Asymptotic Conditional Singular Value Decomposition for High‐Dimensional Genomic Data

Summary High‐dimensional data, such as those obtained from a gene expression microarray or second generation sequencing experiment, consist of a large number of dependent features measured on a small number of samples. One of the key problems in genomics is the identification and estimation of factors that associate with many features simultaneously. Identifying the number of factors is also important for unsupervised statistical analyses such as hierarchical clustering. A conditional factor model is the most common model for many types of genomic data, ranging from gene expression, to single nucleotide polymorphisms, to methylation. Here we show that under a conditional factor model for genomic data with a fixed sample size, the right singular vectors are asymptotically consistent for the unobserved latent factors as the number of features diverges. We also propose a consistent estimator of the dimension of the underlying conditional factor model for a finite fixed sample size and an infinite number of features based on a scaled eigen‐decomposition. We propose a practical approach for selection of the number of factors in real data sets, and we illustrate the utility of these results for capturing batch and other unmodeled effects in a microarray experiment using the dependence kernel approach of Leek and Storey (2008, Proceedings of the National Academy of Sciences of the United States of America 105 , 18718–18723) .     

High-resolution 3-D imaging of living cells in suspension using confocal axial tomography

Conventional flow cytometry (FC) methods report optical signals integrated from individual cells at throughput rates as high as thousands of cells per second. This is further combined with the powerful utility to subsequently sort and/or recover the cells of interest. However, these methods cannot extract spatial information. This limitation has prompted efforts by some commercial manufacturers to produce state-of-the-art commercial flow cytometry systems allowing fluorescence images to be recorded by an imaging detector. Nonetheless, there remains an immediate and growing need for technologies facilitating spatial analysis of fluorescent signals from cells maintained in flow suspension. Here, we report a novel methodological approach to this problem that combines micro-fluidic flow, and microelectrode dielectric-field control to manipulate, immobilize and image individual cells in suspension. The method also offers unique possibilities for imaging studies on cells in suspension. In particular, we report the system's immediate utility for confocal "axial tomography" using micro-rotation imaging and show that it greatly enhances 3-D optical resolution compared with conventional light reconstruction (deconvolution) image data treatment. That the method we present here is relatively rapid and lends itself to full automation suggests its eventual utility for 3-D imaging cytometry.     

Methods for acquisition of quantitative data from confocal images of gene expression in situ

In this review, we summarize original methods for the extraction of quantitative information from confocal images of gene-expression patterns. These methods include image segmentation, the extraction of quantitative numerical data on gene expression, and the removal of background signal and spatial registration. Finally, it is possible to construct a spatiotemporal atlas of gene expression from individual images recorded at each developmental stage. Initially all methods were developed to extract quantitative numerical information from confocal images of segmentation gene expression in Drosophila melanogaster. The application of these methods to Drosophila images makes it possible to reveal new mechanisms in the formation of segmentation gene expression domains, as well as to construct a quantitative atlas of segmentation gene expression. Most image processing procedures can be easily adapted to process a wide range of biological images.     

Enhanced expression of the human chitinase 3-like 2 gene (YKL-39) but not chitinase 3-like 1 gene (YKL-40) in osteoarthritic cartilage

The knowledge of molecular alterations in osteoarthritic cartilage is important to identify novel therapeutic targets or to develop new diagnostic tools. We aimed to characterize the molecular response to cartilage degeneration by identification of differentially expressed genes in human osteoarthritic versus normal cartilage. Gene fragments selectively amplified in osteoarthritic cartilage by cDNA representational difference analysis included YKL-39 and the oesophageal-cancer-related-gene-4 (ECRG4). YKL-39 expression was significantly upregulated in cartilage from patients with osteoarthritis (n=14) versus normal subjects (n=8) according to real-time PCR (19-fold, p=0.009) and cDNA array analysis (mean 15-fold, p<0.001) and correlated with collagen 2 up-regulation. In contrast, the homologous cousin molecule YKL-40 (chitinase 3-like 1), which is elevated in serum and synovial fluid of patients with arthritis, showed no significant regulation in OA cartilage. Enhanced levels of YKL-40 may, therefore, be derived from synovial cells while modulation of YKL-39 and collagen 2 expression reflected the cartilage metabolism in response to degradation.     

Integrating technologies for comparing 3D gene expression domains in the developing chick limb

Chick embryos are good models for vertebrate development due to their accessibility and manipulability. Recent large increases in available genomic data from both whole genome sequencing and EST projects provide opportunities for identifying many new developmentally important chicken genes. Traditional methods of documenting when and where specific genes are expressed in embryos using wholemount and section in-situ hybridisation do not readily allow appreciation of 3-dimensional (3D) patterns of expression, but this can be accomplished by the recently developed microscopy technique, Optical Projection Tomography (OPT). Here we show that OPT data on the developing chick wing from different labs can be reliably integrated into a common database, that OPT is efficient in capturing 3D gene expression domains and that such domains can be meaningfully compared. Novel protocols are used to compare 3D expression domains of 7 genes known to be involved in chick wing development. This reveals previously unappreciated relationships and demonstrates the potential, using modern genomic resources, for building a large scale 3D atlas of gene expression. Such an atlas could be extended to include other types of data, such as fate maps, and the approach is also more generally applicable to embryos, organs and tissues.     

Loss of heterozygosity of the p53 gene and deregulated expression of its mRNA and protein in human brain tumors

Tumor-specific alterations at the p53 gene locus were analyzed in 40 human brain tumor samples. Gliomas were more prevalent in young males and meningiomas in old females. Structural changes at the intron 1 region of the p53 gene were analyzed in these tumors by Southern blotting. Among the 40 tumors, 33 were informative and 21 of these (63.6%) informative cases showed loss of heterozygosity (LOH). This is the first report showing LOH at the intron 1 region of p53 gene in human brain tumors. The level of p53 mRNA, p53 protein and Ser 392 phosphorylated p53 protein were also analyzed in all tumor samples. Normal sized p53 mRNA and protein were present in all the tumor samples; however, their levels were 1.5- to 4-fold higher compared to the control suggesting deregulated p53 pathway in these tumors. No correlation was found between LOH status and the levels of p53 mRNA and protein. In all high-grade glioblastomas majority of the p53 protein existed as Ser 392 phosphorylated form as compared to low-grade gliomas. In addition, the percentage of Ser 392 phosphorylated form of p53 protein was lower in meningiomas and other brain tumor types irrespective of tumor grade. These results suggest involvement of Ser 392 phosphorylated form of p53 protein during the later stages of glioma development. These results also indicate that deregulation of p53 gene could occur at various steps in p53 pathway and suggest an overall deregulation of p53 gene in most brain tumor types.     

Separation of chitooligosaccharides and the potent effects on gene expression of cell surface receptor CR3

Chitooligosaccharides were prepared through hydrolysis of colloidal chitosan by enzyme source from Aspergillus fumigatus BSF114. Chitosan pentamer (COS5) and chitosan hexamer (COS6) were isolated and purified from COS by the ultra-filtration, nano-filtration, ethanol precipitation and the CM-Sephadex C-25 column. COS5 consisted of (GlcN)₄ (59.84%) and (GLcN)₅ (40.16%). COS6, however, mainly consisted of (GLcN)₆ (93.11%) and (GLcN)₅ (6.89%). Effects of COS5 and COS6 in vivo and in vitro on gene expression of cell surface CR3 receptor were investigated by relatively quantitative RT-PCR and ELISA. The results showed that the expression of CR3 mRNA could be promoted by both COS5 and COS6. The promotion effect caused by COS6 was greater than that of COS5.     

管氏肿腿蜂毒液过敏原3基因的克隆及表达分析

为研究管氏肿腿蜂Scleroderma guani毒液过敏原3基因SgA3的功能,通过RT-PCR克隆SgA3基因,采用荧光定量PCR分析其在不同发育阶段和组织中的表达特征,并利用载体pSUMO-Mut对其进行原核表达。克隆获得SgA3基因开放阅读框长699 bp,编码233个氨基酸,其中信号肽位于N端第1~23位氨基酸。多序列比对分析发现,SgA3与丽蝇蛹集金小蜂Nasonia vitripennis、粉蝶盘绒茧蜂Cotesia glomerata、红火蚁Solenopsis invicta和常见黄胡蜂Vespula vulgaris过敏原3的氨基酸序列一致性分别为50.44%、50%、48.89%和47.83%。荧光定量PCR结果表明,SgA3基因在雌成虫中高表达,且在毒液器官中的表达量显著高于雌成虫其他组织和雄成虫中的表达量。利用pSUMO-Mut表达载体,成功表达并纯化得到高纯度的SgA3重组蛋白。该研究结果为进一步研究SgA3基因的功能奠定了基础。     

Combined method of whole mount and block-face imaging: Acquisition of 3D data of gene expression pattern from conventional in situ hybridization

Visualization of spatiotemporal expression of a gene of interest is a fundamental technique for analyzing the involvements of genes in organ development. In situ hybridization (ISH) is one of the most popular methods for visualizing gene expression. When conventional ISH is performed on sections or whole-mount specimens, the gene expression pattern is represented in 2-dimensional (2D) microscopic images or in the surface view of the specimen. To obtain 3-dimensional (3D) data of gene expression from conventional ISH, the “serial section method” has traditionally been employed. However, this method requires an extensive amount of time and labor because it requires researchers to collect a tremendous number of sections, label all sections by ISH, and image them before 3D reconstruction. Here, we proposed a rapid and low-cost 3D imaging method that can create 3D gene expression patterns from conventional ISH-labeled specimens. Our method consists of a combination of whole-mount ISH and Correlative Microscopy and Blockface imaging (CoMBI). The whole-mount ISH-labeled specimens were sliced using a microtome or cryostat, and all block-faces were imaged and used to reconstruct 3D images by CoMBI. The 3D data acquired using our method showed sufficient quality to analyze the morphology and gene expression patterns in the developing mouse heart. In addition, 2D microscopic images of the sections can be obtained when needed. Correlating 2D microscopic images and 3D data can help annotate gene expression patterns and understand the anatomy of developing organs. These results indicated that our method can be useful in the field of developmental biology.     

Characterization and expression of AmphiBMP3 /3b gene in amphioxus Branchiostoma japonicum

Bone morphogenetic proteins (BMPs) are responsible for regulating embryo development and tissue homeostasis beyond osteogenesis. However, the precise biological roles of BMP3 and BMP3b remain obscure to a certain extent. In the present study, we cloned an orthologous gene (AmphiBMP3/3b) from amphioxus (Branchiostoma japonicum) and found its exon/intron organization is highly conserved. Further, in situ hybridization revealed that the gene was strongly expressed in the dorsal neural plate of the embryos. The gene also appeared in Hatschek’s left diverticulum, neural tube, preoral ciliated pit and gill slit of larvae, and adult tissues including ovary, neural tube and notochordal sheath. Additionally, real‐time quantitative polymerase chain reaction (RTqPCR) analysis revealed that the expression displayed two peaks at gastrula and juvenile stages. These results indicated that AmphiBMP3/3b, a sole orthologue of vertebrate BMP3 and BMP3b, might antagonize ventralizing BMP2 orthologous signaling in embryonic development, play a role in the evolutionary precursors of adenohypophysis, as well as act in female ovary physiology in adult.     

鲤鱼肥胖基因的分子克隆及在大肠杆菌中的表达

为了研究鲤鱼肥胖基因的结构特点和体外表达产物的生物学活性 ,利用RT PCR技术从鲤鱼肠系膜脂肪组织中扩增出鲤鱼肥胖基因的cDNA编码序列 ,分析表明该cDNA序列由 4 38个核苷酸组成 ,编码 14 6个氨基酸组成的多肽 ,鲤鱼肥胖基因与人、猪、鼠的相比 ,核苷酸同源性分别为 :84 %、 86 %、 95 % ;氨基酸的同源性分别为 84 %、 82 %、 96 %。构建了原核表达载体 pET 2 8a li,利用IPTG在大肠杆菌中进行了诱导表达 ,并对表达产物进行了初步纯化和生物活性检测 ,结果表明 ,鲤鱼肥胖基因在大肠杆菌中进行了高效特异性融合表达 ,融合蛋白质分子量约为 2 0kD ,经薄层扫描分析 ,目的蛋白占菌体总蛋白的 2 0 3%。表达产物经过纯化和复性能够明显抑制小鼠的摄食和生长 ,说明表达产物Leptin具有明显的生物学活性