Intra-individual variation of plasma lipids and lipoproteins in prepubescent children

Estimates of the average intra-individual biological variability for plasma lipids and lipoproteins differs substantially among published studies. Moreover, this topic does not appear to have received consideration in exercise and health literature with normal, healthy children as subjects. The purpose of this study was, therefore, to determine the short-term, day-to-day variability of the lipid-lipoprotein profile from 19 children [mean (SD), 11.5 (0.8) years] from 3 separate venous blood samples. Intra-individual standard deviations, variances and coefficients of variance were determined for total cholesterol (TC), triacylglycerol (TG), high-density lipoprotein-cholesterol (HDL-C), HDL-C sub-fractions HDL2 and HDL3, and low-density lipoprotein-cholesterol (LDL-C). The intra-individual variation for TC and LDL-C was used to calculate the 95% confidence intervals (CIs) around the National Cholesterol Education Programme (NCEP 1991) cut-off points. The main finding was that all of the measured blood analytes including TC, TG, HDL-C, HDL2, HDL3, and LDL-C varied considerably from day-to-day. Coefficients of total variation ranged from 3.5% for HDL3 to 25.4% for TG. Classification of individuals using NCEP guidelines was difficult based on only one or two blood samples. The magnitude of variation for LDL-C meant that a 95% CI could not be constructed around the NCEP borderline-high classification from either one or two samples. However, averaging three TC and LDL-C measurements increased the likelihood of classification within the 95% CI. The results indicate that when using the NCEP guidelines for children and adolescents, true concentrations for TC and LDL-C should be based on the mean of multiple samples.     

干燥综合征合并特发性肺纤维化miRNA表达谱的变化及生物标志物的研究*

目的: 探究干燥综合征合并特发性肺纤维化患者血清中miRNA表达谱之间的差异关系。方法: 选择在云南省第一人民医院确诊为干燥综合征的3例患者作为对照组,平均年龄为(55.67±4.78) 岁,病程为(10.67±1.70)月;3例干燥综合征合并特发性肺纤维化患者作为观察组,平均年龄为(57.67±3.68) 岁,病程为(11.00±2.45) 月;6例患者均为女性。两组基本资料没有差异(P＞0.05)。利用芯片检测两组患者血清中miRNA表达谱的差异。通过GO富集分析,筛选出13个免疫细胞功能相关的聚类;表达差异明显的基因集中于免疫调节的信号通路。采用qRT-PCR验证其中5个表达有差异的miRNA。结果: 芯片检测结果共筛选出差异表达基因13个,其中6个miRNA上调:hsa-miR-6740-5p,hsa-miR-4507,hsa-miR-6775-5p,hsa-miR-4281,hsa-miR-4459,hsa-miR-6089,7个miRNA下调:hsa-miR-6873-3p,hsa-miR-4290,hsa-miR-6858-3p,hsa-miR-574-3p,hsa-miR-92b-3p,hsa-miR-3151-3p,hsa-miR-6886-3p。qRT-PCR结果验证了5个最明显的差异miRNA,结果和芯片趋势一致,差异显著,具有统计学意义。结论: 干燥综合征与干燥综合征合并特发性肺纤维化血清中miRNA表达存在差异,其中miR-6886-3p,miR-6873-3p,miR-574-3p,miR-6740-5p和miR-4507特异性和敏感度较高,可能作为干燥综合征特发性肺纤维化区别于原发性干燥综合征的生物标志物。     

Multiplexing RT-PCR for the detection of multiple miRNA species in small samples

MicroRNAs are short (approximately 22 nucleotides), non-coding RNAs that play critical roles in gene regulation and may be used as rapid precise diagnostic indicators of early stages of cancer. The small size of these RNAs makes detection of multiple microRNA species in very small samples problematic. Here we investigate the parameters associated with multiplexing RT-PCR to obtain relative abundance profiles of multiple microRNAs in small sample sizes down to the amount of RNA found in a single cell.     

循环miRNA 表达谱在肺癌早期诊断、判断预后和指导化疗中的应用

MicroRNA(miRNA)是一种高保守,长度大概21-23个核苷酸,非蛋白编码RNA,起着调节基因表达的作用。近年来有关miRNA与肺癌的关系已经得到证实,并且成为当前研究的热点。miRNA能整体调节基因表达,这使得miRNA表达谱在作为生物信号方面比蛋白编码基因更具有提示作用。最近发现miRNA以被保护的状态存在于循环血液中,这使得miRNA表达的发现具有非侵袭性、重现性以及易检测性。研究显示血浆miRNA表达谱可作为肺癌生物信号分子,在肺癌早期诊断、判断预后和指导化疗药物应用等方面具有重要作用。本文将对血浆miRNA与肺癌的研究进展,以及在肺癌早期诊断、判断预后和指导化疗药物应用等方面作一综述。     

一种利用分泌型荧光素酶基因表达变化监测活细胞中miRNA活性的新方法

建立了一种以分泌型的荧光素酶Gluc为报告基因的miRNA传感器质粒(命名为Gsensor)监测活细胞中miRNA(microRNA)活性的方法。首先构建了pAAV2neo-Gluc-MCS-polyA质粒作为Gsensor的空载体,同时其中的MCS位点可供插入miRNA的靶序列。以miR142-3p为检测对象,将1个和3个拷贝的与miR142-3p完全互补靶序列分别插入pAAV2neo-Gluc-MCS-polyA中,构建成miR142-3p Gsensor和miR142-3p Gsensor-3。将它们分别转染至U937细胞中,检测培养上清中Gluc的表达水平。结果显示二者均可有效反映U937细胞中miR142-3p的抑制活性(分别与Gsensor空载体相比),提示Gsensor中采用一个拷贝的miRNA靶序列即可满足检测要求。并且miR142-3p Gsensor也能有效地反映出Anti-miR142对miR142-3p活性的抑制作用。随后,分析了时间、转染剂量对Gsensor检测结果的影响。结果表明,在U937细胞中miR142-3p Gsensor表现的miR142-3p活性在48h后趋于稳定;Gsensor转染剂量在0.001~0.05pg/cell范围内不影响其功能。最后,利用miR142-3p Gsensor检测了HEK293、U937、K562、SP2/0和P815细胞内miR142-3p活性,结果发现miR142-3p活性在U937、K562、SP2/0和P815细胞中均较高,而在HEK293中几乎没有活性。用QRT-PCR方法检测miR142-3p的相对拷贝数。结果表明,在HEK293、U937和K562细胞中,miR142-3p活性与其相对拷贝数呈正相关。本研究表明Gsensor可作为一种有效的miRNA活性检测工具,为体外实时动态监测miRNA活性提供了一种新方法。     

四种乡土珍贵阔叶树种叶功能性状的种内和种间变异

植物功能性状种间性状变异反映不同物种的生活史对策,种内性状变异反映了同一物种不同个体应对不同环境的性状应答.人工林均一的环境有利于深入分析不同树种种内和种间变异.该研究以南宁良凤江林场的四种乡土珍贵阔叶树种[观光木(Tsoongiodendron odorum)、红锥(Castanopsis hystrix)、灰木莲(...     

Selenium levels in human plasma and hair in northern poland

The aim of this study was to (1) estimate the concentration of selenium in the plasma of 146 residents (65 men and 81 women) and in the hair of 34 persons from the Gdańsk region in northern Poland, aged 19–70 and (2) compare the obtained results with data corresponding to healthy populations living in different European countries. Selenium in plasma was determined by atomic absorption spectrometry using the hydride generation method. The mean selenium concentration in plasma of the investigated persons was 73.3 ± 14.1 μg/L, 76.7 ± 13.2 μg/L in men, and 70.4 ± 14.7 μg/L in women. No age — dependent differences in plasma selenium were found in the investigated population. In 20% of the investigated persons, the selenium level in plasma was lower than 60 μg/L. The mean selenium concentration in hair was 0.30 ± 0.11 μg/g. A positive, statistically significant correlation between selenium concentrations in the plasma and hair of the investigated persons was found. The obtained results indicate that the selenium level in significant part of this population is suboptimal and should be elevated by supplementation with this element.     

Large-scale information entropy analysis of important sites in mature and precursor miRNA sequences

In order to find evidence of consistent sequence conservation or the base correlation degree in miRNA, some important sites in the sequences of reported miRNA and their precursors (pre-miRNA) were in-vestigated via information entropy analysis. Twelve different groups of sites were obtained from special locations (head, tail) in miRNAs of different sources according to taxonomy (animal, plant and virus) and then analyzed by measuring the single base information redundancy (D₁(L)) and the adjacent base related information redundancy (D₂(L)). The results showed that D₂(L) has more information than D₁(L), though D₁(L) changes roughly consistently with D₂(L) in each group. Viral pre-miRNAs are more con-servative than those belonging to animals or plants. In addition, U is dominant in most sites compared with other nucleotides. It was also found that in the middle of several groups, there were sites where miRNAs were cut down from pre-miRNAs by Enzyme Dicer which were significantly conservative. This phenomenon shows that the conservatism is an aspect of the of miRNA and may be involved in the recognition and cutting by the Dicer. Those results provided another perspective for understanding more about the primary structure of pre-miRNA.     

Composition of the peptide fraction in human blood plasma: database of circulating human peptides

A database was established from human hemofiltrate (HF) that consisted of a mass database and a sequence database, with the aim of analyzing the composition of the peptide fraction in human blood. To establish a mass database, all 480 fractions of a peptide bank generated from HF were analyzed by MALDI-TOF mass spectrometry. Using this method, over 20 000 molecular masses representing native, circulating peptides were detected. Estimation of repeatedly detected masses suggests that approximately 5000 different peptides were recorded. More than 95% of the detected masses are smaller than 15 000, indicating that HF predominantly contains peptides. The sequence database contains over 340 entries from 75 different protein and peptide precursors. 55% of the entries are fragments from plasma proteins (fibrinogen A 13%, albumin 10%, β2-microglobulin 8.5%, cystatin C 7%, and fibrinogen B 6%). Seven percent of the entries represent peptide hormones, growth factors and cytokines. Thirty-three percent belong to protein families such as complement factors, enzymes, enzyme inhibitors and transport proteins. Five percent represent novel peptides of which some show homology to known peptide and protein families. The coexistence of processed peptide fragments, biologically active peptides and peptide precursors suggests that HF reflects the peptide composition of plasma. Interestingly, protein modules such as EGF domains (meprin Aα-fragments), somatomedin-B domains (vitronectin fragments), thyroglobulin domains (insulin like growth factor-binding proteins), and Kazal-type inhibitor domains were identified. Alignment of sequenced fragments to their precursor proteins and the analysis of their cleavage sites revealed that there are different processing pathways of plasma proteins in vivo.     

Odontometric variation within and between taxonomic levels of cercopithecidae: Implications for interpetations of fossil samples

This study examines metric variation of the post-canine permanent dentition of four monkey groups (savannah and forest baboons, savannah and forest guenons) represented by 1,840 individuals.Papio, Mandrillus, Cercopithecus aethiops, andC. mitis each display marked within-group variation in tooth size. Taxonomic status and/or sexual composition of a sample does not correspond to a particular magnitude of variation. Therefore, the use of the coefficient of variation to assess either the specific or sexual composition of fossil samples is untenable.     

Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing

There has been a growing interest in using next-generation sequencing (NGS) to profile extracellular small RNAs from the blood and cerebrospinal fluid (CSF) of patients with neurological diseases, CNS tumors, or traumatic brain injury for biomarker discovery. Small sample volumes and samples with low RNA abundance create challenges for downstream small RNA sequencing assays. Plasma, serum, and CSF contain low amounts of total RNA, of which small RNAs make up a fraction. The purpose of this study was to maximize RNA isolation from RNA-limited samples and apply these methods to profile the miRNA in human CSF by small RNA deep sequencing. We systematically tested RNA isolation efficiency using ten commercially available kits and compared their performance on human plasma samples. We used RiboGreen to quantify total RNA yield and custom TaqMan assays to determine the efficiency of small RNA isolation for each of the kits. We significantly increased the recovery of small RNA by repeating the aqueous extraction during the phenol-chloroform purification in the top performing kits. We subsequently used the methods with the highest small RNA yield to purify RNA from CSF and serum samples from the same individual. We then prepared small RNA sequencing libraries using Illumina’s TruSeq sample preparation kit and sequenced the samples on the HiSeq 2000. Not surprisingly, we found that the miRNA expression profile of CSF is substantially different from that of serum. To our knowledge, this is the first time that the small RNA fraction from CSF has been profiled using next-generation sequencing.     

CID-miRNA: a web server for prediction of novel miRNA precursors in human genome

microRNAs (miRNA) are a class of non-protein coding functional RNAs that are thought to regulate expression of target genes by direct interaction with mRNAs. miRNAs have been identified through both experimental and computational methods in a variety of eukaryotic organisms. Though these approaches have been partially successful, there is a need to develop more tools for detection of these RNAs as they are also thought to be present in abundance in many genomes. In this report we describe a tool and a web server, named CID-miRNA, for identification of miRNA precursors in a given DNA sequence, utilising secondary structure-based filtering systems and an algorithm based on stochastic context free grammar trained on human miRNAs. CID-miRNA analyses a given sequence using a web interface, for presence of putative miRNA precursors and the generated output lists all the potential regions that can form miRNA-like structures. It can also scan large genomic sequences for the presence of potential miRNA precursors in its stand-alone form. The web server can be accessed at http://mirna.jnu.ac.in/cidmirna/.     

Mechanical stretch regulates the expression of specific miRNA in extracellular vesicles released from lung epithelial cells

The underlying mechanism of normal lung organogenesis is not well understood. An increasing number of studies are demonstrating that extracellular vesicles (EVs) play critical roles in organ development by delivering microRNAs (miRNA) to neighboring and distant cells. miRNAs are important for fetal lung growth; however, the role of miRNA–EVs (miRNAs packaged inside the EVs) during fetal lung development is unexplored. The aim of this study was to examine the expression of miRNA–EVs in MLE-12, a murine lung epithelial cell line subjected to mechanical stretch in vitro with the long-term goal to investigate their potential role in the fetal lung development. Both cyclic and continuous mechanical stretch regulate miRNA differentially in EVs released from MLE-12 and intracellularly, demonstrating that mechanical signals regulate the expression of miRNA–EVs in lung epithelial cells. These results provide a proof-of-concept for the potential role that miRNA–EVs could play in the development of fetal lung.     

Cross‐sectional relations of whole‐blood miRNA expression levels and hand grip strength in a community sample

MicroRNAs (miRNAs) regulate gene expression with emerging data suggesting miRNAs play a role in skeletal muscle biology. We sought to examine the association of miRNAs with grip strength in a community‐based sample. Framingham Heart Study Offspring and Generation 3 participants (n = 5668 54% women, mean age 55 years, range 24, 90 years) underwent grip strength measurement and miRNA profiling using whole blood from fasting morning samples. Linear mixed‐effects regression modeling of grip strength (kg) versus continuous miRNA ‘Cq’ values and versus binary miRNA expression was performed. We conducted an integrative miRNA–mRNA coexpression analysis and examined the enrichment of biologic pathways for the top miRNAs associated with grip strength. Grip strength was lower in women than in men and declined with age with a mean 44.7 (10.0) kg in men and 26.5 (6.3) kg in women. Among 299 miRNAs interrogated for association with grip strength, 93 (31%) had FDR q value < 0.05, 54 (18%) had an FDR q value < 0.01, and 15 (5%) had FDR q value < 0.001. For almost all miRNA–grip strength associations, increasing miRNA concentration is associated with increasing grip strength. miR‐20a‐5p (FDR q 1.8 × 10^?6) had the most significant association and several among the top 15 miRNAs had links to skeletal muscle including miR‐126‐3p, miR‐30a‐5p, and miR‐30d‐5p. The top associated biologic pathways included metabolism, chemokine signaling, and ubiquitin‐mediated proteolysis. Our comprehensive assessment in a community‐based sample of miRNAs in blood associated with grip strength provides a framework to further our understanding of the biology of muscle strength.     

Sources of individual variation in plasma testosterone levels

The steroid hormone testosterone (T) plays a central role in the regulation of breeding in males, because many physiological, morphological and behavioural traits related to reproduction are T dependent. Moreover, in many seasonally breeding vertebrates, male plasma T levels typically show a pronounced peak during the breeding season. While such population-level patterns are fairly well worked out, the sources and the implications of the large variability in individual T levels within the seasonal cycle remain surprisingly little understood. Understanding the potential sources of individual variation in T levels is important for behavioural and evolutionary ecologists, for at least two reasons. First, in 'honest signalling' theory, T is hypothesized to play a critical role as the assumed factor that enforces honesty of the expression of sexually selected quality indicators. Second, T is often considered a key mediator of central life-history trade-offs, such as investment in survival versus reproduction or in mating versus parental care. Here, we discuss the patterns of within- and between-individual variation in male plasma T levels in free-living populations of birds. We argue that it is unclear whether this variability mainly reflects differences in underlying individual quality (intrinsic factors such as genetic or maternal effects) or in the environment (extrinsic factors including time of day, individual territorial status and past experience). Research in avian behavioural endocrinology has mainly focused on the effects of extrinsic factors, while other sources of variance are often ignored. We suggest that studies that use an integrative approach and investigate the relative importance of all potential sources of variation are essential for the interpretation of data on individual plasma T levels.     

应用双荧光报告系统检测斑马鱼microRNA的动态表达

microRNA(miRNA)是一类细胞内源表达的小分子非编码RNA, 主要通过降解靶基因的mRNA或者抑制靶基因的翻译, 在动植物的发育以及其他重要的生理过程中起调控作用。miRNA的功能跟它的表达位置与时间密切相关, 但是目前尚缺乏一个能够在活体与个体水平稳定、持续地实时观察miRNA动态表达的方法。文章以斑马鱼为模式, 建立了一个双荧光报告系统(我们称之为miRNA Tracer), 用于在斑马鱼整体胚胎中追踪特定miRNA的表达谱及动态变化过程。该系统以Tol2转座子为基础, 采用来自斑马鱼hsp70基因的热激启动子分别驱动eGFP和mRFP1荧光报告基因, 同时在其中一个报告基因的3′-UTR区连接待测miRNA的互补序列, 构成Tracer质粒。该互补序列与斑马鱼胚胎中相应的内源miRNA结合后能够使对应报告基因的荧光信号强度减弱, 通过比较两个报告基因在表达谱上的差异辨别miRNA的表达区域, 检测斑马鱼胚胎中miRNA起作用的位置和时间。文章选择在肌肉系统特异表达的miR-206以及在神经系统特异表达的miR-219, 分别在显微注射瞬时表达和转基因稳定整合等两个层次上验证了上述Tracer系统。结果表明, 所用的方法能够如实地在单细胞水平和整体水平检测到目标miRNA的时空表达动态变化。miRNA Tracer系统为在斑马鱼发育过程中对miRNA进行活体、实时的时空定位提供了一个独特而有效的方法, 也为对miRNA进行功能与作用机制等更深入的研究奠定了基础。

补充资料

s219mRFP1-dF转基因胚胎的3-D图像 [视频]