Direct adenylation from 5′-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase

5′-Adenylated oligonucleotides (AppOligos) are widely used for single-stranded DNA/RNA ligation in next-generation sequencing (NGS) applications such as microRNA (miRNA) profiling. The ligation between an AppOligo adapter and target molecules (such as miRNA) no longer requires ATP, thereby minimizing potential self-ligations and simplifying library preparation procedures. AppOligos can be produced by chemical synthesis or enzymatic modification. However, adenylation via chemical synthesis is inefficient and expensive, while enzymatic modification requires pre-phosphorylated substrate and additional purification. Here we cloned and characterized the Pfu RNA ligase encoded by the PF0353 gene in the hyperthermophilic archaea Pyrococcus furiosus. We further engineered fusion enzymes containing both Pfu RNA ligase and T4 polynucleotide kinase. One fusion enzyme, 8H-AP, was thermostable and can directly catalyze 5′-OH-terminated DNA substrates to adenylated products. The newly discovered Pfu RNA ligase and the engineered fusion enzyme may be useful tools for applications using AppOligos.     

Lithocholic acid inhibits dendritic cell activation by reducing intracellular glutathione via TGR5 signaling

Dendritic cells (DCs) are the major antigen-presenting cells and play an important role in autoimmune uveitis. Emerging evidence suggests that bile acids (BAs) regulate DCs maturation. However, the underlying mechanisms by which BAs regulate the function of DCs still need to be clarified. Here, we demonstrate that lithocholic acid (LCA) inhibits the production of pro-inflammatory cytokines and the expression of surface molecules in bone marrow-derived dendritic cells (BMDCs). LCA attenuates the severity of EAU by modulating the maturation of splenic CD11C⁺MHCII^high DCs. Notably, Takeda G-protein coupled receptor 5 (TGR5) deficiency partially reverses the inhibitory effect of LCA on DCs in vitro and in vivo. TGR5 activation also downregulates the NF-κB and MAPK pathways by inhibiting glutathione production and inducing oxidative stress in DCs, which leads to apoptosis and autophagy in DCs. In addition, LCA or INT-777 treatment increases the TGR5 expression in monocyte-derived dendritic cells (MD-DCs) of patients with active BD, whereas both LCA and TGR5 agonists inhibit the activation of MD-DCs. These results suggest that LCA and TGR5 agonists might be potential therapeutic drugs for the treatment of autoimmune uveitis.     

三种杀虫菌超氧化物歧化酶电泳图型和同源性

本文报道了球形芽孢杆菌、苏云金芽孢杆菌和森田芽孢杆菌三种昆虫病原菌的超氧化物歧化酶（SOD）的电泳图型及其同源性。实验证明B．S和B．tSOD分别具有Ef25～30、56．8和52．9特征酶带,B．mSOD也具有与B．T相同的特征酶带;免疫琼脂双扩散实验证明B．sSOD与B．T和B．mSOD无同源性,B．tSOD抗血清与B.sSOD抗原无沉淀反应,但与B．mSOD抗原产生沉淀反应,证明B．S与B.t和B．M的亲缘关系远,B．T和B．M的亲缘关系近。     

球形芽孢杆菌C3-41超氧化物岐化酶的特性

本文研究了球形芽孢杆菌（Bacillussphaericus）C3－41超氧化物歧化酶（SOD）的产生条件和部分特性。当C3－41菌株处于孢子囊中期时为产SOD酶高峰期,在30℃下的平板培养物及培养基起始pH为中性（pH7．0）时产生的SOD酶比活最高,经硫酸铵分级沉淀,DEAE－32离子交换层析和SephadexG-100凝胶过滤提纯了SOD酶。此酶属Mn－SOD,在25～35℃和pH5～9范围内较稳定,但在55℃下10min完全失活。     

曲酸菌选育及发酵工艺研究

筛选获得产曲酸菌株米曲霉（Aspergillusoryzae）MSA进行⁶⁰Co诱变,并进行了发酵工艺条件研究,以葡萄糖为碳源、豆饼粉为氮源,在pH3.0、33℃摇瓶培养,可达到5d产酸5％以上水平。发酵液采用直接浓缩结晶工艺,脱色与重结晶后获得无色针状晶体,红外图谱检测确证为曲酸。     

Distinct basolateral amygdala excitatory inputs mediate the somatosensory and aversive-affective components of pain

Pain is a multidimensional perception that includes unpleasant somatosensory and affective experiences; however, the underlying neural circuits that mediate different components of pain remain elusive. Although hyperactivity of basolateral amygdala glutamatergic (BLA^Glu) neurons is required for the somatosensory and emotional processing of pain, the precise excitatory inputs to BLA^Glu neurons and their roles in mediating different aspects of pain are unclear. Here, we identified two discrete glutamatergic neuronal circuits in male mice: a projection from the insular cortex glutamatergic (IC^Glu) to BLA^Glu neurons, which modulates both the somatosensory and affective components of pain, and a projection from the mediodorsal thalamic nucleus (MD^Glu) to BLA^Glu neurons, which modulates only the aversive-affective component of pain. Using whole-cell recording and fiber photometry, we found that neurons within the IC→BLA and MD→BLA pathways were activated in mice upon inflammatory pain induced by injection of complete Freund’s adjuvant (CFA) into their paws. Optical inhibition of the IC^Glu→BLA pathway increased the nociceptive threshold and induced behavioral place preference in CFA mice. In contrast, optical inhibition of the MD^Glu→BLA pathway did not affect the nociceptive threshold but still induced place preference in CFA mice. In normal mice, optical activation of the IC^Glu→BLA pathway decreased the nociceptive threshold and induced place aversion, while optical activation of the MD^Glu→BLA pathway only evoked aversion. Taken together, our results demonstrate that discrete IC^Glu→BLA and MD^Glu→BLA pathways are involved in modulating different components of pain, provide insights into its circuit basis, and better our understanding of pain perception.     

经济学视角下的流域生态补偿制度——基于一个污染赔偿的算例

近年来,随着流域水污染问题日益严重,流域上下游之间的利益冲突日趋明显,流域生态补偿成为协调流域上下游利益冲突的重要途径。虽然我国一些地区已经开展了流域生态补偿的实践,并取得了一定成效,但在具体执行中还存在许多问题。通过分析不同制度安排下流域上下游利益相关者的福利变化,来说明制度的重要作用。在介绍有关制度的重要概念后,通过比较"囚徒困境"和"囚徒梦想"两个博弈案例,指出了制度安排对效率的重要影响,即效率和公平的是由现有制度安排决定的,在每一种制度安排下,效率和公平的含义会有所不同。基于一个流域污染赔偿的算例,分析了四种制度安排下的效率差异。在这个例子中,甲代表上游的排放污水的企业,乙代表下游受害的居民。在第一种制度安排下,甲不用考虑对乙造成的损失,双方的总收益实现了最大化,但大多数人会认为这种制度安排对乙不公平。在后三种"更公平"的制度安排下,存在着对乙的利益进行补偿的不同方案,且在第三种制度安排下,双方的总收益同样达到了最大化,只有在对甲进行强制的制度安排下(即强制要求甲安装污水处理设施),双方的总收益均少于其它三种制度。对案例的分析表明:(1)通过设计合理的制度,公平和效率可以"兼得";(2)如果利益相关者拥有影响制度安排的机会,将能够促进社会公平。研究为决策者提供一个分析框架,对缓解目前流域生态补偿中上下游的利益冲突具有重要的意义。