Clustering Time-Series Gene Expression Data Using Smoothing Spline Derivatives

Microarray data acquired during time-course experiments allow the temporal variations in gene expression to be monitored. An original postprandial fasting experiment was conducted in the mouse and the expression of 200 genes was monitored with a dedicated macroarray at 11 time points between 0 and 72 hours of fasting. The aim of this study was to provide a relevant clustering of gene expression temporal profiles. This was achieved by focusing on the shapes of the curves rather than on the absolute level of expression. Actually, we combined spline smoothing and first derivative computation with hierarchical and partitioning clustering. A heuristic approach was proposed to tune the spline smoothing parameter using both statistical and biological considerations. Clusters are illustrated a posteriori through principal component analysis and heatmap visualization. Most results were found to be in agreement with the literature on the effects of fasting on the mouse liver and provide promising directions for future biological investigations.     

Cell Cycle Regulation Targets of MYCN Identified by Gene Expression Microarrays

Background: We have previously shown that MYCN knockdown causes a G1 arrest in MYCN amplified (MNA), p53 wild type (wt) and p53 mutant MNA neuroblastoma cell lines, with increases in p21WAF1 and hypo RB in p53 wt cell lines. 1 Hypothesis: MYCN acts by inhibiting p21WAF1, and also by p21 independent mechanisms to override the G1 checkpoint in exponentially growing cells. Methods: Genes potentially regulated by MYCN were identified using gene expression microarrays in p53 wt MNA IMR-32 and p53 mutant MNA SKNBE(2c) neuroblastoma cell lines treated with MYCN or scrambled siRNA. Results were validated using qRT-PCR and confirmed using the regulatable MYCN expression system (SHEP Tet21N). Results: MYCN knockdown altered the expression of several cell cycle related genes. SKP2 was down regulated in both cell lines, and up regulated in MYCN+ Tet21N cells. Expression of the WNT antagonist DKK3 increased in both cell lines and decreased in MYCN+ Tet21N cells. Expression of CDKN1C (p57cip2) and TP53INP1 also increased after MYCN knockdown. Conclusions: MYCN may override the G1 checkpoint through down-regulation of SKP2 and TP53INP1 resulting in reduced p21WAF1 expression in p53 wt cell lines, and in addition may act through the WNT signalling pathway in a p53 independent manner.     

Matching Patterns of Gene Expression to Mechanical Stiffness at Cell Resolution through Quantitative Tandem Epifluorescence and Nanoindentation

Cell differentiation has been associated with changes in mechanical stiffness in single-cell systems, yet it is unknown whether this association remains true in a multicellular context, particularly in developing tissues. In order to address such questions, we have developed a methodology, termed quantitative tandem epifluorescence and nanoindentation, wherein we sequentially determine cellular genetic identity with confocal microscopy and mechanical properties with atomic force microscopy. We have applied this approach to examine cellular stiffness at the shoot apices of Arabidopsis (Arabidopsis thaliana) plants carrying a fluorescent reporter for the CLAVATA3 (CLV3) gene, which encodes a secreted glycopeptide involved in the regulation of the centrally located stem cell zone in inflorescence and floral meristems. We found that these CLV3-expressing cells are characterized by an enhanced stiffness. Additionally, by tracking cells in young flowers before and after the onset of GREEN FLUORESCENT PROTEIN expression, we observed that an increase in stiffness coincides with this onset. This work illustrates how quantitative tandem epifluorescence and nanoindentation can reveal the spatial and temporal dynamics of both gene expression and cell mechanics at the shoot apex and, by extension, in the epidermis of any thick tissue.Morphogenesis is a complex process that results from the coordinated actions of many genes and gene products across developing tissues and organs. Because shape is a function of the structural elements of cells, the molecular and genetic control of growth and morphogenesis must rely on the regulation of the mechanics of these elements. In this context, cell differentiation has been linked with mechanical stiffness in animal single-cell systems (Collinsworth et al., 2002; Balland et al., 2006; Engler et al., 2006; Darling et al., 2008), although the direct measurement of cell mechanics in growing animal tissues remains elusive (Blanchard and Adams, 2011; Davidson, 2011).In plants, growth involves a delicate mechanical balance: it is powered by turgor pressure and contained by cell wall stiffness (Cosgrove, 1986). Several groups have recently achieved mechanical measurements made at a subcellular resolution in plants (Milani et al., 2011; Peaucelle et al., 2011; Fernandes et al., 2012; Radotić et al., 2012; Routier-Kierzkowska et al., 2012) using scaled-down indentation methods (Geitmann, 2006; Hayot et al., 2012; Milani et al., 2013; Routier-Kierzkowska and Smith, 2013), wherein one quantifies the force needed to push down on a sample to a prescribed depth. These studies have revealed spatiotemporal patterns of stiffness, notably in tissues (Milani et al., 2011; Peaucelle et al., 2011; Fernandes et al., 2012; Routier-Kierzkowska et al., 2012).However, these measurements have not been associated directly with cell identity. This association would become feasible if mechanical measurements were combined with optical imaging of fluorescent reporters. Such a combination, termed nanoindentation coupled to inverted optical microscopy, has already been developed for single animal cells and for thin plant tissues, (Rotsch and Radmacher, 2000; Routier-Kierzkowska and Smith, 2014), but it cannot be extended to thick tissues because they are opaque, making it impossible to simultaneously observe the tissue surface optically with an inverted microscope and probe it mechanically. To circumvent this difficulty, we have developed a methodology involving the use of three microscopes to image the same sample: (1) an atomic force microscope (AFM), which is a nanoindentation system for obtaining stiffness maps of the surface of a sample; (2) an AFM-coupled upright epifluorescence macroscope to precisely identify the points to be probed; and (3) a confocal microscope to determine cell fate at cellular resolution, which may in turn be correlated with the stiffness maps. We call this methodology quantitative tandem epifluorescence and nanoindentation (qTEN), and we use it to probe the shoot apical meristem (SAM) of Arabidopsis (Arabidopsis thaliana), which is a good model system in which to investigate morphogenesis.The SAM is located at the growing tip of the shoot and consists of distinct functional zones (Ha et al., 2010). One of these zones is the slow-dividing central zone (CZ), which can be defined by the expression of the CLAVATA3 (CLV3) signaling glycopeptide. Through cell division, cells exit the CZ into the surrounding peripheral zone (PZ). In the PZ, cells proliferate rapidly, and some become incorporated into organ primordia, thus yielding all aerial organs of the plant. Recent work on the SAM has revealed patterns of mechanical properties (Milani et al., 2011; Peaucelle et al., 2011; Kierzkowski et al., 2012; Braybrook and Peaucelle, 2013), but it is still unclear how these patterns are related to the activity of the SAM or to its functional zonation. Here, we analyze the dynamics of such a mechanical pattern in vivo and show that it is spatially and temporally related to stem cell fate.     

proUK-KGDW融合基因在CHO细胞中的高表达

利用常规分子生物学技术,构建了新型高效的proUK-KGDW融合基因的分泌型哺乳动物细胞表达载体。将该载体线性化后转染CHO/dhfr-细胞,经G418筛选获得阳性克隆,然后挑取表达水平较高的克隆进行MTX加压扩增,以提高proUK-KGDW杂合体的表达水平,经2～3轮MTX加压扩增,获得多株表达水平超过10μg/(106细胞·24h)的稳定的高表达细胞株,为proUK-KGDW杂合体的制备及功能研究奠定了基础。     

Expression of Ribosomal Cistrons of Human Chromosomes at High Resolution

The expression of ribosomal cistrons in the nucleolar organizer regions (NORs) has been studied with high resolution banding in the acrocentric chromosomes of 10 normal individuals. It was found that if a particular chromosome did not stain with silver nitrate at metaphase, then it did not stain at prophase either. Therefore, it is concluded that some of the acrocentric chromosomes have variable expression of NORs.     

Inference of Gene Regulatory Networks Using Time-Series Data: A Survey

The advent of high-throughput technology like microarrays has provided the platform for studying how different cellular components work together, thus created an enormous interest in mathematically modeling biological network, particularly gene regulatory network (GRN). Of particular interest is the modeling and inference on time-series data, which capture a more thorough picture of the system than non-temporal data do. We have given an extensive review of methodologies that have been used on time-series data. In realizing that validation is an impartible part of the inference paradigm, we have also presented a discussion on the principles and challenges in performance evaluation of different methods. This survey gives a panoramic view on these topics, with anticipation that the readers will be inspired to improve and/or expand GRN inference and validation tool repository.     

HMGB1对小鼠系膜细胞细胞周期及细胞周期相关蛋白表达的影响

该实验以小鼠系膜细胞MMC为研究对象,以重组HMGB1为刺激物,通过检测细胞周期的变化及细胞PCNA、CyclinD1、CDK4和p16的表达水平,初步探讨HMGB1对系膜细胞的细胞周期及其相关调控因子的影响。选取小鼠系膜细胞MMC为研究对象,随机分为对照组及0.05mg/LHMGB1刺激组,经流式细胞术检测发现HMGB1能够上调小鼠系膜细胞中S期细胞所占比例;免疫细胞化学检测显示,PCNA蛋白在小鼠系膜细胞中的表达上调;通过RT-PCR技术及Western blot技术检测到小鼠系膜细胞中CyclinD1 mRNA和蛋白以及CDK4蛋白的高表达情况,而p16蛋白的表达呈时间依赖性降低。由此可见,HMGB1可能是通过上调CyclinD1/CDK4的表达,并下调p16的表达,促进细胞从G_0/G_1期进入S期,介导了小鼠系膜细胞的异常增殖,可能是HMGB1参与狼疮性肾炎发病的可能机制之一。     

Study of Integrated Heterogeneous Data Reveals Prognostic Power of Gene Expression for Breast Cancer Survival

BackgroundStudies show that thousands of genes are associated with prognosis of breast cancer. Towards utilizing available genetic data, efforts have been made to predict outcomes using gene expression data, and a number of commercial products have been developed. These products have the following shortcomings: 1) They use the Cox model for prediction. However, the RSF model has been shown to significantly outperform the Cox model. 2) Testing was not done to see if a complete set of clinical predictors could predict as well as the gene expression signatures.Methodology/FindingsWe address these shortcomings. The METABRIC data set concerns 1981 breast cancer tumors. Features include 21 clinical features, expression levels for 16,384 genes, and survival. We compare the survival prediction performance of the Cox model and the RSF model using the clinical data and the gene expression data to their performance using only the clinical data. We obtain significantly better results when we used both clinical data and gene expression data for 5 year, 10 year, and 15 year survival prediction. When we replace the gene expression data by PAM50 subtype, our results are significant only for 5 year and 15 year prediction. We obtain significantly better results using the RSF model over the Cox model. Finally, our results indicate that gene expression data alone may predict long-term survival.Conclusions/SignificanceOur results indicate that we can obtain improved survival prediction using clinical data and gene expression data compared to prediction using only clinical data. We further conclude that we can obtain improved survival prediction using the RSF model instead of the Cox model. These results are significant because by incorporating more gene expression data with clinical features and using the RSF model, we could develop decision support systems that better utilize heterogeneous information to improve outcome prediction and decision making.