Amyloid Formation in Nanoliter Droplets

Processes that monitor the nucleation of amyloids and characterize the formation of amyloid fibrils are vital to medicine and pharmacology. In this study, we observe the nucleation and formation of lysozyme amyloid fibrils using a facile microfluidic system to generate nanoliter droplets that can control the flow rate and movement of monomer-in-oil emulsion droplets in a T-junction microchannel. Using a fluorescence assay, we monitor the nucleation and growth process of amyloids based on the volume of droplets. Using the microfluidic system, we demonstrate that the lag phase, which is vital to amyloid nucleation and growth, is reduced at a lower droplet volume. Furthermore, we report a peculiar phenomenon of high amyloid formation at the edge of a bullet-shaped droplet, which is likely due to the high local monomer concentration. Moreover, we discovered that amyloid fibrils synthesized in the nanoliter droplets are shorter and thicker than fibrils synthesized from a bulk solution via the conventional heating method. Herein, a facile procedure to observe and characterize the nucleation and growth of amyloid fibrils using nanoliter droplets is presented, which is beneficial for investigating new features of amyloid fibril formation as an unconventional synthetic method for amyloid fibrils.     

Key Residues Affecting Binding Affinity of Sirex noctilio Fabricius Odorant-Binding Protein (SnocOBP9) to Aggregation Pheromone

Sirex noctilio Fabricius (Hymenoptera Siricidae) is a major quarantine pest responsible for substantial economic losses in the pine industry. To achieve better pest control, (Z)-3-decen-ol was identified as the male pheromone and used as a field chemical trapping agent. However, the interactions between odorant-binding proteins (OBPs) and pheromones are poorly described. In this study, SnocOBP9 had a higher binding affinity with Z3D (Ki = 1.53 ± 0.09 μM) than other chemical ligands. Molecular dynamics simulation and binding mode analysis revealed that several nonpolar residues were the main drivers for hydrophobic interactions between SnocOBP9 and Z3D. Additionally, computational alanine scanning results indicated that five amino acids (MET54, PHE57, PHE71, PHE74, LEU116) in SnocOBP9 could potentially alter the binding affinity to Z3D. Finally, we used single-site-directed mutagenesis to substitute these five residues with alanine. These results imply that the five residues play crucial roles in the SnocOBP9-Z3D complex. Our research confirmed the function of SnocOBP9, uncovered the key residues involved in SnocOBP9-Z3D interactions, and provides an inspiration to improve the effects of pheromone agent traps.     

Lectin Conjugated Gold Nanoparticle-based Colorimetric Assay for Studying the Interactions of Antibiotic with Living Cell

The interactions of antibiotic with living cells were studied by lectin conjugated gold nanoparticles(GNPs)based colorimetric assay. Because of the high affinity of lectin for saccharides, the lectin conjugated GNPs are able to employ as indicators for monitoring the antibiotic induced changes of glycosyl complexes. The interactions of a well known antibiotiC., tunicamycin, with two different cell lines, HeLa and SHG-44, were selected to establish this assay.In the presence of tunicamycin, the dose-and time-dependence on the decreasing of binding affinity of lectin conjugated GNPs with living cells were demonstrated by conventional microscopic and UV-Vis spectroscopic studies. The experimental result demonstrates that our approach can be used to identify antibiotic induced expression difference of glycosyl complexes on different cellular surfaces and determine drug activity quantitatively. For further confirming the capability of the GNP-based assay, the system was also studied by confocal laser scanning microscopy(CLSM)and classic flow cytometry(FCM) assay, and satisfactory results were obtained.     

Activity and Tissue Expression of Tyrosine Phosphatase PTP-MEG2

Protein tyrosine phosphatases(PTPs) are crucial regulators of signal transduction. Among them,PTP-MEG2 is an intracellular enzyme of 593 amino acid residues with a putative lipid-binding domain at the N-terminus. In the present study, we cloned the full-length form of the enzyme and expressed it in E. coli cells as a 6xHis-tagged protein. The majority of the expressed enzyme was found in the inclusion body of E. coli cell extracts.Upon extraction with a buffer containing urea, the recombinant enzyme was purified to near homogeneity on a single Ni-NTA-agarose column. This procedure resulted in the production of over 100 mg of purified recombinant PTP-MEG2 from 1 L E. coli cell culture. The purified protein displayed a single polypeptide band with expected molecular size on SDS-polyacrylamide gel electrophoresis under reducing conditions. Isolated under denatured conditions in urea, the purified enzyme was re-natured by dialyzing against a refolding buffer. The re-natured enzyme effectively dephosphorylated the common PTP substrate para-nitrophenylphosphate with a specific activity of 2000units/mg. Meanwhile, the denatured enzyme was used to immunize a rabbit to produce antibodies. The resulting antiserum had extremely high sensitivity and specificity. When used for Western blot analysis, the anti-serum revealed a wide expression of PTP-MEG2 in many tissues of mice. Together, we developed a highly effective way to purify a large amount of PTP-MEG2 and generated highly sensitive antibodies that can specifically detect endogenous expression of the enzyme in tissues.     

Quantitation of estragole by stable isotope dilution assays

Various calibration strategies for the quantitation of the phenylpropane estragole by gas chromatography-mass spectrometry were developed and compared. For application in stable isotope dilution assays, two deuterium labelled estragole isotopologues were synthesized. Of these, [3′,3′-²H₂]estragole was prepared by Wittig reaction of 4-methoxy-phenylacetaldehyde with [²H₃]methyl-triphenyl-phosphonium bromide, whereas [1″,1″,1″-²H₃]estragole was obtained by demethylation of estragole and deuteromethylation of the resulting 4-allylphenole.Besides estragole isotopologues, 1,2,4-trimethoxybenzene and 4-propylanisole were also tested as internal standards (I.S.) for the determination of estragole in fennel tea.[1″,1″,1″-²H₃]Estragole, 1,2,4-trimethoxybenzene, and 4-propylanisole revealed linear calibration functions and, therefore, were suitable for estragole quantitation. In contrast to this, [3′,3′-²H₂]estragole could only be applied as I.S. if it was added to the extracts in stoichiometric deficiency compared to unlabelled estragole. Moreover, due to its different chemical and physical properties, 1,2,4-trimethoxybenzene showed a recovery as low as 77%, whereas the other I.S. revealed recovery rates close to 100%. Considering the “real” values of estragole in fennel tea, the choice of the I.S. obviously is less important than the way of preparing the tea. In contrast to the common method for tea preparation, squeezing of the teabags increased the estragole content significantly by 50%.     

Lactoperoxidase-mediated degradation of single-walled carbon nanotubes in the presence of pulmonary surfactant

Carbon nanotubes (CNTs) may elicit inflammatory responses following pulmonary exposure. Conversely, enzymatic biodegradation of CNTs by inflammatory cells has also been reported. The aim of this study was to study the degradation of oxidized single-walled CNTs (ox-SWCNTs) by lactoperoxidase (LPO), a secreted peroxidase present in the airways, and whether pulmonary surfactant affects this biodegradation. To this end, ox-SWCNTs were incubated in vitro with recombinant bovine LPO + H₂O₂ + NaSCN in the presence and absence of porcine lung surfactant (Curosurf®) and biodegradation was monitored using UV–Vis–NIR spectroscopy, Raman spectroscopy, and scanning electron microscopy. The interaction of recombinant LPO with bundles of ox-SWCNTs was confirmed by atomic force microscopy. Cell-free biodegradation of ox-SWCNTs was also observed ex vivo in murine bronchoalveolar lavage fluid in the presence of H₂O₂ + NaSCN. Our study provides evidence for biodegradation of ox-SWCNTs with a lung surfactant ‘bio-corona’ and expands the repertoire of mammalian peroxidases capable of biodegradation of ox-SWCNTs. These findings are relevant to inhalation exposure to these materials, as LPO serves as an important component of the airway defense system.     

Radio-resistance induced by nitric oxide to heavy ion irradiation in A172 human glioma cells

To investigate effects of nitric oxide on cellular radio-sensitivity, three human glioma cell lines, i.e. A172, A172 transfected green fluorescence protein (EGFP) gene (EA172) and A172 transfected inducible nitric oxide synthesis (iNOS) gene (iA172), were irradiated by 12C6 ions to 0, 1 or 2Gy. Productions of nitric oxide and glutathione (GSH) in A172, EA172 and iA172 were determined by chemical methods, cell cycle was analyzed by flow cytometry at the 24th hour after irradiation, and survival fraction of the cells was measured by colorimetric MTT assay at the 5th day after irradiation. The results showed that the concentrations of nitric oxide and GSH in iA172 were significantly higher than in A172 and EA172; the G2/M stage arrest induced by the 12C6 ion irradiation was observed in A172 and EA172 but not in iA172 at the 24th hour after exposure; and the survival fraction of iA172 was higher than that of EA172 and iA172. Data suggest that the radio-sensitivity of the A172 was reduced after the iNOS gene transfection. The increase of GSH production and the change of cellular signals such as the cell cycle control induced by nitric oxide may be involved in this radio-resistance.     

HPLC测定盐酸奥洛他定滴眼液中苯扎氯铵的含量

目的建立HPLC测定盐酸奥洛他定滴眼液中苯扎氯铵含量的方法。方法以氰基键合硅胶为填充剂;乙腈-水-三乙胺为440∶560∶1,用磷酸调pH 3.4为流动相,流速1.0 mL/min;检测波长210 nm。结果苯扎氯铵在0.05～0.15 mg/mL范围内呈良好的线性关系(r=0.999 8);平均回收率99.7%(n=9,RSD=0.69%)。结论此方法简便灵敏准确,重复性好,可用于测定盐酸奥洛他定滴眼液中苯扎氯铵的含量。