A Novel Minimally-Invasive Method to Sample Human Endothelial Cells for Molecular Profiling

ObjectiveThe endothelium is a key mediator of vascular homeostasis and cardiovascular health. Molecular research on the human endothelium may provide insight into the mechanisms underlying cardiovascular disease. Prior methodology used to isolate human endothelial cells has suffered from poor yields and contamination with other cell types. We thus sought to develop a minimally invasive technique to obtain endothelial cells derived from human subjects with higher yields and purity.MethodsNine healthy volunteers underwent endothelial cell harvesting from antecubital veins using guidewires. Fluorescence-activated cell sorting (FACS) was subsequently used to purify endothelial cells from contaminating cells using endothelial surface markers (CD34 / CD105 / CD146) with the concomitant absence of leukocyte and platelet specific markers (CD11b / CD45). Endothelial lineage in the purified cell population was confirmed by expression of endothelial specific genes and microRNA using quantitative polymerase chain reaction (PCR).ResultsA median of 4,212 (IQR: 2161 – 6583) endothelial cells were isolated from each subject. Quantitative PCR demonstrated higher expression of von Willebrand Factor (vWF, P<0.001), nitric oxide synthase 3 (NOS3, P<0.001) and vascular cell adhesion molecule 1 (VCAM-1, P<0.003) in the endothelial population compared to similarly isolated leukocytes. Similarly, the level of endothelial specific microRNA-126 was higher in the purified endothelial cells (P<0.001).ConclusionThis state-of-the-art technique isolates human endothelial cells for molecular analysis in higher purity and greater numbers than previously possible. This approach will expedite research on the molecular mechanisms of human cardiovascular disease, elucidating its pathophysiology and potential therapeutic targets.     

《中国植被志》的植被分类系统、植被类型划分及编排体系

植被志(vegegraphy)是基于植被调查资料, 全面记叙植被的群落外貌、种类组成、结构和功能、生境条件, 以及地理分布等特征, 并对同类植被进行归纳和总结的志书。“植被志”的英文译为“vegegraphy”, 是本文的新造词, 它是由“vegetation”的词头“vege-”和英文后缀“-graphy” (记叙之意)组成的合成词。《中国植被志》的研编是一项时间紧迫、内容复杂、工作量浩繁的重大科学工程。它的完成将极大提升中国植被科学和生态学的研究水平, 并为中国植被资源的合理利用、生物多样性保护及生态环境治理等提供不可或缺的基础资料。本文首先简述了植被的基本特征(主要包括外貌特征、种类组成、群落结构以及动态变化等)和国内外植被调查的进展情况, 简要回顾了中国植被分类系统的研究历史, 并对以往的分类系统进行了若干修订。在此基础上, 着重讨论并提出了《中国植被志》卷册编排体系和用于《中国植被志》研编的植被类型划分方案。在对植被分类系统的修订方面, 主要对高级分类单位及相对应的英文名称进行了讨论和修订。按照本文修订的分类系统, 中国植被的分类单位及其对应的英文名称分别是: 植被型组(Vegetation Formation Group)、植被型(Vegetation Formation)和植被亚型(Vegetation Subformation)、群系组(Alliance Group)、群系(Alliance)和亚群系(Suballiance)、群丛组(Association Group)以及群丛(Association)。在植被型组中, 划分为9类: 森林、灌丛、草本植被、荒漠、高山冻原与稀疏植被、沼泽与水生植被、农业植被、城市植被及无植被地段。关于《中国植被志》的卷册编排和“植被类型”划分, 首先按高级分类单位——植被型划分相应的“卷”; 在此框架下, 模糊“植被亚型”、“群系组”和“群系”的概念, 确定“植被类型” (Vegetation type), 并将其作为植被志各卷中的“册”。这样处理不仅保证了研编工作的可操作性, 也保持了同一卷册中特定植被类型的完整性。《中国植被志》编排体系中的“植被类型”的划分很重要, 它是指具有相同建群种及相同优势类群(如种、属)的植被组合, 但它不是严格意义上的植被分类单位。“植被类型”的划分遵循“优势类群及生活型的同一性, 生境条件的相对重要性, 植被特征及用途的差异性, 以及突出植被志的应用性”等原则。按该编排体系, 《中国植被志》将由48卷约110册组成。     

《中国植被志》研编内容与规范

植被志是基于植被(或植物群落)调查资料, 全面记叙植被的外貌、物种组成、结构和功能, 以及地理分布和生境条件等特征, 并对同类植被进行归纳和总结的志书。《中国植被志》是第一部对中国植被进行全面记述的志书, 预计完成约48卷110册。在借鉴《中国植被》(1980)(简称“80方案”)植被分类基本原则的基础上,《中国植被志》将对中国植被分类系统的高级分类单位(植被型组、植被型和植被亚型)进行归纳和总结, 对中级和低级分类单位(群系组、群系、亚群系, 群丛组、群丛)进行详细描述。植被高级分类单位的描述具有概括性质, 是在中国植被分类系统中级和低级分类单位描述的基础上, 对其在全球和中国境内的地理分布、自然环境、群落外貌、植被类型及多样性、优势种或共优势种、生物多样性保育价值以及资源现状等进行概述, 并对“80方案”相关内容进行修订和拓展, 将提供对中国植被基本特征客观、准确的记述。在植被中级和低级分类单位中, 群系组描述的内容包括地理分布、自然环境、群落外貌、植被类型以及价值与保育等内容; 群系描述的主要内容包括地理分布、自然环境、生态特征、物种组成、群落结构、群丛组和群丛的分类与描述、优势种的生物学特性、生物量与生产力、植被动态与演替以及价值与保育等方面。作为植被志研编的核心内容, 群丛组和群丛的分类与描述主要基于植被调查资料, 采用数量分类方法, 根据群落结构和物种组成的差异划分出不同的植被类型, 并对其基本特征进行定量描述和归纳。其中, 群落的层片结构、特定植被分类单元的特征种或特征种组的筛选与甄别是植被类型划分的关键环节; 而群落外貌, 群落结构, 物种组成, 各类物种的生长习性、生境的偏适性等是群丛组和群丛描述与归纳的重点内容。该文提出了中国植被中级和低级分类单位的命名方案, 其特点在于植被类型的科学名称中同时体现了植被分类单元特征种或优势种的名称及其所属的高级植被分类单位(植被型组或植被型)的名称, 兼顾了植被名称的规范性与实用性。《中国植被志》的研编工作由文献整编、群落调查、数据分析与整理、文本撰写等环节组成。该文对植被样方的调查与收集, 文献收集与整编, 气候、土壤、地形等相关数据的来源及其整理方法, 植被分类方法, 植被命名, 植被分类单元描述的内容, 植被志章节编写大纲、体裁及撰写等多个规范进行了详细的阐述或示例。     

Revisiting delta-6 desaturase regulation by C18 unsaturated fatty acids,depending on the nutritional status

Dietary polyunsaturated fatty acids (PUFA) play a key role in regulating delta-6 desaturase (D6D), the key enzyme for long-chain PUFA biosynthesis. Nevertheless, the extent of their effects on this enzyme remains controversial and difficult to assess. It has been generally admitted that C18 unsaturated fatty acids (UFAs) regulate negatively delta-6 desaturase (D6D). This inhibition has been evidenced in regard to a high glucose/fat free (HG/FF) diet used in reference. However, several nutritional investigations did not evidence any inhibition of desaturases when feeding fatty acids.Because the choice of the basal diet appeared to be of primary importance in such experiments, our goal was to reconsider the specific role of dietary UFAs on D6D regulation, depending on nutritional conditions. For that, sixteen adult Wistar rats were fed purified linoleic acid, α-linolenic acid or oleic acid, included in one of two diets at 4% by weight: an HG/FF or a high starch base (HS) where the pure UFAs replaced a mixed vegetable oil. Our results showed first that D6D specific activity was significantly greater when measured in presence of an HG/FF than with an HS/4% vegetable oil diet. Secondly, we found that linoleic and alpha-linolenic acids added to HG/FF reduced the specific activity of D6D. In contrast, when pure UFAs were added to an HS base, D6D specific activities remained unchanged or increased. Concordant results were obtained on D6D mRNA expression.Altogether, this study evidenced the importance of the nutritional status in D6D regulation by C18 UFAs: when used as control, HG/FF diet stimulates D6D compared with a standard control diet containing starch and 4% fats, leading to an overestimation of the D6D regulation by UFAs. Then, UFAs should be considered as repressors for unsaturated fatty acid biosynthesis only in very specific nutritional conditions.     

Ethanol increases cytochromes P450IIE, IIB1/2, and IIIA in cultured rat hepatocytes

In intact rats, ethanol treatment has been associated with increases in hepatic levels of both P450IIB1/2 and P450IIE. When rat hepatocytes were cultured on an extracellular tumor matrix (Matrigel), exposure to ethanol from 48 to 96 h in culture resulted in increases in cytochromes P450IIE, IIB1/2, and IIIA. Cytochrome P450IIE was detected immunologically and enzymatically, using two activities associated with cytochrome P450IIE, p-nitrophenol hydroxylation, and acetaminophen activation to a metabolite that binds to glutathione. The content of cytochrome P450IIE in freshly isolated cells decreased when the cells were placed in culture. Exposure of the cultured hepatocytes to ethanol from 48 to 96 h after inoculation resulted in an increase in cytochrome P450IIE compared to untreated cultured cells. In addition, in culture, the amount of enzymatically active protein after ethanol treatment was equal to that in hepatocytes freshly isolated from intact animals. Ethanol treatment resulted in increases in cytochrome P450IIB1/2 compared to untreated cells, as shown immunologically and by increased benzyloxyresorufin dealkylase activity. However, phenobarbital induced cytochrome P450IIB1/2 to higher levels, compared to ethanol. Ethanol and phenobarbital treatments both increased P450IIIA, as determined immunologically and by the amount of propoxycoumarin depropylase activity that is inhibited by triacetyloleandomycin. However, the amount of P450IIIA increased after ethanol treatment was less than that increased after treatment with dexamethasone in these cells. The ethanol-mediated increases in all four forms of cytochrome P450 in culture suggest that these increases in the intact animal result from direct effects of ethanol on the liver.     

Multiple developmental processes underlie sex differentiation in angiosperms

The production of unisexual flowers has evolved numerous times in dioecious and monoecious plant taxa. Based on repeated evolutionary origins, a great variety of developmental and genetic mechanisms underlying unisexual flower development is predicted. Here, we comprehensively review the modes of development of unisexual flowers, test potential correlations with sexual system, and end with a synthesis of the genetics and hormonal regulation of plant sex determination. We find that the stage of organ abortion in male and female flowers is temporally correlated within species and also confirm that the arrest of development does not tend to occur preferentially at a particular stage, or via a common process.     

Microbial contamination in kitchens and bathrooms of rural Cambodian village households

Aims: To quantify microbial contamination on kitchen and bathroom surfaces (fomites) in rural Cambodian homes and to compare these concentrations to similar data from the United States and Japan. Methods and Results: This study monitored the numbers of faecal coliforms (i.e. thermotolerant coliforms), total coliforms, Escherichia coli and heterotrophic plate count bacteria on household surfaces in a rural village of Cambodia. Faecal coliform levels in Cambodia were highest on moist locations such as the plastic ladle used for sink water, the toilet seat surface and the cutting board surface with 100‐fold higher levels of faecal coliform bacteria than E. coli and 100‐fold higher levels of faecal coliforms than the US and Japanese studies. Conclusions: A single public health intervention barrier, such as an improved latrine, is only partially effective for household sanitation. For complete sanitation, multiple environmental barriers may be necessary. These barriers occur in a house constructed with easily washable surfaces, a chlorinated water distribution system, house climate control and cleaning product availability. Significance and Impact of the Study: Results of this study can be used to emphasize the importance of increasing household environmental sanitation barriers.     

Quantitative profiling of serum samples using TMT protein labelling, fractionation and LC-MS/MS

Blood-borne biomarkers are urgently required for the early detection, accurate diagnosis and prognosis of disease. Additionally, improved methods of profiling serum and plasma proteins for biomarker discovery efforts are needed. Herein, we report a quantitative method based on amino-group labelling of serum proteins (rather than peptides) with isobaric tandem mass tags (TMT) and incorporating immune-based depletion, gel-based and strong anion exchange separation of proteins prior to differential endoproteinase treatment and liquid chromatography tandem mass spectrometry. We report a generally higher level of quantitative coverage of the serum proteome compared to other peptide-based isobaric tagging approaches and show the potential of the method by applying it to a set of unique samples that pre-date the diagnosis of pancreatic cancer.     

SIRT1 deacetylase in SF1 neurons protects against metabolic imbalance

Chronic feeding on high-calorie diets causes obesity and type 2 diabetes mellitus (T2DM), illnesses that affect hundreds of millions. Thus, understanding the pathways protecting against diet-induced metabolic imbalance is of paramount medical importance. Here, we show that mice lacking SIRT1 in steroidogenic factor 1 (SF1) neurons are hypersensitive to dietary obesity owing to maladaptive energy expenditure. Also, mutant mice have increased susceptibility to developing dietary T2DM due to insulin resistance in skeletal muscle. Mechanistically, these aberrations arise, in part, from impaired metabolic actions of the neuropeptide orexin-A and the hormone leptin. Conversely, mice overexpressing SIRT1 in SF1 neurons are more resistant to diet-induced obesity and insulin resistance due to increased energy expenditure and enhanced skeletal muscle insulin sensitivity. Our results unveil important protective roles of SIRT1 in SF1 neurons against dietary metabolic imbalance.     

Mitotic arrest in teratoma susceptible fetal male germ cells

Formation of germ cell derived teratomas occurs in mice of the 129/SvJ strain, but not in C57Bl/6 inbred or CD1 outbred mice. Despite this, there have been few comparative studies aimed at determining the similarities and differences between teratoma susceptible and non-susceptible mouse strains. This study examines the entry of fetal germ cells into the male pathway and mitotic arrest in 129T2/SvJ mice. We find that although the entry of fetal germ cells into mitotic arrest is similar between 129T2/SvJ, C57Bl/6 and CD1 mice, there were significant differences in the size and germ cell content of the testis cords in these strains. In 129T2/SvJ mice germ cell mitotic arrest involves upregulation of p27(KIP1), p15(INK4B), activation of RB, the expression of male germ cell differentiation markers NANOS2, DNMT3L and MILI and repression of the pluripotency network. The germ-line markers DPPA2 and DPPA4 show reciprocal repression and upregulation, respectively, while FGFR3 is substantially enriched in the nucleus of differentiating male germ cells. Further understanding of fetal male germ cell differentiation promises to provide insight into disorders of the testis and germ cell lineage, such as testis tumour formation and infertility.