克隆差异表达基因的新策略

基因表达的变化有两种,即新出现的基因表达与表达量差异的基因表达.表达量差异的基因克隆技术主要有mRNA差异展示,此技术是目前筛选差异表达基因最有效的方法之一,但主要存在假阳性率高的不足,针对此缺点,近几年提出了新的策略与方法,如差异消减展示、基于PCR和减法杂交基础上的差异表达基因克隆技术,这些技术具有显著优势.     

Leukocyte isolation by sedimentation: the effect of rouleau-promoting agents on leukocyte differential count.

The rouleau-promoting agents dextran and polyvinylpyrrolidone (PVP) were used to accelerate erythrocyte sedimentation in order to harvest the leukocyte rich plasma (LRP). The objective of the work was to determine if agent concentration or blood: agent ratio had any effect on the leukocyte differential count and if so at what agent concentration and agent:blood ratio did the LRP leukocyte differential count most closely match the whole blood leukocyte differential count. With both sedimentation agents the data clearly indicate that both parameters effect LRP differential counts and that low concentrations of sedimentation agents are most important in obtaining LRP differential counts which most closely match the whole blood differential counts.     

Modelling inert gas exchange in tissue and mixed-venous blood return to the lungs

Inert gas exchange in tissue has been almost exclusively modelled by using an ordinary differential equation. The mathematical model that is used to derive this ordinary differential equation assumes that the partial pressure of an inert gas (which is proportional to the content of that gas) is a function only of time. This mathematical model does not allow for spatial variations in inert gas partial pressure. This model is also dependent only on the ratio of blood flow to tissue volume, and so does not take account of the shape of the body compartment or of the density of the capillaries that supply blood to this tissue. The partial pressure of a given inert gas in mixed-venous blood flowing back to the lungs is calculated from this ordinary differential equation. In this study, we write down the partial differential equations that allow for spatial as well as temporal variations in inert gas partial pressure in tissue. We then solve these partial differential equations and compare them to the solution of the ordinary differential equations described above. It is found that the solution of the ordinary differential equation is very different from the solution of the partial differential equation, and so the ordinary differential equation should not be used if an accurate calculation of inert gas transport to tissue is required. Further, the solution of the PDE is dependent on the shape of the body compartment and on the density of the capillaries that supply blood to this tissue. As a result, techniques that are based on the ordinary differential equation to calculate the mixed-venous blood partial pressure may be in error.     

Differential fertility as a major mode of selection

Population genetic models have focused Primarily on the differential survival of genotypes as the vehicle through which selection can lead to evolutionary change. A growing body of diverse evidence suggests that differential fertility is an equally important mode of selective action. Laboratory studies with Drosophila and field studies in natural populations of plants and animals have provided direct demonstrations. Experimental ecology and reproductive biology have offered indirect but compelling evidence for the importance of differential fertility. Selection through differential fertility is not always interchangeable with selection through differential survival: the genetic dynamics can be far more complicated and can lead to unpredictable results when driven by fertility differences. The study of fertility selection in natural populations poses several problems: selection differentials can be difficult to estimate; they are likely to be very sensitive to changes in environmental conditions, and when generations overlap the fertility differential cannot be estimated correctly without demographic data. Describing the role of differential fertility in natural populations represents an emerging challenge for theoreticians and empiricists alike.     

Circular differential scattering and circular differential absorption of DNA-protein condensates and of dyes bound to DNA-protein condensates

DNA-protein condensates that give positive and negative psi-type circular dichroism (CD) spectra (psi condensates) bind intercalative and nonintercalative dyes. CD depends both on circular differential scattering and on circular differential absorption; scattering-corrected CD measurements are approximations to circular differential absorption. The circular differential scattering and scattering-corrected CD patterns observed in the DNA absorption band of psi condensates are mimicked in the induced CD band of intercalators bound to psi condensates. The induced scattering-corrected CD and circular differential scattering patterns of the groove-binding dye Hoechst 33342 bound to psi condensates are the inverse of the patterns seen with intercalative dyes, whereas the groove-binding dye manganese(III) meso-tetrakis(4-N-methylpyridyl)porphine [MnIIITMpyP-4] shows no significant induced CD patterns. The large circular differential scattering and scattering-corrected CD bands are interpreted as resulting from long-range chiral packing, rather than near-neighbor short-range interactions. Dyes intercalated into the DNA of the psi condensates have the same type of long-range chiral packing as the DNA bases. Therefore, the psi-type CD spectra seen in the UV spectra originating from the long-range packing of the DNA bases are also observed in the visible spectra when dyes are intercalated in the DNA of the psi condensates. Our interpretation comes from the observation that the induced circular differential scattering and circular differential absorption of the dye bound to the psi condensates depend only upon the sign of the circular differential absorption and the pattern of the circular differential scattering of the psi condensates without bound dye.(ABSTRACT TRUNCATED AT 250 WORDS)     

芥蓝不定芽发生过程的基因表达差异分析

采用cDNA-AFLP技术和双向电泳技术对不同培养基上的‘中花’芥蓝(Brassicaoleraceavar.alboglabracv.Zhonghua)下胚轴外植体愈伤组织和不定芽发生的基因表达差异进行研究和分析。结果表明:芥蓝不定芽发生基因差异在RNA水平上有所体现,特异条带主要出现在产生不定芽的外植体上,且主要集中在200～600bp之间;芥蓝不定芽发生的基因表达差异在蛋白质水平有很大差异,发生不定芽的外植体和不发生不定芽的外植体出现了160种蛋白质的差异点,差异蛋白的pI值多在5～7之间,在这个范围内的差异蛋白点达到了122个,占差异蛋白总数的76.25%,差异蛋白的分子量多在40～70kDa之间,在这个范围内的差异蛋白点达到了124个,占差异蛋白总数的77.5%。     

生物信息学基因表达差异分析

基因的差异化表达由多种因素共同导致,并且与许多疾病的发生和发展有密切联系,对差异化表达的基因进行生物信息学以及生物统计学的分析对于研究细胞调节机制和疾病机理有着重要意义。目前,对差异化表达的基因有以下几种主流的研究方法：DNA微阵列（DNA microarray）,抑制性消减杂交（SSH）,基因表达连续性分析（SAGE）,代表性差异分析（RDA）,以及mRNA差异显示PCR（mRNA DDRT-PCR）。目前许多基因差异化表达数据是建立在时段（time series）基础上,因此对基于时间变化的基因差异化表达分析变得尤为重要。本文将对差异化表达基因的几种主流方法进行详细阐述,并介绍一种基于傅里叶函数的时段基因差异化表达分析。     

Different effects of 33258 Hoechst and DAPI in fluorescent staining of sister chromatids differentially substituted with bromodeoxyuridine

Summary After substitution with 5-bromodeoxyuridine (BrdUrd) for two rounds of replication, chromosomes in cytological preparations stained with 33258 Hoechst show upon epiluminescence an immediate differential sister chromatid fluorescence. When stained with DAPI, however, which has a structural resemblance to part of the 33258 Hoechst molecule, such a differential pattern of fluorescence was only induced after some delay. Upon restaining with the same dye the differential fluorescence appeared instantly. In preparations double stained with ethidium bromide and 33258 Hoechst the induction of a differential staining of sister chromatids with 33258 Hoechst was not accompanied by a differential staining with ethidium bromide. Once a differential staining was obtained with DAPI in preparations double stained with ethidium bromide and DAPI, the ethidium bromide pattern also appeared to be differential upon subsequent observation. No differentiation could be obtained with ethidium bromide alone. The observations described in the case of 33258 Hoechst staining are in agreement with a molecular quenching by BrdUrd without gross structural consequences for the DNA. In the case of DAPI staining, however, there occurs a differential photolysis of BrdUrd-substituted DNA. Besides the nature, most likely the size, of the fluorochrome molecules themselves, the state of the fixed chromatin appeared also to play a role in determining the mechanism of the sister chromatid differentiation: after prolonged incubation in buffer, BrdUrd-containing chromosomes stained with 33258 Hoechst showed a differential staining evidently caused by photolysis, indicating that they had become more susceptible to light.     

The routine differential leukocyte count vs automated differential counts

The clinical use of the proposed performance standards for differential leukocyte counts is determined by multiple factors. Their use must be considered according to the specific use of the differential count, the sources of variability in differential counting, the relation between specific use and sources of variability, the role of analytic errors in the detection of nonspecific changes, the use of qualitative vs quantitative data, the sensitivity and specificity of the routine eye count, the role of disease or specific cell prevalence in determination of predictive value, the effect on use of automated instruments for screening, and whether abnormal specimen flagging can be done. The routine eye-count differential method, as performed by a well-trained technologist or technician, seems to lack both sensitivity and specificity. Because of the magnitude of technique- and method-related and biologic sources of variability, the 100- or 200-cell eye-count differential method is not a good screening method for detection of hematologic illnesses, particularly those that are uncommon. The automated differential leukocyte instruments address many of the technique- and method-related errors and are thus able to equal or exceed the performance of the routine eye-count differential method.     

Differential-Evolution Control Parameter Optimization for Unmanned Aerial Vehicle Path Planning

The differential evolution algorithm has been widely applied on unmanned aerial vehicle (UAV) path planning. At present, four random tuning parameters exist for differential evolution algorithm, namely, population size, differential weight, crossover, and generation number. These tuning parameters are required, together with user setting on path and computational cost weightage. However, the optimum settings of these tuning parameters vary according to application. Instead of trial and error, this paper presents an optimization method of differential evolution algorithm for tuning the parameters of UAV path planning. The parameters that this research focuses on are population size, differential weight, crossover, and generation number. The developed algorithm enables the user to simply define the weightage desired between the path and computational cost to converge with the minimum generation required based on user requirement. In conclusion, the proposed optimization of tuning parameters in differential evolution algorithm for UAV path planning expedites and improves the final output path and computational cost.     

差异显示的改进及其在木本植物研究中的应用

从差异显示的基本原理出发,概述了差显的优缺点及其近年来在实验方法上的改进,并简要介绍了差显在植物尤其是木本植物研究中的应用。     

DHPLC对猪肌肉组织差异表达EST的鉴定

采用DHPLC系统和Labwork4．0图像分析软件,对大白猪和广东地方品种蓝塘猪眼肌组织差异显示EST进行鉴定,两种方法均证实了差异显示条带的真实性。结果表明：用DHPLC系统能够准确而简捷的检测不同组织中mRNA表达的差异,是1种鉴定基因表达差异的有效方法,在研究基因表达,比较不同环境条件下动物组织的mRNA表达差异等方面具有广阔的应用前景。     

Identification of lncRNA‐associated differential subnetworks in oesophageal squamous cell carcinoma by differential co‐expression analysis

Differential expression analysis has led to the identification of important biomarkers in oesophageal squamous cell carcinoma (ESCC). Despite enormous contributions, it has not harnessed the full potential of gene expression data, such as interactions among genes. Differential co‐expression analysis has emerged as an effective tool that complements differential expression analysis to provide better insight of dysregulated mechanisms and indicate key driver genes. Here, we analysed the differential co‐expression of lncRNAs and protein‐coding genes (PCGs) between normal oesophageal tissue and ESCC tissues, and constructed a lncRNA‐PCG differential co‐expression network (DCN). DCN was characterized as a scale‐free, small‐world network with modular organization. Focusing on lncRNAs, a total of 107 differential lncRNA‐PCG subnetworks were identified from the DCN by integrating both differential expression and differential co‐expression. These differential subnetworks provide a valuable source for revealing lncRNA functions and the associated dysfunctional regulatory networks in ESCC. Their consistent discrimination suggests that they may have important roles in ESCC and could serve as robust subnetwork biomarkers. In addition, two tumour suppressor genes (AL121899.1 and ELMO2), identified in the core modules, were validated by functional experiments. The proposed method can be easily used to investigate differential subnetworks of other molecules in other cancers.     

Male specific genes from dioecious white campion identified by fluorescent differential display

Fluorescent differential display (FDD) has been used to screen for cDNAs that are differentially up-regulated in male flowers of the dioecious plant Silene latifolia in which an X/Y chromosome system of sex determination operates. To adapt FDD to the cloning of large numbers of differential cDNAs, a novel method of confirming the differential expression of these has been devised. FDD gels were Southern electro-blotted and probed with mixtures of individual cDNA clones derived from different FDD product ligation reactions. These Southern blots were then stripped and re-probed with further mixtures of individual cloned FDD products to identify the maximum number of recombinant clones carrying the true differential amplification products. Of 135 differential bands identified by FDD, 56 differential amplification products were confirmed; these represent 23 unique differentially expressed genes as determined by virtual Northern analysis and two genes expressed at or below the level of detection by virtual Northern analysis. These two low expressed genes show bands of hybridization on genomic Southern blots that are specific to male plants, indicating that they are derived from, or closely related to, Y chromosome genes.     

Differential display: Identifying genes involved in cardiomyocyte proliferation

An estimated 15,000 different mRNA species are expressed in a typical mammalian cell. The differential expression of mRNAs in both a temporal and cell-specific manner determines the fate of the cell and creates the organism. Analysis of this differential gene expression has become a central aim of many laboratories attempting to understand the mechanisms underlying various biological processes. Currently, we are using a technique called differential display to analyze the differential expression of genes in cardiomyocytes. Differential display is a rapid and powerful technique that was introduced by Liang and Pardee in 1992. Since that time, it has been successfully applied by several groups, and it is quickly becoming a standard method for studying differential gene expression. Here, we present a detailed article discussing the differential display methodology and how we have utilized it to identify potential genes involved in cardiomyocyte proliferation. Furthermore, we have provided a list of materials and supplied examples of data obtained, in an effort to allow the reader to perform the technique with success in their own laboratory.     

A quantitative dynamical systems approach to differential learning: self-organization principle and order parameter equations

Differential learning is a learning concept that assists subjects to find individual optimal performance patterns for given complex motor skills. To this end, training is provided in terms of noisy training sessions that feature a large variety of between-exercises differences. In several previous experimental studies it has been shown that performance improvement due to differential learning is higher than due to traditional learning and performance improvement due to differential learning occurs even during post-training periods. In this study we develop a quantitative dynamical systems approach to differential learning. Accordingly, differential learning is regarded as a self-organized process that results in the emergence of subject- and context-dependent attractors. These attractors emerge due to noise-induced bifurcations involving order parameters in terms of learning rates. In contrast, traditional learning is regarded as an externally driven process that results in the emergence of environmentally specified attractors. Performance improvement during post-training periods is explained as an hysteresis effect. An order parameter equation for differential learning involving a fourth-order polynomial potential is discussed explicitly. New predictions concerning the relationship between traditional and differential learning are derived.     

含扩散和时滞的偏微分方程解的振动性

研究一类含扩散和时滞的偏微分方程解的振动性,利用平均法,通过使用偏泛函微分方程上、下解思想和泛函微分方程振动性理论,获得了其解的非负性和关于正平衡态振动的充分条件．     

Comparison of brush border membrane vesicles prepared by three methods from larval Manduca sexta midgut

Brush border membrane vesicles (BBMV) were prepared from freshly isolated posterior larval Manduca sexta midguts by differential calcium precipitation, differential magnesium precipitation and differential ultrasonication. BBMV were also prepared from frozen posterior larval M. sexta midguts by differential calcium precipitaion and differential magnesium precipitation. The yield of BBMV by both differential precipitation methods was 5–6 times greater than that by the differential ultrasonification method. Enrichments of the brush border membrane marker enzymes alkaline phosphatase, γ-glutamyl transferase, and aminopeptidase were similar in all preparations. The polypeptide composition of all preparations was also similar. The specific activity of mitochondrial and microsomal marker enzymes was higher in BBMV prepared from freshly isolated midguts by the differential precipitation methods than in BBMV prepared by the ultrasonication method. The specific activity of cytochrome-c oxidase was 2.5–7 times higher in BBMV prepared from frozen midguts than in BBMV prepared from fresh tissue.