Polyclonal Antibodies in Microplates to Predict the Maximum Adsorption Activities of Enzyme/Mutants from Cell Lysates

With microplate-immobilized polyclonal antibodies against a starting enzyme or its active mutant bearing consistent accessible epitopes, the maximum activity of an adsorbed enzyme/mutant (Vs) was predicted for comparison to recognize weakly-positive mutants. Rabbit antisera against Escherichia coli alkaline phosphatase (ECAP) were fractionated with 33% ammonium sulfate to yield crude polyclonal antibodies for conventional immobilization in 96-well microplates. The response curve of the activities of ECAP/mutant adsorbed by the immobilized polyclonal antibodies to protein quantities from a cell lysate was fit to an approximation model to predict Vs. With 0.4 μg crude polyclonal antibody for immobilization, Vs was consistent for ECAP in cell lysates bearing fourfold differences in its apparent specific activities when its abundance was greater than 0.9%. The ratio of Vs of the mutant R168K to that of ECAP was 1.5?±?0.1 (n?=?2), consistent with that of their specific activities after affinity purification. Unfortunately, the prediction of Vs with polyclonal antibodies that saturated microplate wells was ineffective to Pseudomonas aeruginosa arylsulfatase bearing less than 2% specific activity of ECAP. Therefore, with microplate-immobilized polyclonal antibodies to adsorb enzyme/mutants from cell lysates, high-throughput prediction of Vs was practical to recognize weakly-positive mutants of starting enzymes bearing fairly-high activities.     

Intensified plant N and C pool with more available nitrogen under experimental warming in an alpine meadow ecosystem

Nitrogen (N) availability is projected to increase in a warming climate. But whether the more available N is immobilized by microbes (thus stimulates soil carbon (C) decomposition), or is absorbed by plants (thus intensifies C uptake) remains unknown in the alpine meadow ecosystem. Infrared heaters were used to simulate climate warming with a paired experimental design. Soil ammonification, nitrification, and net mineralization were obtained by in situ incubation in a permafrost region of the Qinghai‐Tibet Plateau (QTP). Available N significantly increased due to the stimulation of net nitrification and mineralization in 0–30 cm soil layer. Microbes immobilized N in the end of growing season in both warming and control plots. The magnitude of immobilized N was lower in the warming plots. The root N concentration significantly reduced, but root N pool intensified due to the significant increase in root biomass in the warming treatment. Our results suggest that a warming‐induced increase in biomass is the major N sink and will continue to stimulate plant growth until plant N saturation, which could sustain the positive warming effect on ecosystem productivity.     

兼性肠球菌Enterococcus hirae AUH-HM195对黄豆苷原的开环转化

摘要：【目的】从褐马鸡粪样中分离对大豆异黄酮黄豆苷原具有转化作用的功能微生物菌株。【方法】在厌氧工作站内对褐马鸡新鲜粪样进行梯度稀释后涂板,从板上挑取单菌落与底物黄豆苷原厌氧混合培养,用高效液相色谱检测底物被转化情况。【结果】分离出一株对黄豆苷原具开环转化作用的革兰氏阳性兼性好氧菌株AUH-HM195（EU919863）,经BLAST比对,该菌株的16S rDNA基因全序与肠球菌属菌株Enterococcus hirae (DSM20160) 的相似性为100%。根据保留时间、代谢产物最大紫外吸图谱以及核     

五爪金龙乙醇提取物对福寿螺毒杀和水稻苗生长的影响

福寿螺和五爪金龙是两种危害严重的入侵生物,利用五爪金龙防治稻田福寿螺是以害治害的方法.盆栽条件下研究了五爪金龙乙醇提取物毒杀福寿螺的效果,以及其对水稻苗生长的影响.在砂培条件下五爪金龙乙醇提取物及其分离得到的甲醇相、乙酸乙酯相、乙醚相水溶液不仅对福寿螺起到毒杀作用, 而且对水稻秧苗生长有促进作用, 毒杀和促进作用由强到弱的顺序均为:乙醇提取物>甲醇相>乙酸乙酯相>乙醚相.在实验浓度范围内五爪金龙乙醇提物对福寿螺的毒杀效果及其对水稻苗促进作用强度均随浓度的增大而增强,在水稻土盆栽条件下抑制福寿螺取食水稻苗的效果与密达差异不显著(p>0.05).五爪金龙乙醇提取物能毒杀福寿螺,对水稻苗的生长没有不利的影响.因此, 利用五爪金龙防治稻田福寿螺危害是环境友好的非化学防治措施,具有良好的应用前景.     

孕妇肠道微生物组成与孕妇及新生儿糖脂代谢相关性研究

目的研究孕妇肠道微生物组成与孕妇以及新生儿糖脂代谢的相关性。方法选择2017年6月至2018年9月在大连市妇幼保健院定期产检的诊断为妊娠期糖尿病(GDM)的孕妇89例为GDM组,血糖正常孕妇96例为对照组。收集孕妇一般资料,孕妇和新生儿糖脂代谢物、孕妇粪便进行微生物检测。结果 GDM组孕妇空腹血糖、甘油三酯明显高于对照组,高密度脂蛋白水平明显低于对照组(均P<0.05)。GDM新生儿脂联素和血糖明显低于对照组,胰岛素和糖化血红蛋白明显高于对照组(均P<0.05)。对照组孕妇肠道微生物中拟杆菌(Bacteroides)丰富度最高(36.6%),其次是普雷沃菌(Prevotella)(15.3%)、柔嫩梭菌(Faecalibacterium)(10.2%)、考拉杆菌(Phascolarctobacterium)(7.2%)。GDM组孕妇肠道微生物中拟杆菌(Bacteroides)丰富度最高(29.4%),其次是柔嫩梭菌(Faecalibacterium)(19.7%)、普雷沃菌(Prevotella)(11.1%)、考拉杆菌(Phascolarctobacterium)(9.3%)。柔嫩梭菌(Faecalibacterium)与孕妇空腹血糖呈正相关(r=0.377 6,P=0.027 6),拟杆菌(Bacteroides)、埃希菌(Escherichia)与孕妇甘油三酯呈负相关(r=-0.027 5,-0.585 8,P=0.023 7,0.041 3)。柔嫩梭菌(Faecalibacterium)与新生儿空腹血糖呈负相关(r=-0.397 6,P=0.027 6)。结论 GDM孕妇肠道微生物构成与血糖正常孕妇不同,菌群丰富度和多样性较低,并且孕妇肠道菌群与其及新生儿的糖脂代谢相关,调节孕妇肠道菌群或有利于改善其与新生儿的糖脂代谢功能。     

Evaluation of an immune-privileged scaffold for In vivo implantation of tissue-engineered trachea

Forty tracheas were harvested from donor New Zealand rabbits. Thirty of the tracheas were randomly divided into four treatment groups corresponding to 4, 5, 6, or 7% NaClO₄ and one untreated group (n = 6 each group). Scanning electron microscopy distinctly revealed the cilium of epithelial cells in the fresh trachea. The internal surface of the trachea was rough in the 4% treatment group and smooth in the 5% treatment group, whereas the matrix was fractured in the 6% treatment group and highly fractured in the 7% treatment group. We observed that the number of nuclei in the cells of the 4, 5, 6, and 7% treatment groups decreased compared to the cells of the untreated group (p < 0.05). Although there was a significant decrease in maximum tensile strength, tensile strain at fracture and the elastic modulus (p < 0.05) with increasing concentrations of NaClO₄, the content of glycosaminoglycans (GAGs) did not significantly decline (p > 0.05) in the 5% treatment group. In addition, histopathological analysis showed that the fiber component and basement membrane of the matrix in the 5% treatment group were retained after optimal decellularization. Despite the preserved cartilage, in vitro immunohistochemical analysis revealed that the matrix did not show the presence of major histocompatibility complex (MHC) antigens. The remaining ten donor tracheas, which were divided into a positive control group and an optimal decellularized group, were used for allogeneic transplantation. Blood samples were taken regularly, and histologic examinations were performed at 30 days postimplantation, which showed no significant immune rejection. In conclusion, we surveyed the structural integrity through morphological observation and compared the biomechanical and immunogenic changes in the tracheal matrix under the different treatments. The optimal decellularized tracheal matrix with preserved cartilage, which was acquired via 5% NaClO4 treatment, exhibited structural integrity, antigen cell removal and immune privilege and would be suitable for use as a tissue-engineered trachea for in vivo transplantation in rabbit models.     

Genetic Variation Analysis of Mugil cephalus in China Sea Based on Mitochondrial COI Gene Sequences

In this study, genetic diversity and population genetic structure of flathead grey mullet, Mugil cephalus, among four China Sea populations were investigated by COI sequences. All the populations studied had high values of haplotype and nucleotide diversity, except for the Yellow Sea population. In the phylogenetic tree, these haplotypes clustered in two groups, one for the populations from the Bohai and East China seas, and the other from the Yellow and South China seas. Analysis of molecular variance indicated that the northern populations (Bohai and East China) had lower genetic divergence (0.0725, P > 0.05) than that of the southern population (South China) (0.4530-0.6827, P < 0.001), suggesting that two distinct genetic groups exist in Chinese waters. Tests of neutral evolution and mismatch distribution indicated that no historical demographic expansion occurred in these populations. The results provide new information for genetic assessment, fishery management, and conservation of this species.