新型聚球藻噬藻体Yong-L2-223的分离及基因组分析

【目的】噬藻体(cyanophages)是特异性侵染蓝藻(cyanobacteria)的病毒，广泛分布于各类水体中，在调节蓝藻种群动态和密度、推动生物地球水生生态系统循环中起着重要作用。本研究的目的在于分离、鉴定噬藻体。【方法】本研究以海洋聚球藻(Synechococcus sp.) PCC 7002为指示宿主，从淡水水样中分离培养一株新型噬藻体Yong-L2-223，对其进行了宿主范围实验、全基因组测序、基因功能注释和系统进化分析。【结果】针对31株供试蓝藻的宿主范围实验，结果除指示藻PCC 7002 [属于聚球藻目(Synechococcales)]外，Yong-L2-223能够感染2株淡水蓝藻，分别是来源于滇池的绿色微囊藻(Microcystis viridis) FACHB-1342 [属于色球藻目(Chroococcales)]和水华束丝藻(Aphanizomenon flos-aquae)FACHB-1209[属于念珠藻目(Nostocales)]。既可在高盐条件下感染海洋蓝藻，又可在低盐条件下感染淡水蓝藻，Yong-L2-223具有广盐性。透射电镜观察表明，Yong-L2...     

新型淡水微囊藻噬藻体vB_MweS-yong2的分离与基因组分析

【目的】蓝藻(cyanobacteria)水华频繁暴发,引起水质恶化,使水生生物大量死亡,给水产养殖业造成巨大的经济损失;其代谢产物藻毒素具有肝毒性、神经毒性、生殖毒性、遗传毒性和肿瘤促进作用,并可在水生生物中富集,造成饮用水安全风险和水产品食用安全风险。噬藻体(cyanophages)是一类特异性侵染蓝藻的病毒,参与调控蓝藻的种群密度和丰度,被认为是极具潜力的蓝藻水华生物防控工具。以往的研究报道多集中于海水噬藻体,有关淡水噬藻体的报道寥寥无几,迄今尚无惠氏微囊藻(Microcystis wesenbergii)噬藻体的研究报道。本研究的目的在于分离、鉴定惠氏微囊藻噬藻体。【方法】以惠氏微囊藻FACHB-1112为指示宿主,采用双层平板法从淡水中分离出噬藻体vB_MweS-yong2,对其进行全基因组测序、基因功能注释和系统进化分析。【结果】vB_MweS-yong2的基因组长44 530 bp,G+C含量为71.6%,有61个开放阅读框(ORF)、1个tRNA基因。成对序列比较 (pairwise sequence comparison,PASC)表明,vB_MweS-yong2与所有已知噬菌体间的全基因组核苷酸序列相似度最高只有20.21%,小于<50%的属边界值。没有在vB_MweS-yong2基因组中发现抗生素耐药基因和毒力因子基因,显示该噬藻体在基因水平上的安全性。【结论】vB_MweS-yong2在有尾目的长尾科中代表一个新的属。本研究丰富了淡水噬藻体库、基因库,并为以后研发该噬藻体的功能基因、进一步研发用于治理以惠氏微囊藻为优势种引起的水华的产品与技术奠定了基础。     

Identification of Auxin Activity Like 1, a chemical with weak functions in auxin signaling pathway

Key message

A new synthetic auxin AAL1 with new structure was identified. Different from known auxins, it has weak effects. By AAL1, we found specific amino acids could restore the effects of auxin with similar structure.

Abstract

Auxin, one of the most important phytohormones, plays crucial roles in plant growth, development and environmental response. Although many critical regulators have been identified in auxin signaling pathway, some factors, especially those with weak fine-tuning roles, are still yet to be discovered. Through chemical genetic screenings, we identified a small molecule, Auxin Activity Like 1 (AAL1), which can effectively inhibit dark-grown Arabidopsis thaliana seedlings. Genetic screening identified AAL1 resistant mutants are also hyposensitive to indole-3-acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D). AAL1 resistant mutants such as shy2-3c and ecr1-2 are well characterized as mutants in auxin signaling pathway. Genetic studies showed that AAL1 functions through auxin receptor Transport Inhibitor Response1 (TIR1) and its functions depend on auxin influx and efflux carriers. Compared with known auxins, AAL1 exhibits relatively weak effects on plant growth, with 20 µM and 50 µM IC50 (half growth inhibition chemical concentration) in root and hypocotyl growth respectively. Interestingly, we found the inhibitory effects of AAL1 and IAA could be partially restored by tyrosine and tryptophan respectively, suggesting some amino acids can also affect auxin signaling pathway in a moderate manner. Taken together, our results demonstrate that AAL1 acts through auxin signaling pathway, and AAL1, as a weak auxin activity analog, provides us a tool to study weak genetic interactions in auxin pathway.

     

Astrocytic YAP prevents the demyelination through promoting expression of cholesterol synthesis genes in experimental autoimmune encephalomyelitis

Cholesterols are the main components of myelin, and are mainly synthesized in astrocytes and transported to oligodendrocytes and neurons in the adult brain. It has been reported that Hippo/yes-associated protein (YAP) pathways are involved in cholesterol synthesis in the liver, however, it remains unknown whether YAP signaling can prevent the demyelination through promoting cholesterol synthesis in experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of multiple sclerosis characterized by neuroinflammation and demyelination. Here, we found that YAP was upregulated and activated in astrocytes of spinal cords of EAE mice through suppression of the Hippo pathway. YAP deletion in astrocytes aggravated EAE with earlier onset, severer inflammatory infiltration, demyelination, and more loss of neurons. Furthermore, we found that the neuroinflammation was aggravated and the proliferation of astrocytes was decreased in YAP^GFAP-CKO EAE mice. Mechanically, RNA-seq revealed that the expression of cholesterol-synthesis pathway genes such as HMGCS1 were decreased in YAP^−/− astrocytes. qPCR, western blot, and immunostaining further confirmed the more significant reduction of HMGCS1 in spinal cord astrocytes of YAP^GFAP-CKO EAE mice. Interestingly, upregulation of cholesterol-synthesis pathways by diarylpropionitrile (DPN) (an ERβ-ligand, to upregulate the expression of HMGCS1) treatment partially rescued the demyelination deficits in YAP^GFAP-CKO EAE mice. Finally, activation of YAP by XMU-MP-1 treatment promoted the expression of HMGCS1 in astrocytes and partially rescued the demyelination and inflammatory infiltration deficits in EAE mice. These findings identify unrecognized functions of astrocytic YAP in the prevention of demyelination through promoting cholesterol synthesis in EAE, and reveal a novel pathway of YAP/HMGCS1 for cholesterol synthesis in EAE pathology.Subject terms: Multiple sclerosis, Astrocyte, Multiple sclerosis     

一株枯草芽孢杆菌原噬菌体Bsu-yong1的基因组分析

【目的】枯草芽孢杆菌（Bacillus subtilis）是在自然界中广泛存在的革兰氏阳性菌，其抗逆性极强，能抑制大多数有害菌的繁殖，是常用的产酶菌，用其生产的蛋白酶、淀粉酶占全球工业酶产量的50%。原噬菌体（prophage）整合在宿主基因组中，可为宿主提供基因和生物学功能，非常具有研究价值。以往，有关B. subtilis原噬菌体的报道主要集中于缺陷型原噬菌体（defective prophage），本研究对一株非缺陷型活性原噬菌体（active prophage）的基因组进行解析，以扩充对非缺陷型原噬菌体的认知。【方法】使用丝裂霉素C从枯草芽孢杆菌中诱导一株噬菌体，命名为Bacillus phage Bsu-yong1（简称Bsu-yong1）。对Bsu-yong1进行负染、透射电镜（transmission electron microscopy，TEM）观察，用Illumina MiSeq测定其基因组序列、综合运用生物信息学工具对其进行基因功能注释和系统进化分析。【结果】Bsu-yong1与PBSX类缺陷型原噬菌体在形态上相似，但Bsu-yong1具有完整的噬菌体基因组，这与缺陷型原噬菌体不同，后者在包装过程中不能正确包裹自身的基因组，而是随机包裹一段宿主染色体。Bsu-yong1基因组全长为43 590 bp，G+C含量为41%，含有62个开放阅读框（open reading frame，ORF），呈模块化分布。Bsu-yong1拥有基因编码T7SS效应器LXG多态性毒素（T7SS effector LXG polymorphic toxin）、ImmA/IrrE蛋白和SMI1/KNR4蛋白。前二者为细菌毒素（toxin），后者为抗毒素（antitoxin），toxin-antitoxin是细菌免疫系统重要成员，参与菌间竞争与环境适应。此前，尚未有编码LXG polymorphic toxin的基因在噬菌体中被发现和报道。在基于全基因组比对构建的蛋白谱进化树（proteomic tree）中，Bsu-yong1与噬菌体sv105、rho14、vB_BteM-A9Y聚集形成一个独立的进化支（clade），基因组比对显示它们基因组的复制与调控模块具有高度保守性，它们共享29个核心基因（core gene），均具有PBSX样形态特征。Bsu-yong1与其他噬菌体的进化距离较远。将Bsu-yong1与所有噬菌体进行比对，得到的成对序列比较（pairwise sequence comparison，PASC）最大值为46.72%，小于属边界值（70%）。【结论】vB_Bsu-yong1在有尾纲中代表一个新的未知的属；建议构建一个新的科（family），该科由Bsu-yong1与噬菌体sv105、rho14、vB_BteM-A9Y组成。vB_Bsu-yong携带免疫相关基因，它可能有利于宿主在菌间竞争中获胜和适应环境。本研究丰富了噬菌体基因数据库，拓展了对芽孢杆菌活性原噬菌体的认知。     

热灭活植物乳杆菌ATCC 8014对UVB暴露皮肤细胞的抗光老化和抗黑色素生成作用研究北大核心

【目的】中波紫外(ultraviolet B,UVB)照射是导致皮肤光老化形成和色素沉着的主要环境原因之一。植物乳杆菌是一种广泛研究的益生菌。本研究利用UVB照射皮肤细胞[正常人皮肤成纤维细胞(normal human dermal fibroblast,NHDF)和小鼠B16F10黑色素瘤细胞],探讨热灭活植物乳杆菌(Lactobacillus plantarum,LP)ATCC 8014的抗光老化和抗黑色素生成作用。【方法】以暴露于UVB的NHDF细胞和小鼠B16F10黑色素瘤细胞为研究对象,用甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTT)法、末端脱氧核苷酸转移酶dUTP缺口末端标记(terminal-deoxynucleoitidyl transferase dUTP nick end labeling,TUNEL)染色分析和2’,7’-二氯荧光素二乙酸酯(2’,7’-dichlorofluorescin diacetate DCFH-DA)染色分析实验检测2组细胞的细胞活力、DNA损伤及相对活性氧(reactive oxygen species,ROS)水平;利用酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)法检测NHDF细胞中的I型胶原蛋白水平;通过NaOH裂解法和多巴氧化法对B16F10细胞黑色素生成和酪氨酸酶活性进行测定;通过qRT-PCR分析和Western blotting分析检测光老化和黑色素生成相关基因和蛋白表达水平变化。【结果】(1)LP抑制UVB诱导的NHDF和B16F10细胞毒性,这与减少ROS介导的细胞DNA损伤有关;(2)LP下调基质金属蛋白酶(matrix metalloproteinase,MMP)-1、MMP-3和MMP-9(而不是MMP-2)mRNA水平[与抑制细胞外信号调节激酶(extracellular signal-regulated kinase,ERK),p38(而不是JNK)/c-Fos(而不是c-Jun)信号通路有关],增加I型前胶原(procollagen type-1 alpha 1,COL1A1)蛋白水平,从而提高I型胶原蛋白含量;(3)LP作为自噬诱导剂(增加LC3-Ⅱ和Beclin 1蛋白水平以及LC3-Ⅱ/LC3-Ⅰ比率)抑制酪氨酸酶、酪氨酸相关蛋白(tyrosinase-related protein,TYRP)-1和TYRP-2活性和/或表达(与压制PKA/CREB/MITF信号通路有关),从而降低黑色素含量。【结论】LP在UVB暴露的皮肤细胞中具有潜在的抗光老化和抗黑色素生成作用。