A Practical System for High-Throughput Screening of Mutants of <Emphasis Type="Italic">Bacillus fastidiosus</Emphasis> Uricase

For high-throughput screening (HTS) of Bacillus fastidiosus uricase mutants, a practical system was proposed. By error-prone PCR with final 1.5 mM MnCl₂, two focused libraries of mutants for A1-V158 and V150-D212 were generated separately. After induced expression of individual clones in 48-well microplates, Escherichia coli cells (BL21) were lyzed by 1.0 M Tris-HCl at pH 9.0 in 96-well microplates at 25 °C for 7.5 ~ 10.5 h; uricase reaction was continuously monitored with 0.15 mM uric acid in 96-well plates by absorbance at 298 nm to estimate V _m/K _m by kinetic analysis of reaction curve for comparison. V _m/K _m was resistant to initial uric acid levels with an upper limit 3-fold over that of initial rates. By receiver-operator-characteristic analysis of the recognition of the one of higher activity in uricase pair whose specific activity ratio was 1.8 or 3.3, the area-under-the-curve was comparable to that with cell lysates prepared by sonication treatment. A cutoff for the maximum Youden index was thus developed to recognize positive mutants of 1-fold higher activity. Indeed, mutant L171I/Y182F/Y187F/A193S of higher activity but lower thermostability at pH 7.4 and mutant V144A of higher activity and consistent thermostability were discovered. Therefore, the proposed system was practical for HTS of uricase mutants.     

Temperature effect on the optical properties of two different triphenylamine-based organic dyes

In this paper, we present the absorption and photoluminescence (PL) spectra of conjugated, metal-free organic dyes, triphenylamine derivatives 3-(4-(bis(4-(5-(4-(hexyloxy)phenyl)thiophenyl)phenyl)amino)phenyl)-2-cyanoacrylic acid (OD3) and (E)-3-(5′-(4-(bis(4-(5-(4-hexyloxy)phenyl)thiophen-2-yl)phenyl)amino)phenyl)-2,2′-bithiophen-5-yl)-2-cyanoacrylic acid (OD3DT), with and without a π-conjugated bridge, respectively, in film at a temperature range from 13 to 400 K. We find that the intensities of absorption and PL of OD3 decrease gradually beyond 200 K, as well as the ratios of integral areas of absorption to PL spectra. However, the other compound, OD3DT, shows a very different behavior. An increase is discovered in the ratio of the absorption to PL integral area when the temperature is higher than 200 K. A π-conjugated bridge gives this compound a higher degree of symmetry, and therefore, a better alignment of the molecules in the film, which causes a stacking aggregation such that the temperature effect on this compound is different than OD3.     

Facile synthesis of novel [1,3]oxazino[2,3-c][1,2,4] thiadiazin-12-one derivatives

Several novel fused heterocyclic systems which have rings containing N/O or N/S have been synthesized through a facile one-pot method by the treatment of substituted 2-(benzo[d]thiazol-2-yl)phenol and cyanogen bromide in THF and NEt₃. This one-pot method contains tough ring-opening of thiazole, special intermolecular rearrangement and ring-closing reactions. The newly synthesized compounds were characterized by HR-MS, ¹H-NMR, ¹³C-NMR spectral data and DFT calculation analysis. 2a was also determined by X-ray crystallographic analysis.     

Three-dimensionally ordered macroporous metal oxide–silica composite for removal of mercaptan

A series of 3DOM and non-3DOM metal oxide–silica composites were prepared and tested dynamically in a packed-bed reactor at room temperature to remove ethanethiol from a gas stream containing ethyl mercaptan in moist N_2.The obtained sorbents were characterized using X-ray diffraction, scanning electron microscopy, transmission electron microscopy, and nitrogen adsorption–desorption techniques. The experimental results showed that the adsorption ability of different kinds of metal oxide–silica composites with 3DOM structure decreased in the sequence: 3D-CuO/SiO₂ > 3D-NiO/SiO₂ > 3D-Co₃O₄/SiO₂ > 3D-ZnO/SiO₂. The best ratio of CuO to SiO₂ of 3DOM copper–silicon oxide sorbents for ethanethiol removal was found to be 1:2. The 3DOM structure could improve the removal activity of sorbents remarkably because of the high porosity with ordered interconnected macropores as well as the large surface area and high dispersion of CuO. It was also found that a moist atmosphere greatly benefited the adsorption of ethanethiol at ambient condition.     

具有高灵敏性且稳定可重复的正温度系数效应的PVDF/CF导电复合材料

以碳纤维(CF)为填料,聚偏氟乙烯(PVDF)为基体,通过熔融共混法制备PVDF/CF导电复合材料.所得复合材料具有显著的正温度系数(PTC)效应,温度上升到聚合物熔点附近时,电阻率对温度变化敏感.在转折温度区间(155.5~171.0oC,(35)(28)15.5oC)内,其体积电阻率的增加速率约为1.3×105?cm K-1.在不同CF含量下,复合材料表现出不同的PTC行为.随着CF含量的增加,其峰值电阻略有下降.高导电粒子含量下,无负温度系数(NTC)效应.在冷却循环过程,导电网络的重构性良好.复合材料即使经过多次热循环,依然表现出良好的PTC特性重现性.     

Determination of Polycyclic Aromatic Hydrocarbons in Mosses by Ultrasonic-Assisted Extraction and Gas Chromatography–Tandem Mass Spectrometry

A facile method for the determination of polycyclic aromatic hydrocarbons in mosses is reported using ultrasonic-assisted extraction and gas chromatography–tandem mass spectrometry. The efficiency of ultrasonic-assisted extraction and the reagents was optimized. Tandem mass spectrometry with selective reaction monitoring was used to enhance the selectivity to reduce matrix interferences and simplify the purification protocol. The detection limits were from 0.1 to 2.0?ng/mL. The linear calibration range was two orders of magnitude and the coefficients of linear correlation exceeded 0.9992 for all analytes. The relative standard deviations within 1 day and 3 days were less than 9.0%. Recoveries from 56.8 to 109.0% were obtained in fortified mosses. The rapid, low solvent gas chromatography–tandem mass spectrometry method was used to determine polycyclic aromatic hydrocarbons in mosses.     

Efficient copper-catalyzed N-arylation of NH-containing heterocycles and sulfonamides with arenediazonium tetrafluoroborates

A practical copper-catalyzed N-arylation of NH-containing heterocycles with arenediazonium tetrafluoroborates has been developed using CuCl as catalyst and K₂CO₃ as additive under ligand-free conditions. This reaction system has wide substrate scope including imides, 1H-pyrazole, 1H-tetrazoles, 1,2-benzisothiazol-3(2H)-one, and related sulfonamides and gives moderate to excellent yields (up to 95%) of the desired products. This strategy is very general, simple, environmentally friendly, and tolerant of oxygen.     

Ring-contraction from benzo[b][1,4]thiazines to benzo[d]thiazolines induced by oxygen

An unexpected ring-contraction from benzo[b]pyrazino[1,2-d][1,4]thiazine-1,4-diones (6) to benzo[4,5]thiazolo[3,2-a]pyrazine-1,4-diones (7) has been developed. The preliminary mechanistic studies showed the transformation contained two independent steps: the first step is the formation of a Michael adduct upon the addition of the protic solvent, in the presence of base, to the C-2-C-3 double bond of compound 6, and the second step is a ring-contraction induced by oxygen via the migration of sulfur atom from C-2 to C-3 position. And its scope is also studied.     

Transition-metal-free trifluoromethylthiolation and difluoromethylthiolation of thiols with trifluoromethanesulfonyl chloride and difluoromethanesulfonyl chloride

Triphenylphosphine-mediated metal-free trifluoromethylthiolation and difluoromethylthiolation of thiols by CF₃SO₂Cl and CHF₂SO₂Cl to synthesize trifluoromethyl disulfides and difluoromethyl disulfides, respectively, was achieved at room temperature. Iodine generated in situ from iodide facilitated this reaction via the formation of iodotriphenylphosphonium iodide which could serve as a reducing agent in this transformation. Readily available reagents and mild reaction conditions without transition-metals allow this protocol to be more practical than traditional methods.     

Development of a light-up fluorescent probe for HRAS G-quadruplex DNA

The development of small-molecule G-quadruplex DNA probes has attracted significant attention in recent years. However, G-quadruplexes can display a wide variety of topologies, which process different structures and functions. Therefore, selective discrimination one G-quadruplex structure over another is promising. Herein, we reported the design, synthesis and biological evaluation of a long-chain fatty amine functionalized triphenylamine-quinolinium conjugate 1b. Significant enhancement of the fluorescence intensity (over 180 fold) was observed when 1b bound with HRAS G-quadruplex DNA, while much weaker enhancements were presented in the presence of other G-quadruplexes (45–90-fold) and single/double-stranded DNAs (less than 20-fold), indicating 1b had an excellent selectivity to HRAS. The details of the interactions were investigated by UV–Vis, FID and CD analysis. The results show 1b could interact and stabilize HRAS structure mainly by π-π stacking binding mode. The introduced amine chain of the structure core was found to be better in the terms of inducing selectivity toward G-quadruplex structure. In addition, the application of 1b as a fluorescent agent for living cell imaging was also demonstrated.