Quantification of the oxidative damage biomarker 2,3-dinor-8-isoprostaglandin-F2α in human urine using liquid chromatography-tandem mass spectrometry

F₂-isoprostanes are useful biomarkers of oxidative status in humans. We developed an ultraperformance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify 2,3-dinor-8-iso prostaglandin F_2α, a urinary metabolite of 8-iso-prostaglandin F_2α. Urine was purified by solid-phase extraction and analyzed by UPLC-MS/MS with negative-ion electrospray ionization. The method was robust with a mean inaccuracy of 9%, interday and intraday imprecision of 7.5% or lower, and a lower limit of quantification of 0.5 μg/L, equivalent to 0.04 pmol injected onto the column. An analysis time of 6 min was shorter than previously published methods and amenable to large studies.     

大肠杆菌CusS的生物信息学分析及对银离子胁迫的响应

【目的】解析大肠杆菌(Escherichia coli) K-12菌株同型二聚体内膜传感器组氨酸激酶(sensor histidine kinase, CusS)蛋白在细菌应答金属银离子胁迫中的调控机制,为该菌的防治提供重要科学依据。【方法】利用ProtParam、ProtScale、Protein-Sol、TMHMM、SignalP、LocTree3、NetNGlyc-1.0、NetPhosBac-3.0、SOPMA、I-TASSERF、STRING和MEGA分别预测CusS的理化性质、亲水性、可溶性、跨膜域、信号肽、亚细胞定位、糖基化位点、磷酸化位点、二级结构、三级结构、蛋白互作的关系网络和蛋白在革兰阴性杆菌中的同源性。采用Red同源重组技术构建大肠杆菌ΔcusS,在不同培养基中连续监测ΔcusS的生长情况,观察该基因缺失后的细菌生长活性;通过最小抑菌浓度(minimal inhibitory concentration, MIC)试验评价该缺失株对金属铜、银离子和临床常见抗生素的敏感性变化;运用RT-qPCR检测cusS缺失后其下游基因cusCFBA和cusR转录水平。【结果】CusS蛋白由480个氨基酸组成,相对分子质量为53 738.05,原子总数为7 624,等电点为6.02,具有稳定性,是一种亲水性、不溶性蛋白;含有跨膜域;不存在信号肽,定位于细胞内膜中;存在2个糖基化位点、24个丝氨酸磷酸化位点、14个苏氨酸磷酸化位点和3个酪氨酸磷酸化位点;二级结构中α-螺旋占比55.42%,β-折叠占比11.67%,β-转角占比3.75%,无规则卷曲占比29.17%;cusS在埃希菌属和志贺菌属中的保守性高;菌落PCR和一代测序验证ΔcusS构建成功;连续检测生长曲线表明cusS缺失并不影响细菌的生长代谢,但CusS蛋白为大肠杆菌抵御金属银胁迫的关键基因。【结论】cusS作为一个关键基因,它的缺失并不影响大肠杆菌的生长活性,但会显著降低细菌抵御银离子胁迫的应答能力。缺失cusS将使下游基因cusCFBA和cusR的mRNA表达水平显著下降。对CusS蛋白进行生物信息学分析及表型初探,为深入了解CusS在大肠杆菌应答银离子胁迫的调控机制奠定了基础。     

Mechanisms of genome instability in Hutchinson-Gilford progeria

Background

Hutchinson-Gilford progeria syndrome (HGPS) is a devastating premature aging disorder. It arises from a single point mutation in the LMNA gene. This mutation stimulates an aberrant splicing event and produces progerin, an isoform of the lamin A protein. Accumulation of progerin disrupts numerous physiological pathways and induces defects in nuclear architecture, gene expression, histone modification, cell cycle regulation, mitochondrial functionality, genome integrity and much more.

Objective

Among these phenotypes, genomic instability is tightly associated with physiological aging and considered a main contributor to the premature aging phenotypes. However, our understanding of the underlying molecular mechanisms of progerin-caused genome instability is far from clear.

Results and Conclusion

In this review, we summarize some of the recent findings and discuss potential mechanisms through which, progerin affects DNA damage repair and leads to genome integrity.

     

Rictor Is Required for Early B Cell Development in Bone Marrow

The development of early B cells, which are generated from hematopoietic stem cells (HSCs) in a series of well-characterized stages in bone marrow (BM), represents a paradigm for terminal differentiation processes. Akt is primarily regulated by phosphorylation at Thr308 by PDK1 and at Ser473 by mTORC2, and Akt signaling plays a key role in hematopoiesis. However, the role of mTORC2 in the development of early B cells remains poorly understood. In this study, we investigated the functional role of mTORC2 by specifically deleting an integral component, Rictor, in a hematopoietic system. We demonstrated that the deletion of Rictor induced an aberrant increase in the FoxO1 and Rag-1 proteins in BM B cells and that this increase was accompanied by a significant decrease in the abundance of B cells in the peripheral blood (PB) and the spleen, suggesting impaired development of early B cells in adult mouse BM. A BM transplantation assay revealed that the B cell differentiation defect induced by Rictor deletion was not affected by the BM microenvironment, thus indicating a cell-intrinsic mechanism. Furthermore, the knockdown of FoxO1 in Rictor-deleted HSCs and hematopoietic progenitor cells (HPCs) promoted the maturation of B cells in the BM of recipient mice. In addition, we revealed that treatment with rapamycin (an mTORC1 inhibitor) aggravated the deficiency in B cell development in the PB and BM. Taken together, our results provide further evidence that Rictor regulates the development of early B cells in a cell-intrinsic manner by modifying the expression of FoxO1 and Rag-1.     

紫花针茅根际和体内真菌群落结构及与土壤环境因子的相关性研究

[目的] 探究青藏高原不同地区高寒草原紫花针茅根际和体内真菌群落的组成、多样性等特征,及与土壤环境因子（理化性质和酶活性）间的相互关系。[方法] 从青藏高原不同地区采集紫花针茅样品,应用土壤化学方法分析根际土壤理化性质和酶活性,并采用Illumina Miseq高通量测序技术,解析根际土壤和体内真菌群落组成和丰度、Alpha多样性和菌群结构,同时分析了紫花针茅根际真菌种群多样性与土壤环境因子的相关性,厘清了影响紫花针茅根际真菌区系的土壤环境因素。[结果] 三个采样地的根际土壤呈中性偏碱,土壤理化性质和酶活性变化各异。高通量测序共得到314801条有效序列和4491个OTUs;XZ样地的紫花针茅真菌多样性和丰富度相对偏低,GS样地最高。在门分类水平上,子囊菌门Ascomycota和担子菌门Basidiomycota是主要内生真菌类群,占总菌群的88.28%。不同采样地区紫花针茅体内真菌群落结构存在明显差异,而根际土壤真菌群落结构差异不大。相关性分析表明,紫花针茅真菌多样性与土壤pH、有效钾、铁、钙、镁、多酚氧化酶、过氧化物酶和脱氢酶呈显著（P<0.05）或极显著（P<0.01）正相关,而与海拔、土壤酸性磷酸酶呈极显著负相关。RDA分析发现,紫花针茅根际土壤真菌不同,影响的土壤环境因子也不同。[结论] 青藏高原高寒草地紫花针茅根际和体内栖息着丰富的真菌群落,其组成和多样性受多种土壤环境因子影响,且影响不同真菌群落的主要土壤环境因子也不同。本研究对于有益微生物资源的开发、利用及保护具有重要意义,并为紫花针茅草原保育和合理开发利用提供科学依据。     

Methylene blue alleviates nuclear and mitochondrial abnormalities in progeria

Hutchinson–Gilford progeria syndrome (HGPS), a fatal premature aging disease, is caused by a single‐nucleotide mutation in the LMNA gene. Previous reports have focused on nuclear phenotypes in HGPS cells, yet the potential contribution of the mitochondria, a key player in normal aging, remains unclear. Using high‐resolution microscopy analysis, we demonstrated a significantly increased fraction of swollen and fragmented mitochondria and a marked reduction in mitochondrial mobility in HGPS fibroblast cells. Notably, the expression of PGC‐1α, a central regulator of mitochondrial biogenesis, was inhibited by progerin. To rescue mitochondrial defects, we treated HGPS cells with a mitochondrial‐targeting antioxidant methylene blue (MB). Our analysis indicated that MB treatment not only alleviated the mitochondrial defects but also rescued the hallmark nuclear abnormalities in HGPS cells. Additional analysis suggested that MB treatment released progerin from the nuclear membrane, rescued perinuclear heterochromatin loss and corrected misregulated gene expression in HGPS cells. Together, these results demonstrate a role of mitochondrial dysfunction in developing the premature aging phenotypes in HGPS cells and suggest MB as a promising therapeutic approach for HGPS.     

植物进化发育生物学的形成与研究进展

植物进化发育生物学是最近十几年来才兴起的一门学科, 它是进化发育生物学的主要分支之一。进化发育生物学的产生经历了进化生物学与胚胎学、遗传学和发育生物学的三次大的综合, 其历史可追溯到19世纪初冯.贝尔所创立的比较胚胎学。相关研究曾沉寂了近一个世纪, 直到20世纪80年代早期, 动物中homeobox基因被发现, 90年代初花发育的 ABC模型被提出, 加之对发育相关基因研究的不断深入, 才使基因型与表型联系了起来, 进而促进了进化发育生物学的飞速发展。目前进化发育生物学已成为21世纪生命科学领域的研究热点之一。本文详细阐述了进化发育生物学产生和发展的历程, 综述了最近十几年来植物进化发育生物学的主要研究进展。文中重点介绍了与植物发育密切相关的MADS-box基因在植物各大类群中的研究现状, 讨论了植物进化发育生物学领域的研究成果对花被演化、花对称性以及叶的进化等重要问题的启示。     

Mouse models of laminopathies

The A‐ and B‐type lamins are nuclear intermediate filament proteins in eukaryotic cells with a broad range of functions, including the organization of nuclear architecture and interaction with proteins in many cellular functions. Over 180 disease‐causing mutations, termed ‘laminopathies,’ have been mapped throughout LMNA, the gene for A‐type lamins in humans. Laminopathies can range from muscular dystrophies, cardiomyopathy, to Hutchinson–Gilford progeria syndrome. A number of mouse lines carrying some of the same mutations as those resulting in human diseases have been established. These LMNA‐related mouse models have provided valuable insights into the functions of lamin A biogenesis and the roles of individual A‐type lamins during tissue development. This review groups these LMNA‐related mouse models into three categories: null mutants, point mutants, and progeroid mutants. We compare their phenotypes and discuss their potential implications in laminopathies and aging.