SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of excessive damages caused by SARS-CoV-2 remains largely unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is able to cause acute respiratory distress syndrome (ARDS)-like damages in vitro and in vivo. 2-E proteins were found to form a type of pH-sensitive cation channels in bilayer lipid membranes. As observed in SARS-CoV-2-infected cells, heterologous expression of 2-E channels induced rapid cell death in various susceptible cell types and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damages in lung and spleen. A dominant negative mutation lowering 2-E channel activity attenuated cell death and SARS-CoV-2 production. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent cell protective activity in vitro and these activities were positively correlated with inhibition of 2-E channel. Importantly, prophylactic and therapeutic administration of the channel inhibitor effectively reduced both the viral load and secretion of inflammation cytokines in lungs of SARS-CoV-2-infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE-2). Our study supports that 2-E is a promising drug target against SARS-CoV-2.Subject terms: Cell death, Molecular biology     

The Crystal Structure of L-Leucine Dehydrogenase from Pseudomonas aeruginosa

Leucine dehydrogenase (LDH, EC 1.4.1.9) catalyzes the reversible deamination of branched-chain L-amino acids to their corresponding keto acids using NAD⁺ as a cofactor. LDH generally adopts an octameric structure with D4 symmetry, generating a molecular mass of approximately 400 kDa. Here, the crystal structure of the LDH from Pseudomonas aeruginosa (Pa-LDH) was determined at 2.5 _Å resolution. Interestingly, the crystal structure shows that the enzyme exists as a dimer with C2 symmetry in a crystal lattice. The dimeric structure was also observed in solution using multiangle light scattering coupled with size-exclusion chromatography. The enzyme assay revealed that the specific activity was maximal at 60°C and pH 8.5. The kinetic parameters for three different amino acid and the cofactor (NAD⁺) were determined. The crystal structure represents that the subunit has more compact structure than homologs’ structure. In addition, the crystal structure along with sequence alignments indicates a set of non-conserved arginine residues which are important in stability. Subsequent mutation analysis for those residues revealed that the enzyme activity reduced to one third of the wild type. These results provide structural and biochemical insights for its future studies on its application for industrial purposes.     

灰飞虱与褐飞虱种内和种间密度效应比较

在人工气候室内比较研究了灰飞虱和褐飞虱在汕优63和秀水11上的种内和种间密度效应对其主要生物学参数的影响,结果表明两种稻飞虱均存在显著的种内竞争,主要表现为随着密度增加,若虫发育历期延长、若虫羽化率下降、雌成虫寿命缩短、每雌产卵量下降。种内密度效应与稻飞虱种类和寄主品种有显著互作关系,灰飞虱种内竞争较褐飞虱明显,二者在适宜寄主上种内竞争更为明显。同时,两种稻飞虱存在着显著的种间密度效应,主要表现为异种存在时的促进作用,即异种共存时若虫历期缩短、若虫羽化率提高、雌虫寿命延长、每雌产卵量增加。种间密度效应也与稻飞虱种类和寄主品种有显著互作关系,两种共存对灰飞虱的有利作用显著大于对褐飞虱的有利作用,在欠适宜寄主上种间互利效应更为明显。最后,本文对稻飞虱种内和种间密度效应的机制和进化意义进行了讨论。     

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.     

荧光假单胞菌抗噬菌体菌株的选育

本实验从荧光假单胞菌（Ｐｓｅｕｄｏｍｏｎａｓｆｌｕｏｒｅｓｃｅｎｓ）ＡＳ—３菌株的不正常发酵液中分离到一种噬菌体,将其命名为ＰＦＡＳ。ＡＳ—３菌株能利用葡萄糖发酵产生Ｄ－异维生素Ｃ的前体物质２－酮基－Ｄ－葡萄糖酸。电镜观察表明ＰＦＡＳ噬菌体呈蝌蚪形,具有直径为６６ｎｍ的六角形头部及长１１７ｎｍ的尾部。通过紫外线诱变及自然选育两种途径,配合简便有效的初筛方法,经多次分离、纯化、复筛最终在摇并发酵试验中获得６株产量稳定地高于对照敏感菌的抗噬菌体菌株,可望用于生产。     

南京农业大学微生物学虚拟仿真实验教学模式的探索

借助虚拟仿真技术构建的虚实结合的虚拟仿真实验,是提高学生实践动手能力的有力补充和有效途径。本文通过对微生物学实验教学现状的分析,探讨南京农业大学微生物学虚拟仿真实验教学模式建设的意义,重点阐述建设思路、建设方案及教学特点。该仿真实验教学体系主要从基础微生物学和应用微生物学两大模块进行构建,同时将本校的污水生物处理技术、食用菌资源调查与利用等科研成果转化为教学资源,以丰富实验教学内容。     

基于扩展的Budyko模型定量评估平江流域森林恢复和气候变异对季节性径流的影响

流域季节性径流变化反映了年内水资源的动态特征。在以森林为主的流域中,森林变化和气候变异被普遍认为是影响流域水文过程的两大驱动因素。因此在全球气候变化背景下,研究流域森林恢复和气候变异对流域季节性径流的影响,可为协调区域碳-水关系和制订可持续的森林经营管理策略提供参考。选择鄱阳湖流域上游的平江流域为研究对象,根据流域历史森林覆盖率变化情况,将研究期划分为参考期（1961-1985）和森林恢复期（1986-2006）,采用Mann-Kendall趋势分析研究流域长时期水文气象数据是否存在显著变化趋势。同时引入月干旱指数（潜在蒸散发和有效降雨的比率）,将一年定义为能量限制季（1-6月）和水分限制季（7-12月）,结合扩展的Budyko模型定量分析平江流域森林恢复和气候变异对季节性径流的相对贡献。在本研究流域整个研究期内（1961-2006）,通过Mann-Kendall趋势分析发现,研究流域水分限制季径流呈现显著增加趋势,而能量限制季水文和气候变量变化趋势均不显著。其次,相较于参考期,流域森林恢复使能量限制季径流降低了11.71 mm/a （24.40%）,使水分限制季径流增加了12.27 mm/a （17.23%）。同时,气候变异导致能量限制季径流减少了36.28 mm/a （75.60%）,而使水分限制季径流增加了58.94 mm/a （82.77%）。上述研究结果表明,森林恢复对径流影响具有累积效应。森林恢复对季节性径流具有积极的调节作用,同时季节性径流对森林恢复的响应存在时间差,而且森林恢复对径流的影响在能量限制季和水分限制季具有相互抵消的作用,气候变异与森林恢复的影响效应类似。此外,本研究也证实,平江流域季节性径流变化主要是受气候变化主导,但森林恢复对季节性径流的贡献也不容忽视。     

Role of PCK2 in the proliferation of vascular smooth muscle cells in neointimal hyperplasia

Vascular smooth muscle cell (VSMC) proliferation is a hallmark of neointimal hyperplasia (NIH) in atherosclerosis and restenosis post-balloon angioplasty and stent insertion. Although numerous cytotoxic and cytostatic therapeutics have been developed to reduce NIH, it is improbable that a multifactorial disease can be successfully treated by focusing on a preconceived hypothesis. We, therefore, aimed to identify key molecules involved in NIH via a hypothesis-free approach. We analyzed four datasets ({"type":"entrez-geo","attrs":{"text":"GSE28829","term_id":"28829"}}GSE28829, {"type":"entrez-geo","attrs":{"text":"GSE43292","term_id":"43292"}}GSE43292, {"type":"entrez-geo","attrs":{"text":"GSE100927","term_id":"100927"}}GSE100927, and {"type":"entrez-geo","attrs":{"text":"GSE120521","term_id":"120521"}}GSE120521), evaluated differentially expressed genes (DEGs) in wire-injured femoral arteries of mice, and determined their association with VSMC proliferation in vitro. Moreover, we performed RNA sequencing on platelet-derived growth factor (PDGF)-stimulated human VSMCs (hVSMCs) post-phosphoenolpyruvate carboxykinase 2 (PCK2) knockdown and investigated pathways associated with PCK2. Finally, we assessed NIH formation in Pck2 knockout (KO) mice by wire injury and identified PCK2 expression in human femoral artery atheroma. Among six DEGs, only PCK2 and RGS1 showed identical expression patterns between wire-injured femoral arteries of mice and gene expression datasets. PDGF-induced VSMC proliferation was attenuated when hVSMCs were transfected with PCK2 siRNA. RNA sequencing of PCK2 siRNA-treated hVSMCs revealed the involvement of the Akt-FoxO-PCK2 pathway in VSMC proliferation via Akt2, Akt3, FoxO1, and FoxO3. Additionally, NIH was attenuated in the wire-injured femoral artery of Pck2-KO mice and PCK2 was expressed in human femoral atheroma. PCK2 regulates VSMC proliferation in response to vascular injury via the Akt-FoxO-PCK2 pathway. Targeting PCK2, a downstream signaling mediator of VSMC proliferation, may be a novel therapeutic approach to modulate VSMC proliferation in atherosclerosis.     

生态系统生态学研究的关键问题及趋势——从“整体论与还原论的争论”看

在生态学领域中,存在着生态系统整体论与还原论的争论。Tansley A.G.提出,生态系统是"准有机体"。Odum兄弟提出的"生态系统能量说"被广泛接受,但也受到质疑,称其为"还原论者的整体论"。基于对上述质疑的回应以及对生态系统整体论的追求,Patten B.C.等提出"生态网络理论",运用"网络‘环境子’分析"方法,试图从物理层面分析解决生物层面的"涌现性"问题。不过,这一理论也受到批判,认为其在探究符号化的现象对生态系统的动态影响时,陷入了还原论困境。Jrgensen S.E.等更进一步,提出"系统论"的生态系统生态学,试图从系统科学的角度研究生态系统的"物质-能量-信息-网络"系统。这一理论受到生态学界高度重视,但是也存在着在具体研究过程中如何平衡能量视角和生物地球化学视角的问题。由上述争论可见,生态系统生态学研究的趋势是从"物质实体"到"能量流动",再到"网络信息",最后到"开放系统"层层递进。目前面临的关键问题是:如何在更好地定义生态系统整体性的基础上,采取相应的能够体现生态系统整体性的方法,去获得更多、更好的生态系统整体性的认识。