Allelic dosage analysis with genotyping microarrays

Genomic alternations, including dosage and allelic imbalance, constitute a major basis of neoplastic and other genetic disorders. Using oligonucleotide genotyping microarrays, here we report the development and usage of an algorithm, called genome imbalance map (GIM) algorithm, for allelic as well as total gene dosage analysis. Using the GIM algorithm, global genome imbalance status at over 100,000 loci was simultaneously analyzed with unprecedented accuracy and allelic discrimination.     

Simultaneous determination of renal clinical analytes in serum using hydrolase- and oxidase-encapsulated optical array biosensors

An optical array biosensor encapsulated with hydrolase and oxidoreductase using sol-gel immobilization technique has been fabricated for simultaneous analysis and screening of multiple samples to determine the presence of multianalytes which are clinically important in relation to renal failure. Urease and creatinine deiminase were used to detect urea and creatinine, while glucose oxidase and uricase were coimmobilized with horseradish peroxidase to quantify glucose and uric acid. Moreover, the concentrations of analytes in fetal calf serum were measured and quantified using the developed sensing system. The array biosensor showed good specificity for the simultaneous analysis of multiple samples for multianalytes without obvious cross-interference. The analytical ranges of the four analytes were between 0.01 and 10mM with detection limits of 2.5-80 microM. High precision with relative standard deviations of 3.8-9.2% (n=45) was also demonstrated. The reproducibility of array-to-array in 3 consecutive months was 5.4% (n=3). Moreover, the concentrations of analytes in fetal calf serum were 5.9 mM for urea, 0.13 mM for creatinine, 3.3mM for glucose, and 0.15 mM for uric acid, which were in good agreement with results obtained using the traditional spectroscopic methods. These results demonstrate the first use of a sol-gel-derived optical array biosensor for simultaneous analysis of multiple samples for the presence of multiple clinically important renal analytes.     

Simultaneous detection of six biohazardous agents using a planar waveguide array biosensor

Recently, we demonstrated that an array biosensor could be used with cocktails of fluorescent antibodies to perform three assays simultaneously on a single substrate, and that multiple samples could be analyzed in parallel. We extend this technology to demonstrate the simultaneous analysis of six samples for six different hazardous analytes, including both bacteria and protein toxins. The level of antibody cross-reactivity is explored, revealing a possible common epitope in two of the toxins. A panel of environmental interferents was added to the samples; these interferents neither prevented the detection of the analytes nor caused false-positive responses.     

利用悬液芯片系统建立一种高通量检测牛奶中头孢氨苄和莱克多巴胺残留的方法

目的:旨在应用基于荧光编码微球技术的悬液芯片系统建立一种方便、稳定性好及高通量的检测牛奶中头孢氨苄和莱克多巴胺残留的免疫检测方法。方法:利用碳二亚胺法将抗生素合成抗原与表面具有羧基的聚苯乙烯微球通过酰胺键偶联成捕获抗原。利用捕获抗原、抗生素的单克隆抗体及抗生素标准品构建竞争性免疫检测体系。荧光标记的羊抗小鼠的IgG作为荧光探针标记与捕获抗原结合的单克隆抗体得到悬液芯片系统的检测物。悬液芯片系统的检测器由两束特殊的激光构成,能够检测荧光探针荧光强度的同时分辨不同型号的微球以实现高通量检测的目的。结果:通过对头孢氨苄和莱克多巴胺合成抗原包被微球的条件进行优化得到包被100μl的微球所需两者合成抗原的量分别是8.4μg 和 87.73μg;实验结果表明抗生素的单克隆抗体特异性良好;在牛奶中,该方法对头孢氨苄和莱克多巴胺的检测限(LOD)分别是20.59 ng/ml 和23.51 ng/ml, 标准添加回收率在70%~110%之间。     

阵列生物传感器研究进展

简要介绍了阵列生物传感器的基本原理和分类。根据换能器的不同,评述了光学、电化学、质量型、磁致阻抗等阵列生物传感器的研究进展,并对目前阵列生物传感器研究中存在的问题进行了分析。     

Transcriptome atlas of glutamine family amino acid metabolism‐related genes in eight regenerating liver cell types

To explore glutamine family amino acid metabolism of eight liver cell types in rat liver regeneration, eight kinds of rat regenerating liver cells were isolated by using the combination of Percoll density gradient centrifugation and immunomagnetic bead methods, then Rat Genome 230 2.0 Array was used to detect the expression profiles of the genes associated with metabolism of glutamine family amino acid in rat liver regeneration and finally how these genes involved in activities of eight regenerating liver cell types were analysed by the methods of bioinformatics and systems biology. The results showed that in the priming stage of liver regeneration, hepatic stellate cells and sinusoidal endothelial cells transformed proline and glutamine into glutamate; hepatocytes, hepatic stellate cells, sinusoidal endothelial cells and dendritic cells catabolized glutamate to 2‐oxoglutarate or succinate; hepatic stellate cells and sinusoidal endothelial cells catalysed glutamate into glutamyl‐tRNA for protein synthesis; urea cycle, which degraded from arginine, was enhanced in biliary epithelia cells, sinusoidal endothelial cells and dendritic cells; synthesis of polyamines from arginine was enhanced in biliary epithelia cells, sinusoidal endothelial cells, Kupffer cells and dendritic cells; the content of NO was increased in sinusoidal endothelial cells and dendritic cells; degradation of proline was enhanced in hepatocytes and biliary epithelia cells. In the progress stage, biliary epithelia cells converted glutamine into GMP and glucosamine 6‐phosphate; oval cells converted glutamine into glucosamine 6‐phosphate; hepatic stellate cells converted glutamine into NAD; the content of NO, which degraded from arginine, was increased in biliary epithelia cells, oval cells, pit cells and dendritic cells. In the termination stage, oval cells converted proline into glutamate; glutamate degradation, which degraded from arginine, was enhanced in hepatocytes and dendritic cells; the content of NO was increased in oval cells, sinusoidal endothelial cells, pit cells and dendritic cells. The synthesis of creatine phosphate was enhanced in hepatocytes, biliary epithelia cells, pit cells and dendritic cells in both progress and termination stages. In summary, glutamine family amino acid metabolism has some differences in liver regeneration in different liver cells.     

High-throughput single cell arrays as a novel tool in biopreservation

Microwell array cytometry is a novel high-throughput experimental technique that makes it possible to correlate pre-stress cell phenotypes and post-stress outcomes with single cell resolution. Because the cells are seeded in a high density grid of cell-sized microwells, thousands of individual cells can be tracked and imaged through manipulations as extreme as freezing or drying. Unlike flow cytometry, measurements can be made at multiple time points for the same set of cells. Unlike conventional image cytometry, image analysis is greatly simplified by arranging the cells in a spatially defined pattern and physically separating them from one another. To demonstrate the utility of microwell array cytometry in the field of biopreservation, we have used it to investigate the role of mitochondrial membrane potential in the cryopreservation of primary hepatocytes.Even with optimized cryopreservation protocols, the stress of freezing almost always leads to dysfunction or death in part of the cell population. To a large extent, cell fate is dominated by the stochastic nature of ice crystal nucleation, membrane rupture, and other biophysical processes, but natural variation in the initial cell population almost certainly plays an important and under-studied role. Understanding why some cells in a population are more likely to survive preservation will be invaluable for the development of new approaches to improve preservation yields.For this paper, primary hepatocytes were seeded in microwell array devices, imaged using the mitochondrial dyes Rh123 or JC-1, cryopreserved for up to a week, rapidly thawed, and checked for viability after a short recovery period. Cells with a high mitochondrial membrane potential before freezing were significantly less likely to survive the freezing process, though the difference in short term viability was fairly small. The results demonstrate that intrinsic cell factors do play an important role in cryopreservation survival, even in the short term where extrinsic biophysical factors would be expected to dominate. We believe that microwell array cytometry will be an important tool for a wide range of studies in biopreservation and stress biology.     

Discovering tumor suppressor genes through genome-wide copy number analysis

Classical tumor suppressor gene discovery has largely involved linkage analysis and loss-of-heterozygosity (LOH) screens, followed by detailed mapping of relatively large chromosomal regions. Subsequent efforts made use of genome-wide PCR-based methods to detect rare homozygous deletions. More recently, high-resolution genomic arrays have been applied to cancer gene discovery. However, accurate characterization of regions of genomic loss is particularly challenging due to sample heterogeneity, the small size of deleted regions and the high frequency of germline copy number polymorphisms. Here, we review the application of genome-wide copy number analysis to the specific problem of identifying tumor suppressor genes.     

A Coiled Coil Trigger Site Is Essential for Rapid Binding of Synaptobrevin to the SNARE Acceptor Complex

Exocytosis from synaptic vesicles is driven by stepwise formation of a tight α-helical complex between the fusing membranes. The complex is composed of the three SNAREs: synaptobrevin 2, SNAP-25, and syntaxin 1a. An important step in complex formation is fast binding of vesicular synaptobrevin to the preformed syntaxin 1·SNAP-25 dimer. Exactly how this step relates to neurotransmitter release is not well understood. Here, we combined different approaches to gain insights into this reaction. Using computational methods, we identified a stretch in synaptobrevin 2 that may function as a coiled coil “trigger site.” This site is also present in many synaptobrevin homologs functioning in other trafficking steps. Point mutations in this stretch inhibited binding to the syntaxin 1·SNAP-25 dimer and slowed fusion of liposomes. Moreover, the point mutations severely inhibited secretion from chromaffin cells. Altogether, this demonstrates that the trigger site in synaptobrevin is crucial for productive SNARE zippering.