cDNA芯片阳性对照的制备及在芯片敏感性分析中的应用

cDNA芯片是一种高通量基因表达谱分析技术,在生理病理条件下细胞基因表达谱分析,新基因发现和功能研究等方面具有广阔应用前景。CDNA芯片阳性对照的选取以及CDNA芯片检测敏感性是芯片成功应用的关键问题之一。以在系统发育上与人类基因同源性小的荧火虫荧光素酶基因材料,制备了用于人类和其他动物基因表达谱CDNA芯片的通用型阳性对照探针和相应的mRNA参照物,经反转录对mRNA参照物进行Cy3荧光标记并与DNA芯片杂交后发现,mRNA参照物能特异性地与荧光酶基因cDNA片断杂交,而与人β-肌动蛋白基因,人G3PDH基因以及λDNA/HINDⅢ无杂交反应。把mRNA参照物以不同比例加入HepG2总RNA中,以反转录荧光标记后与CDNA芯片杂交,结果发现当总RNA中的MRNA含量为1/10^4稀释（即mRNA分子个数约为10^8个）时,CDNA芯片基本检测不出mRNA标记产物的杂交信号。而且,cDNA芯片检测的信号强度与芯片上固定的探针浓度密切相关,当探针浓度为2g/L时,杂交信号最强,随着探针浓度下降芯片的杂交信号趋于减弱。CDNA芯片通用型阳性参照物的制备以及应用于CDNA芯片检测敏感性研究为CDNA芯片应用于人和其他动物基因表达谱高通量分析和新基因功能研究提供了技术基础和理论依据。     

OLIN: optimized normalization, visualization and quality testing of two-channel microarray data

SUMMARY: Microarray data are generated in complex experiments and frequently compromised by a variety of systematic errors. Subsequent data normalization aims to correct these errors. Although several normalization methods have recently been proposed, they frequently fail to account for the variability of systematic errors within and between microarray experiments. However, optimal adjustment of normalization procedures to the underlying data structure is crucial for the efficiency of normalization. To overcome this restriction of current methods, we have developed two normalization schemes based on iterative local regression combined with model selection. The schemes have been demonstrated to improve considerably the quality of normalization. They are implemented in a freely available R package. Additionally, functions for visualization and detection of systematic errors in microarray data have been incorporated in the software package. A graphical user interface is also available. AVAILABILITY: The R package can be downloaded from http://itb.biologie.hu-berlin.de/~futschik/software/R/OLIN. It underlies the GPL version 2. CONTACT: m.futschik@biologie.hu-berlin.de SUPPLEMENTARY INFORMATION: Further information about the methods used in the OLIN software package can be found at http://itb.biologie.hu-berlin.de/~futschik/software/R/OLIN.     

Quick and simple: quality control of microarray data

MOTIVATION: Microarrays are high-throughput tools for parallel miniaturized detection of biomolecules. In contrast to experiments using ratios of signals in two channels, experiments with only one fluorescent dye cause special problems for data analysis. The present work compares algorithms for quality filtering on spot level as well as array/slide level. RESULTS: Methods for quantitative spot filtering are discussed and new sets of quality scores for data preprocessing are designed. As measures of spot quality also reflect the quality of protocols, they were employed to find the optimal print buffer in an optimization experiment. In order to determine problematic arrays within a set of replicates we tested methods of outlier detection which can suitably replace the visual inspection of slides. CONTACT: Ursula.Sauer@arcs.ac.at.     

Issues in cDNA microarray analysis: quality filtering, channel normalization, models of variations and assessment of gene effects

We consider the problem of comparing the gene expression levels of cells grown under two different conditions using cDNA microarray data. We use a quality index, computed from duplicate spots on the same slide, to filter out outlying spots, poor quality genes and problematical slides. We also perform calibration experiments to show that normalization between fluorescent labels is needed and that the normalization is slide dependent and non-linear. A rank invariant method is suggested to select non-differentially expressed genes and to construct normalization curves in comparative experiments. After normalization the residuals from the calibration data are used to provide prior information on variance components in the analysis of comparative experiments. Based on a hierarchical model that incorporates several levels of variations, a method for assessing the significance of gene effects in comparative experiments is presented. The analysis is demonstrated via two groups of experiments with 125 and 4129 genes, respectively, in Escherichia coli grown in glucose and acetate.     

Cell surface targeting of VPAC1 receptors: evidence for implication of a quality control system and the proteasome

Like for most transmembrane proteins, translation of G protein-coupled receptors (GPCRs) mRNA takes place at the endoplasmic reticulum (ER) where they are synthesized, folded and assembled. The molecular mechanisms involved in the transport process of GPCRs from ER to the plasma membrane are poorly investigated. Here we studied the mechanisms involved in glycosylation-dependent cell surface expression and quality control of the receptor for Vasoactive Intestinal Polypeptide (VIP) VPAC1, a member of the B family of GPCRs. Using biochemical and pharmacological techniques and fluorescence microscopy, we have shown that only a fraction of newly synthesized VPAC1 attains properly conformation that allows their cell surface targeting. Misfolded or immature VPAC1 are taken in charge by co- and post-translational quality control that involves: 1) calnexin-dependent folding strictly through a glycan-dependent mechanism, 2) BiP-dependant folding, 3) translocation to the cytoplasm and proteasome-dependent degradation of improper proteins, and 4) post-ER quality control check points. Our data suggest that VPAC1 expression/trafficking pathways are under the control of complex and precise molecular mechanisms to ensure that only proper VPAC1 reaches the cell surface.     

IPGWAS: an integrated pipeline for rational quality control and association analysis of genome-wide genetic studies

Alveolar rhabdomyosarcoma (aRMS) is a very aggressive sarcoma of children and young adults. Our previous studies have shown that small molecule inhibition of Pdgfra is initially very effective in an aRMS mouse model. However, slowly evolving, acquired resistance to a narrow-spectrum kinase inhibitor (imatinib) was common. We identified Src family kinases (SFKs) to be potentiators of Pdgfra in murine aRMS primary cell cultures from mouse tumors with evolved resistance in vivo in comparison to untreated cultures. Treating the resistant primary cell cultures with a combination of Pdgfra and Src inhibitors had a strong additive effect on cell viability. In Pdgfra knockout tumors, however, the Src inhibitor had no effect on tumor cell viability. Sorafenib, whose targets include not only PDGFRA but also the Src downstream target Raf, was effective at inhibiting mouse and human tumor cell growth and halted progression of mouse aRMS tumors in vivo. These results suggest that an adaptive Src-Pdgfra-Raf-Mapk axis is relevant to PDGFRA inhibition in rhabdomyosarcoma.     

Patterns of parasitoid host utilization and development across a range of temperatures: implications for biological control of an invasive forest pest

Although climate change frequently has been linked to observed shifts in the distributions or phenologies of species, little is known about the potential effects of varying temperatures on parasitoids and their relationships with hosts. Using the egg parasitoid Oobius agrili (Hymenoptera: Encyrtidae) we examined host utilization patterns of this species across a range of temperatures (20–35 °C) to explore how changing climate could affect the interaction with its host—the emerald ash borer (EAB) (Coleoptera: Buprestidae), a serious invasive forest pest that has killed tens of millions of ash (Fraxinus spp.) trees in North America. Results from our study showed that the window of host susceptibility to O. agrili parasitism declined significantly from 14.8 to 2.6 days in an inverse second-order relationship with increasing exposure temperatures from 20 to 35 °C. In contrast, parasitoid host attack rate changed in a bell-shaped second-order relationship—i.e., increased with temperatures from 20 to 25 °C, but decreased at about the same rate when temperatures increased from 30 to 35 °C. This range of temperatures also significantly affected the development and mortality of immature parasitoids with 35 °C resulting in 100 % mortality. There was little mortality (0–4.5 %) and no significant differences in the percentage (20.9–34.9 %) of immature O. agrili that entered diapause (as mature larvae) at 20, 25, and 30 °C. However, there were significant differences in the time event of adult wasp emergence within this temperature range. The median time for 50 % of immature O. agrili emerging as adults at 20, 25, and 30 °C were 38, 18, and 17 days after parental wasp oviposition, respectively. Together these findings indicate that the non-linear and unequal temperature effects on these host utilization parameters are likely to result in differential host parasitism rates, and thus could reduce the efficacy of this parasitoid in suppressing host populations due to climate change (global warming and extreme heat).     

A molecular switch for targeting between endoplasmic reticulum (ER) and mitochondria: conversion of a mitochondria-targeting element into an ER-targeting signal in DAKAP1

dAKAP1 (AKAP121, S-AKAP84), a dual specificity PKA scaffold protein, exists in several forms designated as a, b, c, and d. Whether dAKAP1 targets to endoplasmic reticulum (ER) or mitochondria depends on the presence of the N-terminal 33 amino acids (N1), and these N-terminal variants are generated by either alternative splicing and/or differential initiation of translation. The mitochondrial targeting motif, which is localized between residues 49 and 63, is comprised of a hydrophobic helix followed by positive charges ( Ma, Y., and Taylor, S. (2002) J. Biol. Chem. 277, 27328-27336 ). dAKAP1 is located on the cytosolic surface of mitochondria outer membrane and both smooth and rough ER membrane. A single residue, Asp(31), within the first 33 residues of dAKAP1b is required for ER targeting. Asp(31), which functions as a separate motif from the mitochondrial targeting signal, converts the mitochondrial-targeting signal into a bipartite ER-targeting signal, without destroying the mitochondria-targeting signal. Therefore dAKAP1 possesses a single targeting element capable of targeting to both mitochondria and ER, with the ER signal overlapping the mitochondria signal. The specificity of ER or mitochondria targeting is determined and switched by the availability of the negatively charged residue, Asp(31).     

Conservation of targeting but divergence in function and quality control of peroxisomal ABC transporters: an analysis using cross-kingdom expression

ABC (ATP-binding cassette) subfamily D transporters are found in all eukaryotic kingdoms and are known to play essential roles in mammals and plants; however, their number, organization and physiological contexts differ. Via cross-kingdom expression experiments, we have explored the conservation of targeting, protein stability and function between mammalian and plant ABCD transporters. When expressed in tobacco epidermal cells, the mammalian ABCD proteins ALDP (adrenoleukodystrophy protein), ALDR (adrenoleukodystrophy-related protein) and PMP70 (70?kDa peroxisomal membrane protein) targeted faithfully to peroxisomes and P70R (PMP70-related protein) targeted to the ER (endoplasmic reticulum), as in the native host. The Arabidopsis thaliana peroxin AtPex19_1 interacted with human peroxisomal ABC transporters both in vivo and in vitro, providing an explanation for the fidelity of targeting. The fate of X-linked adrenoleukodystrophy disease-related mutants differed between fibroblasts and plant cells. In fibroblasts, levels of ALDP in some 'protein-absent' mutants were increased by low-temperature culture, in some cases restoring function. In contrast, all mutant ALDP proteins examined were stable and correctly targeted in plant cells, regardless of their fate in fibroblasts. ALDR complemented the seed germination defect of the Arabidopsis cts-1 mutant which lacks the peroxisomal ABCD transporter CTS (Comatose), but neither ALDR nor ALDP was able to rescue the defect in fatty acid β-oxidation in establishing seedlings. Taken together, our results indicate that the mechanism for trafficking of peroxisomal membrane proteins is shared between plants and mammals, but suggest differences in the sensing and turnover of mutant ABC transporter proteins and differences in substrate specificity and/or function.     

Identifying actives from HTS data sets: practical approaches for the selection of an appropriate HTS data-processing method and quality control review

High-throughput screening (HTS) has achieved a dominant role in drug discovery over the past 2 decades. The goal of HTS is to identify active compounds (hits) by screening large numbers of diverse chemical compounds against selected targets and/or cellular phenotypes. The HTS process consists of multiple automated steps involving compound handling, liquid transfers, and assay signal capture, all of which unavoidably contribute to systematic variation in the screening data. The challenge is to distinguish biologically active compounds from assay variability. Traditional plate controls-based and non-controls-based statistical methods have been widely used for HTS data processing and active identification by both the pharmaceutical industry and academic sectors. More recently, improved robust statistical methods have been introduced, reducing the impact of systematic row/column effects in HTS data. To apply such robust methods effectively and properly, we need to understand their necessity and functionality. Data from 6 HTS case histories are presented to illustrate that robust statistical methods may sometimes be misleading and can result in more, rather than less, false positives or false negatives. In practice, no single method is the best hit detection method for every HTS data set. However, to aid the selection of the most appropriate HTS data-processing and active identification methods, the authors developed a 3-step statistical decision methodology. Step 1 is to determine the most appropriate HTS data-processing method and establish criteria for quality control review and active identification from 3-day assay signal window and DMSO validation tests. Step 2 is to perform a multilevel statistical and graphical review of the screening data to exclude data that fall outside the quality control criteria. Step 3 is to apply the established active criterion to the quality-assured data to identify the active compounds.     

Local control of 1–5 fraction radiotherapy regimens for spinal metastases: an analysis of the impacts of biologically effective dose and primary histology

BackgroundThis analysis evaluates the impacts of biologically effective dose (BED) and histology on local control (LC) of spinal metastases treated with highly conformal radiotherapy to moderately-escalated doses.Materials and methodsPatients were treated at two institutions from 2010–2020. Treatments with less than 5 Gy per fraction or 8 Gy in 1 fraction were excluded. The dataset was divided into three RPA classes predictive of survival (1). The primary endpoint was LC.Results223 patients with 248 treatments met inclusion criteria. Patients had a median Karnofsky Performance Status (KPS ) of 80, and common histologies included breast (29.4%), non-small cell lung cancer (15.7%), and prostate (13.3%). A median 24 Gy was delivered in 3 fractions (BED: 38.4 Gy) to a median planning target volume (PTV) of 37.3 cc. 2-year LC was 75.7%, and 2-year OS was 42.1%. Increased BED was predictive of improved LC for primary prostate cancer (HR = 0.85, 95% CI: 0.74–0.99). Patients with favorable survival (RPA class 1) had improved LC with BED ≥ 40 Gy (p = 0.05), unlike the intermediate and poor survival groups. No grade 3–5 toxicities were reported.ConclusionsModerately-escalated treatments were efficacious and well-tolerated. BED ≥ 40 Gy may improve LC, particularly for prostate cancer and patients with favorable survival.     

Function of Semliki Forest virus E3 peptide in virus assembly: replacement of E3 with an artificial signal peptide abolishes spike heterodimerization and surface expression of E1.

The Semliki Forest virus spike glycoproteins E1 and p62 form a heterodimeric complex in the endoplasmic reticulum (ER) and are transported as such to the cell surface. In the mature virus particle, the heterodimeric association of E1 and E2 (the cleavage product of p62) is maintained, but as a more labile and acid-sensitive oligomer than the E1-p62 complex. The E3 peptide forms the N-terminal part of the p62 precursor and carries the signal for the translocation of p62 into the lumen of the ER. The question of whether E3 is also important in the formation and stabilization of the E1-p62 heterodimer has been addressed here with the aid of an E3 deletion mutant cDNA. In this construct, the entire E3 was replaced with a cleavable, artificial signal sequence which preserved the membrane topology of an authentic E2. The E3 deletion, when expressed via a recombinant vaccinia virus, abolished heterodimerization of the spike proteins. It also resulted in the complete retention of E1 in the ER and almost total inhibition of E2 transport to the plasma membrane. The oligomerization and transport defect of E1 expressed from the E3 deletion mutant could be complemented with a wild-type p62 provided from a separate coding unit in double infections. These results point to a central role of E3 in complex formation and transport of the viral structural components to the site of budding. In conjunction with earlier work (M. Lobigs and H. Garoff, J. Virol. 64:1233-1240, 1990; J. Wahlberg, W. A. M. Boere, and H. Garoff, J. Virol. 63:4991-4997, 1989), the data support a model of spike protein oligomerization control of Semliki Forest virus assembly and disassembly which may be mediated by the presence of E3 in the uncleaved p62 precursor and release of E3 after cleavage.     

Characterization of an ERAD gene as VPS30/ATG6 reveals two alternative and functionally distinct protein quality control pathways: one for soluble Z variant of human alpha-1 proteinase inhibitor (A1PiZ) and another for aggregates of A1PiZ

The Z variant of human alpha-1 proteinase inhibitor (A1PiZ) is a substrate for endoplasmic reticulum-associated protein degradation (ERAD). To identify genes required for the degradation of this protein, A1PiZ degradation-deficient (add) yeast mutants were isolated. The defect in one of these mutants, add3, was complemented by VPS30/ATG6, a gene that encodes a component of two phosphatidylinositol 3-kinase (PtdIns 3-kinase) complexes: complex I is required for autophagy, whereas complex II is required for the carboxypeptidase Y (CPY)-to-vacuole pathway. We found that upon overexpression of A1PiZ, both PtdIns 3-kinase complexes were required for delivery of the excess A1PiZ to the vacuole. When the CPY-to-vacuole pathway was compromised, A1PiZ was secreted; however, disruption of autophagy led to an increase in aggregated A1PiZ rather than secretion. These results suggest that excess soluble A1PiZ transits the secretion pathway to the trans-Golgi network and is selectively targeted to the vacuole via the CPY-to-vacuole sorting pathway, but excess A1PiZ that forms aggregates in the endoplasmic reticulum is targeted to the vacuole via autophagy. These findings illustrate the complex nature of protein quality control in the secretion pathway and reveal multiple sites that recognize and sort both soluble and aggregated forms of aberrant or misfolded proteins.