the direction from 3' to 5' terminus, however its activity on the duplex DNAs with 3'-overhang and single-strand DNA is limited. In response to the specific features of Exo III, the proposed E-DNA sensor is designed such that, in the presence of target DNA, the electrode self-assembled signaling probe hybridizes with the target DNA to form a duplex in the form of a 3'-blunt end at signaling probe and a 3'-overhang end at target DNA. In this way, Exo III specifically recognizes this structure and selectively digests the signaling probe. As a result, the target DNA dissociates from the duplex and recycles to hybridize with a new signaling probe, leading to the digestion of a large amount of signaling probes gradually. A redox mediator, Ru(NH(3))(6)(3+) (RuHex) is employed to electrostatically adsorbed onto signaling probes, which is directly related to the amount and the length of the signaling probes remaining in the electrode, and provides a quantitative measure of sequence-specific DNA with the experimentally measured (not extrapolated) detection limit as low as 20 fM. Moreover, this E-DNA sensor has an excellent differentiation ability for single mismatches with fairly good stability.     

One-step "green" preparation of graphene nanosheets and carbon nanospheres mixture by electrolyzing graphite rob and its application for glucose biosensing

The graphene nanosheets and carbon nanospheres mixture (GNS–CNS) was prepared by electrolyzing graphite rob in KNO₃ solution under constant current, which was characterized by TEM, AFM, SEM, FT-IR, XRD, XPS, TGA and UV–vis. The nano-mixture can keep stable in water for more than one month. Based on this kind of mixture material, a novel electrochemical biosensing platform for glucose determination was developed. Cyclic voltammetry of glucose oxidase (GOD) immobilized on GNS–CNS/GCE exhibited a pair of well-defined quasi-reversible redox peaks at −0.488 V (E_pa) and −0.509 V (E_pc) by direct electron transfer between the protein and the electrode. The charge-transfer coefficient (α) was 0.51, the electron transfer rate constant was 2.64 s⁻¹ and the surface coverage of HRP was 3.18 × 10⁻¹⁰ mol cm⁻². The immobilized GOD could retain its bioactivity and catalyze the reduction of dissolved oxygen. The glucose biosensor has a linear range from 0.4 to 20 mM with detection limit of 0.1 mM. Moreover, the biosensor exhibits acceptable reproducibility and storage stability. The fabricated biosensor was further used to determine glucose in human plasma sample with the recoveries from 96.83% to 105.52%. Therefore, GOD/GNS–CNS/GCE could be promisingly applied to determine blood sugar concentration in the practical clinical analysis.     

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.     

The title of the article: effects on growth and digestive enzyme activities of the <Emphasis Type="Italic">Hepialus gonggaensis</Emphasis> larvae caused by introducing probiotics

A bacteria strain Hg4-03 of Carnobacterium sp., isolated from the intestine of Hepialus gonggaensis larvae, was fed back to the fourth instars larvae as probiotics to evaluate its impact on the growth performance and digestive enzymes. The larvae were reared in the lab with a natural diet treated with different concentrations of bacterial fermentation and heating killed bacteria, respectively. Compared with the control group, results showed that the growth rates significantly increased and the insect mortality rate decreased significantly after feeding with live probiotics. Meanwhile, the activities of protease, total amylase and trehalase rose significantly in intestinal fluid of the group fed with live probiotics compared with the control treatment. These findings demonstrated that the intestinal bacteria Hg4-03 play an important role for the growth of H. gonggaensis larvae. The bacteria community can improve the growth of H. gonggaensis larvae, indicating that intestinal bacteria may probably be one of the most important factors impacting H. gonggaensis larvae reared in control conditions.     

Enhanced γ-aminobutyric acid-forming activity of recombinant glutamate decarboxylase (gadA) from Escherichia coli

γ-aminobutyric acid (GABA) is an important bioactive regulator, and its biosynthesis is primarily through the α-decarboxylation of glutamate by glutamate decarboxylase (GAD). The procedures to obtain GABA by bioconvertion with high activity recombinant Escherichia coli GAD have been seldom understood. In this study, Escherichia coli GAD (gadA) was highly expressed (about 70–75% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-gadA, which was induced by 0.4 mM IPTG in LB medium, and maximal GABA-forming activity of the recombinant GAD was 40 U/mL at a concentration (0.15 mM) of pyridoxal phosphate (PLP) and a concentration (0.6 mM) of Ca²⁺ at optimal pH of 3.8. The optimal concentration (7.5 mM) of Mn²⁺ can also improve the activity of recombinant enzyme, but the co-effect of Ca²⁺ and Mn²⁺ exhibited antagonism effect when added simultaneously. LB and 0.1% (w/v) lactose were selected as culture medium and inducer, respectively. The relative activity was markedly higher activated by Ca²⁺ (174%), Mn²⁺ (164%) than that by other seven bivalent cations. Finally, the yield of GABA was high of 94 g/L detected by paper chromatography or HPLC in 1 L reaction system with 30 mL crude GAD (12 U/mL). By entrapping Escherichia coli glutamate decarboxylase into sodium alginate and carrageenan gel beads, the activity of immobilized GAD (IGAD) remained 85% during the initial five batches and the activity still remained 50% at the tenth batch, these results indicated that the recombinant Escherichia coli GAD was feasible for the future industrial production of GABA.