Mutational specificity of the dnaE173 mutator associated with a defect in the catalytic subunit of DNA polymerase III of Escherichia coli.

We developed a system to examine forward mutations that occurred in the rpsL gene of Escherichia coli placed on a multicopy plasmid. Using this system we determined the mutational specificity for a dnaE173 mutator strain in which the editing function of DNA polymerase III is impeded. The frequency of rpsL- mutations increased 32,000-fold, due to the dnaE173 mutator, and 87 independent rpsL- mutations in the mutator strain were analyzed by DNA sequencing, together with 100 mutants recovered from dnaE+ strain, as the control. While half the number of mutations that occurred in the wild-type strain were caused by insertion elements, no such mutations were recovered from the mutator strain. A novel class of mutation, named "sequence substitution" was present in mutants raised in the dnaE173 strain; seven sequence substitutions induced in the mutator strain occurred at six sites, and all were located in quasipalindromic sequences, carrying the GTG or CAC sequence at one or both endpoints. While other types of mutation were found in both strains, single-base frameshifts were the most frequent events in the mutator strain. Thus, the mutator effect on this class of mutation was 175,000-fold. A total of 95% of the single-base frameshifts in the mutator strain were additions, most of which occurred at runs of A or C bases so as to increase the number of identical residues. Base substitutions, the frequency of which was enhanced 25,000-fold by the mutator effect, occurred primarily at several hotspots in the mutator strain, whereas those induced in the wild-type strain were more randomly distributed throughout the rpsL sequence. The dnaE173 mutator also increased the frequency of duplications 28,000-fold. Of the three duplications recovered from the mutator strain, one was a simple duplication, the region of which was flanked by direct repeats. The other duplications were complex, one half part of which was in the inverted orientation of a region containing two sets of inverted repeats. The same duplications were also recovered from the wild-type strain. The present data suggest that dnaE173 is a novel class of mutator that sharply induces sequence-directed mutagenesis, yielding high frequencies of single base frameshifts, duplications with inversions, sequence substitutions and base substitutions at hotspots.     

Kinetics of folding and unfolding of alpha alpha-tropomyosin and of nonpolymerizable alpha alpha-tropomyosin.

Stopped flow CD (SFCD) kinetic studies of self-assembly of coiled coils of rabbit alpha alpha-tropomyosin and of nonpolymerizable alpha alpha-tropomyosin (NPTm) are reported. The protein was denatured in 6 M urea buffer, then renatured by 10-fold dilution into benign saline buffer. Folding was monitored by SFCD in the backbone region (222 nm). Protein chains are shown to be totally unfolded (and separated in the reduced species) in the initial denaturing medium and fully folded as two-chain coiled coils in the final benign medium. In all cases of folding in benign buffer of totally unfolded chains, two phases were found in the folding process: a fast phase (less than 0.04 s, the SFCD dead time), in which an intermediate state with about 70% of the equilibrium ellipticity forms; followed by a slower, observable phase that completes the folding. The slow phase is first order (k-1 = 1.6 s at 20 degrees C), signifying that chain association for reduced samples occurs in the fast phase. In contrast, folding in benign buffer from an initial state with 70% of the equilibrium ellipticity is all fast, suggesting that the folding intermediate is not an equilibrium species. Cross-linking at Cys-190 increases the helix content of the fast-formed intermediate state to about 85% of the equilibrium value, but leaves the rate constant of the slow phase unchanged. In NPTm, which does not form high aggregates at low ionic strength, the rate of the observable phase is almost independent of ionic strength in the range of approximately 0.15-0.6 M, but is reduced one to two orders of magnitude by further reduction to 0.026 M. In folding from totally unfolded chains, the rate is reduced less than one order of magnitude by changing the final state to about 50% folded. In contrast to folding, unfolding of alpha alpha-tropomyosin from the native state is all fast.     

The thermal denaturation of nonpolymerizable alpha alpha-tropomyosin and its segments as a function of ionic strength

Nonpolymerizable tropomyosin (NPTm) is found to unfold thermally at high ionic strength almost exactly as the parent protein, but it does not aggregate at low ionic strength. Thus, NPTm can be used as a tropomyosin surrogate whose coiled-coil structural stability can be probed by varying the ionic strength. Studies of NPTm by CD show that increasing ionic strength stabilizes the coiled-coil structure. CD spectra over a wide range of helix content, obtained by varying either temperature or ionic strength, show an isodichroic point at 203 nm, suggesting a local, residue-level, two-state model. At given temperature, such a local helix in equilibrium random equilibrium suggests ln [phi h/(1-phi h)] = A1 + A2In, wherein phi h is the fraction helix, and A1, A2, and n are constants. In the low ionic strength region, theoretical limiting laws for ionic strength mediated charge-charge, dipole-dipole, and apolar-apolar (salting out) interactions give, respectively, n = 0.5, 1.0, and 1.0. Our experimental values for 40 degrees C, where the data span a wide range of helix content, show n = 1.0, suggesting that ionic strength stabilizes either by reducing dipole-dipole repulsions or by enhancing hydrophobic interactions, both probably interhelix in nature. Two segments of tropomyosin, 11Tm127 and 142Tm281, neither of which aggregate at low ionic strength, give results similar to those for NPTm, i.e., n = 0.96 and 0.84, respectively.     

Phosphorene as Cathode Material for High‐Voltage,Anti‐Self‐Discharge Zinc Ion Hybrid Capacitors

Output voltage and self‐discharge rate are two important performance indices for supercapacitors, which have long been overlooked, though these play a very significant role in their practical application. Here, a zinc anode is used to construct a zinc ion hybrid capacitor. Expanded operating voltage of the hybrid capacitor is obtained with novel electrolytes. In addition, significantly improved anti‐self‐discharge ability is achieved. The phosphorene‐based zinc ion capacitor exploiting a “water in salt” electrolyte with a working potential can reach 2.2 V, delivering 214.3 F g^?1 after 5000 cycles. The operating voltage is further extended to 2.5 V through the use of an organic solvent as the electrolyte; the solvent is prepared by adding 0.2 m ZnCl₂ into the tetraethylammonium tetrafluoroborate in propylene carbonate (Et₄NBF₄/PC) solvent, and it exhibits 105.9 F g^?1 even after 9500 cycles. More importantly, the phosphorene‐based capacitors possess excellent anti‐self‐discharge performance. The capacitors retain 76.16% of capacitance after resting for 300 h. The practical application of the zinc ion capacitor is demonstrated through a flexible paper‐based printed microcapacitor. It is believed that the developed zinc ion capacitor can effectively resolve the severe self‐discharge problem of supercapacitors. Moreover, high‐voltage zinc ion capacitors provide more opportunities for the application of supercapacitors.     

In Situ Electrochemical Synthesis of MXenes without Acid/Alkali Usage in/for an Aqueous Zinc Ion Battery

The traditional method to fabricate a MXene based energy storage device starts from etching MAX phase particles with dangerous acid/alkali etchants to MXenes, followed by device assembly. This is a multistep protocol and is not environmentally friendly. Herein, an all‐in‐one protocol is proposed to integrate synthesis and battery fabrication of MXene. By choosing a special F‐rich electrolyte, MAX V₂AlC is directly exfoliated inside a battery and the obtained V₂CT_X MXene is in situ used to achieve an excellent battery performance. This is a one‐step process with all reactions inside the cell, avoiding any contamination to external environments. Through the lifetime, the device experiences three stages of exfoliation, electrode oxidation, and redox of V₂O₅. While the electrode is changing, the device can always be used as a battery and the performance is continuously enhanced. The resulting aqueous zinc ion battery achieves outstanding cycling stability (4000 cycles) and rate performance (97.5 mAh g^?1 at 64 A g^?1), distinct from all reported aqueous MXene‐based counterparts with pseudo‐capacitive properties, and outperforming most vanadium‐based zinc ion batteries with high capacity. This work sheds light on the green synthesis of MXenes, provides an all‐in‐one protocol for MXene devices, and extends MXenes’ application in the aqueous energy storage field.     

不同病理分期肺腺癌患者血清代谢组学研究

肺癌是当今世界最常见的恶性肿瘤之一,其新发率和死亡率多年来都居于各类癌症之首,其中85%的肺癌都是非小细胞癌,而腺癌又是最常见的非小细胞癌。肺癌的高隐匿性是造成其高死亡率的最主要原因,因此为肺癌的早期诊断和病理分期寻求高效可靠的方法是十分必要的。代谢组学揭示了小分子代谢物的一系列变化,反映了生命活动的最终状态,因此也能直接反映疾病不同发展阶段的病理生理变化。本研究利用核磁共振氢谱(1H-NMR),对在我院就诊的27例不同病理分期的肺腺癌患者和13例健康志愿者进行了血清代谢物分析,运用正交偏最小二乘判别分析(OPLS-DA)对1H-NMR数据进行建模,单变量统计分析对模型进行评价。结果表明肺腺癌患者组的血清中有14种代谢物出现明显差异,其中丙酮酸、丙氨酸、NAC1、乳酸、GPC和甘氨酸比起对照组来有显著上升,而葡萄糖、谷氨酰胺、亮氨酸、异亮氨酸、缬氨酸、丙酮、乙酰乙酸和苏氨酸则显著下降。而在不同分期肺腺癌患者间进行比较后发现,异亮氨酸、乙酰乙酸、NAC1和乳酸的变化与肺腺癌的发展有相关性,可能是肺腺癌早期诊断和分期的潜在生物标志物。