人类免疫缺陷病毒潜伏库定量检测技术的研究进展

以静息CD4~+T细胞为主的人类免疫缺陷病毒(human immunodeficiency virus,HIV)潜伏库的清除已成为治愈HIV-1感染的主要障碍,人们迫切需要建立一种高通量、可靠的、高灵敏度的方法来定量检测病毒潜伏库的真实大小。本文就目前关于HIV潜伏库的多种定量检测方法进行综述。     

Protein markers of Bursaphelenchus xylophilus Steiner & Buhrer, 1934 (Nickle, 1970) populations using quantitative proteomics and character compatibility

The Pine Wood Nematode (PWN) Bursaphelenchus xylophilus is a severe forest pathogen in countries where it has been introduced and is considered a worldwide quarantine organism. In this study, protein markers for differentiating populations of this nematode were identified by studying differences among four selected Iberian and one American population. These populations were compared by quantitative proteomics (iTRAQ). From a total of 2860 proteins identified using the public database from the B. xylophilus genome project, 216 were unambiguous and significantly differentially regulated in the studied populations. Comparisons of their pairwise ratio were statistically treated and supported in order to convert them into discrete character states, suggesting that 141 proteins were not informative as population specific markers. Application of the Character Compatibility methodology on the remaining 75 proteins (belonging to families with different biological functions) excludes 27 which are incompatible among them. Considering only the compatible proteins, the method selects a subset of 30 specific unique protein markers which allowed the compared classification of the Iberian isolates. This approach makes it easier search for diagnostic tools and phylogenetic inference within species and populations of a pathogen exhibiting a high level of genetic diversity.     

Evaluation of dental enamel caries assessment using Quantitative Light Induced Fluorescence and Optical Coherence Tomography

An in vitro study of morphological alterations between sound dental structure and artificially induced white spot lesions in human teeth, was performed through the loss of fluorescence by Quantitative Light‐Induced Fluorescence (QLF) and the alterations of the light attenuation coefficient by Optical Coherence Tomography (OCT). To analyze the OCT images using a commercially available system, a special algorithm was applied, whereas the QLF images were analyzed using the software available in the commercial system employed. When analyzing the sound region against white spot lesions region by QLF, a reduction in the fluorescence intensity was observed, whilst an increase of light attenuation by the OCT system occurred. Comparison of the percentage of alteration between optical properties of sound and artificial enamel caries regions showed that OCT processed images through the attenuation of light enhanced the tooth optical alterations more than fluorescence detected by QLF System.

QLF versus OCT imaging of enamel caries: a photonics assessment     

金鱼草中央细胞中线粒体DNA拷贝数的定量

为探究金鱼草(Antirrhinum majus)中央细胞的线粒体DNA拷贝数,采用竞争型定量PCR技术进行了测定。结果表明,金鱼草中央细胞的体积为(61570±732)μm~3,平均携带(783±25)个拷贝的线粒体DNA,并且中央细胞与卵细胞具有相似的体积/线粒体DNA拷贝数比值。推测金鱼草中央细胞包含如此高丰度线粒体DNA可能是为早期胚乳细胞的发育而储备的。     

基于质谱的蛋白质N末端乙酰化程度定量方法的研究

随着质谱技术及各种定量方法的不断完善和发展,定量蛋白质组学的方法不断地被应用到各类生物学研究中。蛋白质组学定性定量数据的处理主要通过一些多功能的商业化或者开源软件来进行,如常用的数据分析软件Proteome Discoverer和Maxquant。但是在通过化学标记对蛋白质N末端乙酰化程度进行定量这一方面,Proteome Discoverer和Maxquant在一定程度上存在准确性不高和完整度不够的问题。于是本研究针对自己的实验特点,通过Java算法编写了相应的定量程序Acequant来完成N末端乙酰化程度的相对定量。本研究将该程序在已有相关报道的He La cell上进行了验证,Acequant共定量到1 587个蛋白质N末端,而Proteome Discoverer和Maxquant分别只定量到42个和306个N末端。同时,手动验证原始图谱也证实了Acequant定量的准确性更好。于是,本研究将此方法进一步应用到秀丽隐杆线虫N末端乙酰化的研究中,并初步发现了线虫整体的N末端乙酰化状态,为进一步的N末端研究提供了支持。     

铁皮石斛GRAS转录因子家族基因SCL3克隆及表达分析

GRAS家族是一类植物特有的转录调控因子,参与调控植物生长发育及抵御逆境胁迫,并且在赤霉素(gibberellins, GAs)信号转导过程中具有重要作用。本研究采用RT-PCR结合RACE方法从铁皮石斛(Dendrobium officinale Kimura et Migo)原球茎中克隆到了一个转录因子GRAS家族基因SCL3 (scarecrow-like 3)并对其进行了表达分析。该基因cDNA序列全长2 278 bp,命名为DoSCL3 (GenBank注册号:MG252261),其编码425个氨基酸,分子量为47.88 kD,等电点(PI)为6.21。DoSCL3基因编码的蛋白不含跨膜域和信号肽,具有GRAS转录因子家族特有的保守结构域(22~422);多序列比对表明,DoSCL3与小兰屿蝴蝶兰的SCL3蛋白高度同源(相似性达到83%),同时进化树分析结果显示其与小兰屿蝴蝶兰亲缘关系非常相近。qRT-PCR实验结果显示,DoSCL3基因在铁皮石斛种子共生萌发中随着萌发进程的变化(1~3级,萌发至原球茎阶段)其表达量逐渐升高,并且在种子萌发至2级时,共生萌发组表达量显著高于无菌萌发组(为无菌组2.2倍),表明DoSCL3在兰科药用植物铁皮石斛种子共生萌发中菌根共生关系的建立过程起到重要的调控作用。     

Identification of putative G-quadruplex DNA structures in S. pombe genome by quantitative PCR stop assay

In order to understand in which biological processes the four-stranded G-quadruplex (G4) DNA structures play a role, it is important to determine which predicted regions can actually adopt a G4 structure. Here, to identify DNA regions in Schizosaccharomyces pombe that fold into G4 structures, we first optimized a quantitative PCR (qPCR) assay using the G4 stabilizer, PhenDC3. We call this method the qPCR stop assay, and used it to screen for G4 structures in genomic DNA. The presence of G4 stabilizers inhibited DNA amplification in 14/15 unexplored genomic regions in S. pombe that encompassed predicted G4 structures, suggesting that at these sites the stabilized G4 structure formed an obstacle for the DNA polymerase. Furthermore, the formation of G4 structures was confirmed by complementary in vitro assays. In vivo, the S. pombe G4 unwinder Pif1 helicase, Pfh1, was associated with tested G4 sites, suggesting that the G4 structures also formed in vivo. Thus, we propose that the confirmed G4 structures in S. pombe form an obstacle for replication in vivo, and that the qPCR stop assay is a method that can be used to identify G4 structures. Finally, we suggest that the qPCR stop assay can also be used for identifying G4 structures in other organisms, as well as being adapted to screen for novel G4 stabilizers.     

Quantitative structure analysis of genetic diversity among spring bread wheats (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) from different geographical regions

Genetic diversity in spring bread wheat (T.␣aestivum L.) was studied in a total of 69 accessions. For this purpose, 52 microsatellite (SSR) markers were used and a total of 406 alleles were detected, of which 182 (44.8%) occurred at a frequency of <5% (rare alleles). The number of alleles per locus ranged from 2 to 14 with an average of 7.81. The largest number of alleles per locus occurred in the B genome (8.65) as␣compared to the A (8.43) and D (5.93) genomes, respectively. The polymorphism index content (PIC) value varied from 0.24 to 0.89 with an average of 0.68. The highest PIC for all accessions was found in the B␣genome (0.71) as compared to the A (0.68) and D␣genomes (0.63). Genetic distance-based method (standard UPGMA clustering) and a model-based method (structure analysis) were used for cluster analysis. The two methods led to analogical results. Analysis of molecular variance (AMOVA) showed that 80.6% of the total variation could be explained by the variance within the geographical groups. In comparison to the diversity detected for all accessions (H _e = 0.68), genetic diversity among European spring bread wheats was H _e = 0.65. A comparatively higher diversity was observed between wheat varieties from Southern European countries (Austria/Switzerland, Portugal/Spain) corresponding to those from other regions.