Downregulation of RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1, and HYAL2 in non-small cell lung cancer

Chromosomal and genome abnormalities of 3p are frequent in many epithelial tumors, including lung cancer. Several critical regions with a high frequency of hemi-and homozygous deletions in tumors are known for 3p, and more than 20 cancer-related genes occur in 3p21.3. Quantitative real-time PCR was used to measure the mRNA level for tumor-suppressor and candidate genes of 3p21.3 (RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1, and HYAL2) in major types of non-small cell lung cancer (NSCLC): squamous cell lung cancer (SCC) and lung adenocarcinoma (AC). A significant (2-to 100-fold) and frequent (44–100%) decrease in mRNA levels was observed in NSCLC. The mRNA level decrease and its frequency depended on the histological type of NSCLC for all genes. The downregulation of RASSF1A and ITGA9 was significantly associated with AC progression; the same tendency was observed for RBSP3/CTDSPL, NPRL2/G21, HYAL1, and HYAL2. In SCC, the downregulation of all genes was not associated with the clinical stage, tumor cells differentiation, and metastasis in lymph nodes. The RBSP3/CTDSPL, NPRL2/G21, ITGA9, HYAL1, and HYAL2 mRNA levels significantly (5-to 13-fold on average) decreased at a high frequency (83–100%) as early as SCC stage I. Simultaneous downregulation of all six genes was observed in some tumor samples and was independent of the gene position in 3p21.3 and the functions of the protein products. The Spearman correlation coefficient r _s was 0.63–0.91, p < 0.001. The highest r _s values were obtained for gene pairs ITGA9-HYAL2 and HYAL1-HYAL2, whose products mediate cell-cell adhesion and cell-matrix interactions; coregulation of the genes was assumed on this basis. Both genetic and epigenetic mechanisms proved to be important for downregulation of RBSP3/CTDSPL and ITGA9. This finding supported the hypothesis that the cluster of cancerrelated genes in the extended 3p21.3 locus is simultaneously inactivated during the development and progression of lung cancer and other epithelial tumors. A significant and frequent decrease in the mRNA level of the six genes in SCC could be important for developing specific biomarker sets for early SCC diagnosis and new approaches to gene therapy of NSCLC.     

phylochipanalyser - a program for analysing hierarchical probe sets

The recent introduction of phylochips that contain molecular probes facilitates environmental microbial identification in a single experiment without previous cultivation. A set of probes recognizing species at different taxonomic levels is denoted as a hierarchical set. Application of hierarchical probe sets on a DNA microarray allows the assessment of biodiversity with different resolutions. It significantly increases the robustness of the results retrieved from phylochip experiments because of the possible consistency checks of hybridization across different taxonomic levels. Here, we present a computer program, phylo-chipanalyser, for the hierarchy editing and the evaluation of phylochip data generated from hierarchical probe sets.     

Tjp3/zo-3 is critical for epidermal barrier function in zebrafish embryos

TJP3/ZO-3 is a scaffolding protein that tethers tight junction integral membrane proteins to the actin cytoskeleton and links the conserved Crumbs polarity complex to tight junctions. The physiological function of TJP3/ZO-3 is not known and mice lacking TJP3/ZO-3 show no apparent phenotype. Here we show that Tjp3/Zo-3 is a component of tight junctions present in the enveloping cell layer of zebrafish embryos. Silencing tjp3/zo-3 using morpholinos leads to edema, loss of blood circulation and tail fin malformations in the embryos. The ultrastructure of tight junctions of the enveloping cell layer is disrupted, without affecting the asymmetric distribution of plasma membrane proteins. Morphants show a loss of the epidermal barrier, as assessed by an increased permeability of the enveloping cell layer to low molecular weight tracers and a higher sensitivity of the embryos to osmotic stress. Subjecting wild-type embryos to osmotic stress mimicks the morphant phenotype, consistent with the phenotype being a direct consequence of failed osmoregulation. Thus, Tjp3/Zo-3 is critical for barrier function of the enveloping cell layer and osmoregulation in early stages of zebrafish development.     

A novel immobilization strategy using oligonucleotide as linker for small molecule microarrays construction

A novel immobilization method based on oligonucleotide as linker has been developed for small molecule microarrays (SMMs) construction. The oligonucleotide tail was employed as a linker in solid-phase synthesis. Small molecules could be easily conjugated at the 5′ end of the oligonucleotide, previously modified with a functional group. Being a reactive species, the oligonucleotide was activated by UV irradiation, for the attachment of the conjugate to the slide surface. The method was successfully applied to structurally distinct small molecules, including biotin, antibiotic and drug. This immobilization strategy showed high efficiency, 1.1 fmol of small molecules in the spotting solution per spot gave a detectable signal (mean S/N = 10.9). The results suggest that it is very promising for exploring interaction between small molecules and proteins, and high throughput detecting the chemical compounds.     

Egr1 and Gas6 facilitate the adaptation of HEK-293 cells to serum-free media by conferring enhanced viability and higher growth rates

Animal-derived serum is an essential media supplement for mammalian cells in cell culture. For a number of reasons including cost, regulatory concerns, lot inconsistency, potential contamination with adventitious agents, and down-stream processing it is desirable to eliminate the use of serum. Existing protocols designed to adapt cells to serum-free media (SFM) are time-consuming and provide little insight into how the cells adapt. To better understand the physiological responses associated with serum withdrawal and to expedite the adaptation process, a Human Embryonic Kidney-293 (HEK-293) cell line was propagated in 10% fetal bovine serum (FBS) and was progressively adapted to SFM and analyzed at specific serum levels by oligonucleotide microarrays. Of the differentially expressed genes two, early growth response 1 (egr1) and growth arrest specific 6 (gas6), were selected for further analysis based on their level of differential expression, overall expression patterns, and proposed functionalities. HEK-293 cells, propagated in 10% FBS were transfected with egr1 or gas6 and then adapted to SFM. Results indicated that higher expression of either gene moderately enhanced the ability of both cell lines to adapt to SFM. Egr1 appeared to have a greater impact on adaptability than gas6. Results also indicated that specific protein production was unaltered when the expression of egr1 was increased. Flow cytometric analysis revealed increased expression of egr1 was associated with an increase in the percentage of cells in the G2/M phases. These results indicate that enhanced expression of egr1 or gas6 facilitate adaptation to SFM by improving growth and viability.     

应用多重标记引物增强寡核苷酸芯片的荧光信号强度

寡核苷酸芯片技术是一种高通量发掘和采集生物信息的强大技术平台,目前已广泛应用于生物科学领域 . 为改善寡核苷酸芯片的分析性能,对影响芯片杂交结果的因素,如片基表面的化学处理、探针的长度、间隔臂的长度、杂交条件等,进行了深入的研究和优化 . 对寡核苷酸芯片而言,仍有待解决的问题是如何产生更强的荧光信号来改善其检测灵敏度 . 利用两种类型的多个荧光分子标记的引物,来增强二维寡核苷酸芯片平面上的荧光信号强度 . 两种引物分别命名为：多标记线性引物和多标记分支引物 . 通过增加标记在目标 DNA 片段上的荧光分子数,可以显著增强寡核苷酸芯片上相应捕获探针的信号强度 . 实验表明,使用多标记引物能将所用的寡核苷酸微阵列的检测限 ( 以能够检测的最低模板量计算 ) 降低至单荧光标记引物的 1/100 以下,多重标记技术是一种有效增强微型化探针矩阵检测灵敏度的信号放大方法 .     

Development of multiplexed protein profiling and detection using near infrared detection of reverse-phase protein microarrays

Protein microarrays have been recently employed for signal pathway profiling and high-throughput protein expression analysis. Reversephase arrays, where the array consists of immobilized analytes and lysates has especially shown promise in low abundance analyte detection and signal pathway profiling using phospho-specific antibodies. A limitation to current reverse phase array methodology is the inability to multiplex proteomic-based endpoints as each array can only report one analyte endpoint. In this study, we report on the use of a dual dye based approach that can effectively double the number of endpoints observed per array allowing, for example, both phosphospecific and total protein levels to be measured and analyzed at once. The method utilizes antibody bound dyes that emit in the infrared spectral region as a means of sensitive and specific detection.     

一种新型细胞微阵列的制作和应用

为了高通量地检测大量培养细胞中基因原位表达, 发明了一种制作细胞微阵列的新方法,成功地制作含20种细胞系共100个供体细胞石蜡混合物点阵的细胞微阵列.免疫组化检测P53, P21, PTEN、P16基因在细胞微阵列中的蛋白质表达.原位杂交检测BRD7、NGX6 基因在细胞微阵列中mRNA原位表达.建立了P53、P21、PTEN、P16蛋白和BRD7、NGX6 mRNA 在不同培养细胞中的原位表达谱.细胞微阵列为基因功能研究提供一种新的高通量工具.细胞微阵列可广泛用于DNA、RNA和蛋白质水平上的基因原位表达研究.细胞微阵列还可用于筛选药物作用靶标的研究.