Oxidative desorption of thiocholine assembled on core-shell Fe3O4/AuNPs magnetic nanocomposites for highly sensitive determination of acetylcholinesterase activity: an exposure biomarker of organophosphates

Acetylcholinesterase (AChE) activity is a well established biomarker for biomonitoring of exposures to organophosphates (OPs) pesticides and chemical nerve agents. In this work, we described a novel electrochemical oxidative desorption-process of thiocholine, the product of enzymatic reaction, for rapid and highly sensitive determination of AChE activity in human serum. This principle is based on self-assembling of produced thiocholine onto core-shell Fe(3)O(4)/Au nanoparticles (Fe(3)O(4)/AuNPs) magnetic nanocomposites and its oxidation at electrode surface. Fe(3)O(4) magnetic core is not only used for magnetic separation from sample solutions, but also carrying more AuNPs due to its large surface-to-volume ratio. The core-shell Fe(3)O(4)/AuNPs nanocomposites were characterized by UV-Vis spectroscopy, field-emission scanning electron microscopy (FE-SEM) and electrochemical measurements. A linear relationship was obtained between the AChE activity and its concentration from 0.05 to 5.0 mU mL(-1) with a detection limit of 0.02 mU mL(-1). The method showed good results for characterization of AChE spiked human serum and detection of OP exposures from 0.05 to 20 nM, with detection limit of 0.02 nM. This new oxidative desorption assay thus provides a sensitive and quantitative tool for biomonitoring of the exposure to OP pesticides and nerve agents.     

Three-step electrodeposition synthesis of self-doped polyaniline nanofiber-supported flower-like Au microspheres for high-performance biosensing of DNA hybridization recognition

A three-step electrodeposition method has been successfully adopted to fabricate morphology-controlled novel Au microspheres on self-doped polyaniline nanofibers (nanoSPAN) modified glassy carbon electrode. The deposition conditions, such as HAuCl(4) concentration and deposition step, have significant influences on the morphologies and electrochemical properties of the resulted Au microspheres. Well hierarchical and homogeneously dispersed flower-like Au microspheres (HHFAu) were obtained under optimal conditions by the three-step electrodeposition strategy in 5.0mM HAuCl(4) solution. HHFAu possess large surface area, excellent electron transfer ability and good biocompatibility. The DNA probe could be effectively attached to HHFAu and thus a high-performance DNA biosensor was constructed by using electrochemical impedance spectroscopy as detection method. A gene fragment of the cauliflower mosaic virus 35S gene, which is related to one of the screening genes for the transgenically modified plants, has been satisfactorily detected. The linear range was from 1.0 × 10(-13)M to 1.0 × 10(-6)M and the detection limit was 1.9 × 10(-14)M. This HHFAu/nanoSPAN-based impedance biosensing platform holds great promise for the detection of other biological and chemical molecules.     

Construction and characterization of a full-length cDNA library from mycobiont of <Emphasis Type="Italic">Endocarpon pusillum</Emphasis> (lichen-forming Ascomycota)

The first full-length cDNA library for lichenized fungi was constructed from cultured mycobiont of the arid desert lichen Endocarpon pusillum. Based on small-scale sequencing results, 111 genes of the lichenized fungi were identified for the first time, among which 11 genes shared no homology with any known fungal genes. Real-time PCR showed that the size of the mycobiont genome is 39.13 Mb and the copy number of ribosomal RNA gene repeat units is 43. The results of this study will be valuable for the ongoing lichen genome-sequencing project and the large-scale identification of functional genes from lichenized fungi.     

Isolation and characterization of an antimicrobial peptide from bovine hemoglobin ��-subunit

In this study, a novel 18-residue linear antimicrobial peptide derived from the central part of the bovine hemoglobin ??-subunit was identified. The peptide was purified by a combination of cationic exchange and reversed-phase high-performance liquid chromatography. The sequence was determined to be VNFKLLSHSLLVTLASHL. The theoretical molecular weight of this peptide was calculated to be 1992.38 Da, which is the same as that determined (1992.401 Da) by matrix-assisted laser desorption ionization mass spectrometry. Sequence analysis showed that there is a high degree of homology in this peptide among hemoglobin ??-subunits of bovine, sheep, deer, porcine, and human. In a radial-diffusion plate assay, this purified peptide exhibited antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Candida albicans.     

Substitution at carbon 2 of 19-nor-1α,25-dihydroxyvitamin D3 with 3-hydroxypropyl group generates an analogue with enhanced chemotherapeutic potency in PC-3 prostate cancer cells

The active form of vitamin D(3), 1α,25-dihydroxyvitamin D(3)(1α,25(OH)(2)D(3)), has anti-proliferative and anti-invasive activities in prostate cancer cells. Because of 1α,25(OH)(2)D(3) therapeutic potential in treating cancers, numerous analogues have been synthesized with an attempt to increase anti-proliferative and/or decrease calcemic properties. Among these analogues, 19-nor-1α,25(OH)(2)D(2) while being less calcemic has equivalent potency as 1α,25(OH)(2)D(3) in several in vitro and in vivo systems. We recently showed that 19-nor-2α-(3-hydroxypropyl)-1α,25(OH)(2)D(3) (MART-10) was at least 500-fold and 10-fold more active than 1α,25(OH)(2)D(3) in inhibiting the proliferation of an immortalized normal prostate PZ-HPV-7 cells and the invasion of androgen insensitive PC-3 prostate cancer cells, respectively. In this study, we further investigated the effects of MART-10 and 1α,25(OH)(2)D(3) on the dose- and time-dependent induction of CYP24A1 gene expression in PC-3 prostate cancer cells. We found that MART-10 induced CYP24A1 gene expression at a lower concentration with a longer duration compared to 1α,25(OH)(2)D(3), suggesting that MART-10 is less susceptible to CYP24A1 degradation. Molecular docking model of human CYP24A1 and MART-10 indicates that its side chain is far away from the heme ion and is less likely to be hydroxylated by the enzyme. Furthermore, MART-10 was a more potent inhibitor of PC-3 cell proliferation and invasion compared to 1α,25(OH)(2)D(3). In addition, MART-10 down-regulated matrix metalloproteinase-9 (MMP-9) expression which could be one mechanism whereby MART-10 influences cancer cell invasion. Finally, we observed that subcutaneous administration of MART-10 up-regulated the CYP24A1 mRNA expression in rat kidneys without affecting their plasma calcium levels. Thus, our findings demonstrate that MART-10 is biologically active in vivo and may be an effective vitamin D analogue for clinical trials to treat prostate cancer.     

Electricity generation in a membrane-less microbial fuel cell with down-flow feeding onto the cathode

A novel membrane-less microbial fuel cell (MFC) with down-flow feeding was constructed to generate electricity. Wastewater was fed directly onto the cathode which was horizontally installed in the upper part of the MFC. Oxygen could be utilized readily from the air. The concentration of dissolved oxygen in the influent wastewater had little effect on the power generation. A saturation-type relationship was observed between the initial COD and the power generation. The influent flow rate could affect greatly the power density. Fed by the synthetic glucose wastewater with a COD value of 3500 mg/L at a flow rate of 4.0 mL/min, the developed MFC could produce a maximum power density of 37.4 mW/m². Its applicability was further evaluated by the treatment of brewery wastewater. The system could be scaled up readily due to its simple configuration, easy operation and relatively high power density.     

Activation of M3 muscarinic receptors inhibits T-type Ca(2+) channel currents via pertussis toxin-sensitive novel protein kinase C pathway in small dorsal root ganglion neurons

Cobrotoxin (CbT), a short-chain postsynaptic α-neurotoxin, has been reported to play a role in analgesia. However, to date, the detailed mechanisms still remain unknown. In the present study, we identify a novel functional role of CbT in modulating T-type Ca²⁺ channel currents (T-currents) in small dorsal root ganglia (DRG) neurons as well as pain behaviors in mice. We found that CbT inhibited T-currents in a dose-dependent manner. CbT at 1 μM reversibly inhibited T-currents by ~ 26.3%. This inhibitory effect was abolished by the non-selective muscarinic acetylcholine receptor (mAChR) antagonist atropine, or the selective M3 mAChR antagonist 4-DAMP, while naloxone, an opioid receptor antagonist had no effect. Intracellular infusion of GDP-β-S or pretreatment of the cells with pertussis toxin (PTX) completely blocked the inhibitory effects of CbT. Using depolarizing prepulse, we found the absence of direct binding between G-protein βγ subunits and T-type Ca²⁺ channels in CbT-induced T-current inhibition. CbT responses were abolished by the phospholipase C inhibitor U73122 (but not the inactive analog U73343). The classical and novel protein kinase C (nPKC) antagonist chelerythrine chlorid or GF109203X abolished CbT responses, whereas the classical PKC antagonist Ro31-8820 or inhibition of PKA elicited no such effects. Intrathecal administration of CbT (5 μg/kg) produced antinociceptive effects in mechanical, thermal, and inflammatory pain models. Moreover, CbT-induced antinociception could be abrogated by 4-DAMP. Taken together, these results suggest that CbT acting through M3 mAChR inhibits T-currents via a PTX-sensitive nPKC pathway in small DRG neurons, which could contribute to its analgesic effects in mice.     

Linking microbial community structure to membrane biofouling associated with varying dissolved oxygen concentrations

In order to elucidate how dissolved oxygen (DO) concentration influenced the generation of extracellular polymeric substance (EPS) and soluble microbial products (SMP) in mixed liquor and biocake, 16S rDNA fingerprinting analyses were performed to investigate the variation of the microbial community in an aerobic membrane bioreactor (MBR). The function of microbial community structure was proved to be ultimately responsible for biofouling. Obvious microbial community succession from the subphylum of Betaproteobacteria to Deltaproteobacteria was observed in biocake. High concentration of EPS in biocake under the low DO concentration (0.5 mg L⁻¹) caused severe biofouling. The correlation coefficient of membrane fouling rate with EPS content in biocake (0.9941-0.9964) was much higher than that in mixed liquor (0.6689-0.8004).