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.     

男性尿道炎尿道多形核白细胞与沙眼衣原体和淋病奈瑟菌感染临床相关性分析

目的：沙眼衣原体和淋病奈瑟氏球菌是引起泌尿生殖系统尿道炎疾病常见的致病菌。临床化验一般采用美蓝染色检查男性尿道多形核白细胞或病原微生物阳性可提示尿道炎症状,指导临床的早期治疗。在缺乏临床症状体征和尿道多形核白细胞的情况下,临床的治疗将是不同的。本研究采用美兰染色检查男性尿道炎患者多形核白细胞数量、沙眼衣原体抗原和淋病奈瑟氏球菌培养,评价非淋菌性尿道炎和淋病的临床意义。方法：3,000例性病患者的尿道分泌物进行美兰涂片染色镜检,衣原体抗原检测和淋病奈瑟氏球菌染色镜检和培养。结果：3,000例性病患者中,387例患者（12.9%）沙眼衣原体抗原阳性。 在沙眼衣原体患者中,242例（62.5%）≥5个多形核白细胞, 59例（15.2%）为1~4个多形核白细胞,86例（22.2%）为0个多形核白细胞,其中36例患者（9.3%）无症状,141例患者（36.4%）无体征。415例（13.8%）淋病奈瑟氏球菌阳性,在淋病患者中,397例（95.7%）≥5个多形核白细胞, 10例（2.4%）1~4个多形核白细胞, 8例（1.9%）为0个多形核白细胞,其中5例（1.2%）患者无症状, 46例(11.1%)无体征。86例沙眼衣原体和淋病奈瑟氏球菌合并感染的患者中,76例阳性患者≥5个多形核白细胞,5例阳性患者为1~4个多形核白细胞, 5例阳性患者无多形核白细胞。结论：本研究分析了尿道炎患者尿道多形核白细胞,沙眼衣原体和淋球菌感染的相互之间的关系,男性尿道炎患者尿道多形核白细胞数量与沙眼衣原体感染和淋球菌感染存在明显的差异（P<0.001）。86例（22.2%）沙眼衣原体感染和8例（1.2%）淋病患者的尿道中无多形核白细胞。因此,加强临床与实验室诊断可提高男性尿道炎的诊断和控制性病的传播。     

Age‐related memory vulnerability to interfering stimuli is caused by gradual loss of MAPK‐dependent protection in Drosophila

Age‐related memory impairment (AMI) is a common phenomenon across species. Vulnerability to interfering stimuli has been proposed to be an important cause of AMI. However, the molecular mechanisms underlying this vulnerability‐related AMI remain unknown. Here we show that learning‐activated MAPK signals are gradually lost with age, leading to vulnerability‐related AMI in Drosophila. Young flies (2‐ or 3‐day‐old) exhibited a significant increase in phosphorylated MAPK levels within 15 min after learning, whereas aged flies (25‐day‐old) did not. Compared to 3‐day‐old flies, significant 1 h memory impairments were observed in 15‐, 20‐, and 30‐day‐old flies, but not in 10‐day‐old flies. However, with post‐learning interfering stimuli such as cooling or electric stimuli, 10‐day‐old flies had worse memory performance at 1 h than 3‐day‐old flies, showing a premature AMI phenomenon. Increasing learning‐activated MAPK signals through acute transgene expression in mushroom body (MB) neurons restored physiological trace of 1 h memory in a pair of MB output neurons in aged flies. Decreasing such signals in young flies mimicked the impairment of 1 h memory trace in aged flies. Restoring learning‐activated MAPK signals in MB neurons in aged flies significantly suppressed AMI even with interfering stimuli. Thus, our data suggest that age‐related loss of learning‐activated neuronal MAPK signals causes memory vulnerability to interfering stimuli, thereby leading to AMI.     

不同年代梭梭同化枝解剖结构特征及其对土壤条件的响应

叶片是植物与外界环境进行能量、水气交换的主要器官,对环境变化较敏感且可塑性较大.该研究以民勤不同年代梭梭同化枝为研究对象,采用石蜡切片法,分析不同年代梭梭同化枝解剖结构变异特点及其与土壤条件的关系,旨在探讨梭梭同化枝在干旱环境的生态适应机制.结果表明:(1)除角质层和导管孔径,不同年代梭梭同化枝解剖结构指标差异显著(P...     

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.     

DLGCN:基于图卷积网络的药物-lncRNA[]关联预测

为实现高通量识别新的药物-长链非编码RNA(Long non-coding RNA, lncRNA)关联,本文提出了一种基于图卷积网络模型来识别潜在药物-lncRNA关联的方法DLGCN(Drug-LncRNA graph convolution network)。首先,基于药物的结构信息和lncRNA的序列信息分别构建了药物-药物和lncRNA-lncRNA相似性网络,并整合实验证实的药物-lncRNA关联构建了药物-lncRNA异质性网络。然后,将注意力机制和图卷积运算应用于该网络中,学习药物和lncRNA的低维特征,基于整合的低维特征预测新的药物-lncRNA关联。通过效能评估,DLGCN的受试者工作特性曲线下面积(Area under receiver operating characteristic, AUROC)达到0.843 1,优于经典的机器学习方法和常见的深度学习方法。此外,DLGCN预测到姜黄素能够调控lncRNA MALAT1的表达,已被最近的研究证实。DLGCN能够有效预测药物-lncRNA关联,为肿瘤治疗新靶点的识别和抗癌药物的筛选提供了重要参考。     

酸胁迫下短乳杆菌NCL912蛋白质的差异表达及其作用

【目的】本文从蛋白质组水平,对本实验室分离的一株高产γ-氨基丁酸的短乳杆菌NCL912(Lactobacillus brevis)在酸胁迫下蛋白质的差异表达及其应激机理进行探讨。【方法】利用双向凝胶电泳技术对pH 5.0和pH 4.0条件下,不含L-谷氨酸钠的培养物的蛋白质组电泳图谱进行了分析,并对酸胁迫下差异表达的蛋白进行了比较。利用质谱检测技术和生物信息学技术对这些差异表达的蛋白进行了鉴定、功能分类和代谢途径分析等。【结果】通过双向凝胶电泳技术,可以得到均匀、背景清晰、分辨率高、重复性好的Lb.brevis NCL912的双向凝胶电泳图谱。对pH 5.0和pH 4.0条件下培养的该菌总蛋白质电泳图谱进行比较,发现有25个差异表达的蛋白点。对这25个差异表达的蛋白进行了质谱鉴定。由于缺乏短乳杆菌NCL912的全基因组,所以其中只有8个蛋白点被质谱鉴定和分析得到。它们分别参与了蛋白质的合成、核苷酸的合成、糖酵解代谢、细胞能量水平的调节等。【结论】酸应激下这些表达蛋白质可通过其相应的功能来保护细胞耐受酸胁迫,从而使菌能够在酸性环境下生存增值。这可能就是Lb.brevis NCL912的酸胁迫应激机理之一。     

Enhancement of Astragalus polysaccharide on the immune responses in pigs inoculated with foot-and-mouth disease virus vaccine

The effects of Astragalus polysaccharides (APS) on the immune response in pigs immunized with foot-and-mouth disease virus (FMDV) vaccine were investigated. Fifteen pigs were randomly divided into five groups. Four groups were vaccinated with a FMDV inactivated vaccine. Pigs in three experimental groups were administered varying doses of APS (APS1, 5 mg/kg; APS2, 10 mg/kg; APS3, 20 mg/kg). The influence of APS on the number of CD3⁺CD4⁻CD8⁺ cytotoxic T cells, CD3⁺CD4⁺CD8⁺ T helper memory cells, and CD3⁻CD4⁻CD8⁺ natural killer cells among peripheral blood lymphocytes (PBL) in the three APS groups were significant compared to the vaccine group. In vitro stimulation of PBL by Con A and LPS in APS groups induced a stronger proliferative response at 2 and 6 weeks post-inoculation (PI). APS markedly increased the titer of FMDV-specific antibody in a dose-dependent manner, and up-regulated mRNA expression of IFN-γ and IL-6. APS could potentially be used as an immunomodulator for a FMDV vaccine and provide better protection against FMDV.     

Activation characteristics of transient receptor potential ankyrin 1 and its role in nociception

Transient receptor potential (TRP) ankyrin 1 (TRPA1) is a Ca(2+)-permeant, nonselective cationic channel. It is predominantly expressed in the C afferent sensory nerve fibers of trigeminal and dorsal root ganglion neurons and is highly coexpressed with the nociceptive ion channel transient receptor potential vanilloid 1 (TRPV1). Several physical and chemical stimuli have been shown to activate the channel. In this study, we have used electrophysiological techniques and behavioral models to characterize the properties of TRPA1. Whole cell TRPA1 currents induced by brief application of lower concentrations of N-methyl maleimide (NMM) or allyl isothiocyanate (AITC) can be reversed readily by washout, whereas continuous application of higher concentrations of NMM or AITC completely desensitized the currents. The deactivation and desensitization kinetics differed between NMM and AITC. TRPA1 current amplitude increased with repeated application of lower concentrations of AITC, whereas saturating concentrations of AITC induced tachyphylaxis, which was more pronounced in the presence of extracellular Ca(2+). The outward rectification exhibited by native TRPA1-mediated whole cell and single-channel currents was minimal as compared with other TRP channels. TRPA1 currents were negatively modulated by protons and polyamines, both of which activate the heat-sensitive channel, TRPV1. Interestingly, neither protein kinase C nor protein kinase A activation sensitized AITC-induced currents, but each profoundly sensitized capsaicin-induced currents. Current-clamp experiments revealed that AITC produced a slow and sustained depolarization as compared with capsaicin. TRPA1 is also expressed at the central terminals of nociceptors at the caudal spinal trigeminal nucleus. Activation of TRPA1 in this area increases the frequency and amplitude of miniature excitatory or inhibitory postsynaptic currents. In behavioral studies, intraplantar and intrathecal administration of AITC induced more pronounced and prolonged changes in nociceptive behavior than those induced by capsaicin. In conclusion, the characteristics of TRPA1 we have delineated suggest that it might play a unique role in nociception.