Two color hybridization analysis using high density oligonucleotide arrays and energy transfer dyes

High density oligonucleotide arrays (DNA chips) have been used in two color mutational analysis of the 3.43 kb exon 11 of the hereditary breast and ovarian cancer gene BRCA1 . Two color analysis allows competitive hybridization between a reference standard and an unknown sample, improving the performance of the assay. Fluorescein and phycoerythrin dyes werepreviously used due to their compatibility with a single line 488 nm excitation source. Here we show that an alternative dye combination, containing the energy transfer dye system phycoerythrin*cy5 along with phycoerythrin, provides more evenly matched signal intensities and decreased spectral overlap between the two fluorophores, while maintaining compatibility with a 488 nm excitation source.     

Use of continuous stacking hybridization in sequencing using modified oligonucleotide matrices

The opportunity of DNA sequencing by hybridization with oligonucleotide matrix (SHOM) with simultaneously use continuous stacking hybridization and gapped-matrices is considered. The analysis of reconstruction efficiency for various combinations matrices and l-oligonucleotides libraries were made. In most cases combine use of continuous stacking hybridization and gapped matrices permits to decrease the number of additional stacking hybridization twice without lost of efficiency.     

Application of robotic technology to automated sequence fingerprint analysis by oligonucleotide hybridisation

We describe our production line for the rapid analysis of large cDNA libraries applying robotic techniques to automatically pick, amplify, array, hybridise and analyse the clones. We also outline the current state of the hybridisation techniques and describe anticipated future developments of the system. Our approach faces the large-scale analysis of cDNA clones with partial sequence analysis by oligonucleotide fingerprinting in the following way: after picking of individual colonies and arraying them automatically in quadruple density (384-well) microtitre plates, the cDNA clones are amplified by an automated waterbath polymerase chain reaction (PCR), which allows us to run about 46,000 reactions in parallel. The PCR products are automatically transferred to nylon membranes in a high density pattern using a robotic device. We routinely produce twelve 22 cm x 22 cm membranes in 90 min. Each membrane contains 20,736 clones, although much higher densities might be feasible using both miniaturized glass matrices and fluorescence based hybridisation techniques. Theoretical analysis and preliminary computer simulations indicate that about 100-200 sequence specific hybridisations of octanucleotides to about 100,000 PCR products of 1000-1500 base-pairs length will generate sufficient information for classifying the clones into groups of identical or related genes and to identify a large number of previously uncharacterized cDNA clones.     

A simple method for synthesizing [gamma-32P]nucleoside triphosphates using [32P]acetylphosphate and acetate kinase

A simple, rapid, and inexpensive method is described for the synthesis of gamma-32P-labeled ribo- or deoxyribonucleoside triphosphates. The procedure involves chemical synthesis of [32P]acetylphosphate and subsequent phosphorylation of nucleoside diphosphates using acetate kinase (EC 2.7.2.1) and a final purification step. The entire procedure is performed 8 h or less.     

High resolution renal diffusion imaging using a modified steady-state free precession sequence

A modified steady-state free precession (SSFP) diffusion sequence is proposed for high resolution renal imaging. A pair of bipolar diffusion gradients was used to minimize the errors in measured apparent diffusion coefficient (ADC) caused by variations in T1, T2, and RF flip angle that have been observed with previously employed SSFP diffusion sequences. Motion sensitivity was reduced by the use of compensated gradients, frame-by-frame averaging, and a repetition time of 22 ms, which for a single-acquisition 128 x 128 image requires only 3 s. High resolution was achieved by signal averaging. The modified sequence was applied to in vivo diffusion measurements. In six normal rat kidneys the ADCs (mean +/- SD; x 10(-3) mm2/s) of the cortex, outer medulla, and inner medulla were 2.28 +/- 0.05, 2.38 +/- 0.10, and 2.95 +/- 0.05, respectively. The technique requires relatively large gradients to achieve adequate diffusion weighting.     

Enhanced cholesterol efflux by tyrosyl radical-oxidized high density lipoprotein is mediated by apolipoprotein AI-AII heterodimers

Myeloperoxidase secreted by phagocytes in the artery wall may be a catalyst for lipoprotein oxidation. High density lipoprotein (HDL) oxidized by peroxidase-generated tyrosyl radical has a markedly enhanced ability to deplete cultured cells of cholesterol. We have investigated the structural modifications in tyrosylated HDL responsible for this effect. Spherical reconstituted HDL (rHDL) containing the whole apolipoprotein (apo) fraction of tyrosylated HDL reproduced the ability of intact tyrosylated HDL to enhance cholesterol efflux from cholesterol-loaded human fibroblasts when reconstituted with the whole lipid fraction of either HDL or tyrosylated HDL. Free apoAI or apoAII showed no increased capacity to induce cholesterol efflux from cholesterol-loaded fibroblasts following oxidation by tyrosyl radical, either in their lipid-free forms or in rHDL. The product of oxidation of a mixture of apoAI and apoAII (1:1 molar ratio) by tyrosyl radical, however, reproduced the enhanced ability of tyrosylated HDL to induce cholesterol efflux when reconstituted with the whole lipid fraction of HDL. HDL containing only apoAI or apoAII showed no enhanced ability to promote cholesterol efflux following oxidation by tyrosyl radical, whereas HDL containing both apoAI and apoAII did. rHDL containing apoAI-apoAIImonomer and apoAI-(apoAII)2 heterodimers showed a markedly increased ability to prevent the accumulation of LDL-derived cholesterol mass by sterol-depleted fibroblasts compared with other apolipoprotein species of tyrosylated HDL. These results indicate a novel product of HDL oxidation, apoAI-apoAII heterodimers, with a markedly enhanced capacity to deplete cells of the regulatory pool of free cholesterol and total cholesterol mass. The recent observation of tyrosyl radical-oxidized LDL in vivo suggests that a similar modification of HDL would significantly enhance its ability to deplete peripheral cells of cholesterol in the first step of reverse cholesterol transport.     

Strategies for mutational analysis of the large multiexon ATM gene using high-density oligonucleotide arrays

Mutational analysis of large genes with complex genomic structures plays an important role in medical genetics. Technical limitations associated with current mutation screening protocols have placed increased emphasis on the development of new technologies to simplify these procedures. High-density arrays of >90,000-oligonucleotide probes, 25 nucleotides in length, were designed to screen for all possible heterozygous germ-line mutations in the 9.17-kb coding region of the ATM gene. A strategy for rapidly developing multiexon PCR amplification protocols in DNA chip-based hybridization analysis was devised and implemented in preparing target for the 62 ATM coding exons. Improved algorithms for interpreting data from two-color experiments, where reference and test samples are cohybridized to the arrays, were developed. In a blinded study, 17 of 18 distinct heterozygous and 8 of 8 distinct homozygous sequence variants in the assayed region were detected accurately along with five false-positive calls while scanning >200 kb in 22 genomic DNA samples. Of eight heterozygous sequence changes found in more than one sample, six were detected in all cases. Five previously unreported sequence changes, not found by other mutational scanning methodologies on these same samples, were detected that led to either amino acid changes or premature truncation of the ATM protein. DNA chip-based assays should play a valuable role in high throughput sequence analysis of complex genes.     

Equilibrium analysis of high affinity interactions using BIACORE

Both asymmetrical sensory blockade and dural puncture are undesirable outcomes of epidural analgesia. Identifying the epidural space with the needle bevel oriented parallel to the longitudinal axis of the patient's back limits the risk of headache in the event of dural puncture. However, rotating the bevel to direct a catheter cephalad may risk dural puncture. We prospectively studied the effects of needle rotation on the success of labor epidural analgesia and on the incidence of dural puncture. One hundred sixty ASA physical status I or II laboring parturients were randomly assigned to one of four groups. The epidural space was identified with the bevel of an 18-gauge Hustead needle directed to the patient's left. It was then rotated as follows: Group 0 = no rotation, final bevel orientation left (n = 39); Group 90 = rotation 90 degrees clockwise, bevel cephalad (n = 43); Group 180 = rotation 180 degrees clockwise, bevel right (n = 36); Group 270 = rotation 270 degrees clockwise, bevel caudad (n = 42). A single-orifice catheter was inserted 3 cm, and analgesia was induced in a standardized fashion. Dural puncture was evenly distributed among the groups (4.4%). There were more dermatomal segments blocked, fewer one-sided blocks, and more patients comfortable at 30 min with the needle bevel directed cephalad. Using a catheter inserted through a needle oriented in the cephalad direction increases the success of epidural analgesia. Implications: This prospective study shows that an epidural catheter inserted through a needle oriented in the cephalad direction increases the success of labor analgesia in the parturient. Carefully rotating the needle cephalad does not increase the risk of dural puncture, intravascular catheters, or failed blocks.     

Protein identification using 20-minute Edman cycles and sequence mixture analysis

We have developed a 20-min Edman cycle and a multiple sample horizontal flow reactor for the sequence analysis of PVDF-electroblotted proteins. The 20-min cycle uses a 12-min C18 phenylthiohydantoin separation. This cycle and separation is compatible with most Applied Biosystems sequencers. Using this rapid cycle, 10 residues on six different proteins can be completed within a 24-h period. We also demonstrate the use of an algorithm that can sort mixture sequences derived from PVDF bands that contain coeluting proteins.     

模具润滑温压成形法制作的高密度铁基烧结体的磁性与组织

烧结铁是一种好的软磁材料,但其磁通密度比铸造材料的低。低的磁通密度是由于密度低所引起的。本研究开发了一种温压与硬脂酸锂模壁润滑技术(WC-DWL)。使用该技术,得到了密度非常高的成形体。纯铁粉在1176MPa的压力下成形,经1523K烧结后的性能如下:密度=7.76Mg/m3,μm=5300,B160=1.16T,B240=1.28T,B400=1.40T,B2k=1.60T,bHc=110A/m。一些烧结铁表现出各向异性的变化与组织,这导致异常晶粒长大到长度为数毫米。这种异常长大在试样成形密度较高时更为明显。     

Enhanced retroviral transduction of 5-fluorouracil-resistant human bone marrow (stem) cells using a genetically modified packaging cell line

Pluripotent hematopoietic stem cells (PHSC) are rare cells capable of multilineage differentiation, long-term reconstituting activity and extensive self-renewal. Such cells are the logical targets for many forms of corrective gene therapy, but are poor targets for retroviral mediated gene transfer owing to their quiescence, as retroviral transduction requires that the target cells be cycling. To try and surmount this problem we have constructed a retroviral producer line that expresses the membrane-bound form of human stem cell factor (SCF) on its cell surface. These cells are capable, therefore, of delivering a growth signal concomitant with recombinant retroviral vector particles. In this report we describe the use of this cell line to transduce a highly quiescent population of cells isolated from adult human bone marrow using the 5-fluorouracil (FU) resistance technique of Berardi et al. Quiescent cells selected using this technique were transduced by cocultivation with retroviral producers expressing surface bound SCF or with the parent cell line that does not. Following coculture, the cells were plated in long-term bone marrow culture for a further 5 weeks, before plating the nonadherent cells in semisolid media. Colonies forming in the semisolid media over the next 14 days were analyzed by polymerase chain reaction for the presence of the retroviral vector genome. Over six experiments, the transduction frequency of the quiescent 5-FU resistant cells using the SCF-expressing producer line averaged about 20%, whereas those transduced using the parent producer line showed evidence of reduced levels or no transduction.     

Clinical analysis of human urinary proteins using high resolution electrophoretic methods

The application of isoelectric focusing (IEF), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional electrophoresis (2-DE) and capillary electrophoresis (CE) for high resolution electrophoretic analysis of human urinary proteins is reviewed. In each case, the information is tabulated chronologically with details of sample preparation, electrophoretic system, detection method and clinical application. The text includes an historical perspective of the use of each method for urinalysis and a detailed review of the application of the methods to the investigation of renal disease, renal transplantation, Bence Jones proteinuria (BJP), diabetes mellitus, cadmium toxicity, nephrolithiasis and cancers of the urogenital tract.     

Anterior cervical fusion using a modified tricortical bone graft: a radiographic analysis of outcome

A technique was developed to resist graft extrusion by means of mechanical interlocking between graft and vertebrae. A 4-mm burr is used to create a transverse trough (mortise) across the posterior aspect of each endplate. A reversed tricortical graft is shaped to have a ridge (tenon) composed of cortical bone on the upper and lower surfaces of its posterior portion. The ridges lock into the troughs to form a double mortise and tenon joint. Of 117 discs (102 patients) treated, adequate radiographs were available of 106 discs (in 92 patients) of which 89% were fused at last follow-up. Radiographic analysis showed significant (p < 0.001) increases in disc height and extension immediately postoperatively and a return to the preoperative values by the time of fusion. This technique completely eliminated graft extrusion without any reduction in fusion rate compared with most other reported results with the standard tricortical technique.     

BS960在骨架车大梁上的轻量化CAE分析

为了骨架车大梁的轻量化,首先对某大梁车架原始方案进行了静态强度有限元分析,结果显示最大的Mises应力位置位于纵梁鹅颈位置.然后针对纵梁的最大应力和位移区域,对上翼板、下翼板、腹板厚度的关系进行了灵敏度分析,得到影响最大应力和位移的关键部件.最后纵梁材料升级为BS960并减小厚度与原始方案进行对比,根据不同目的需要,推荐了上翼板—腹板—下翼板的板厚组合,推荐的纵梁板厚组合轻量化率最大为21％.     

Differentiation of Actinobacillus pleuropneumoniae strains by sequence analysis of 16S rDNA and ribosomal intergenic regions, and development of a species specific oligonucleotide for in situ detection

The aims of this study were to characterize and determine intraspecies and interspecies relatedness of Actinobacillus pleuropneumoniae to Actinobacillus lignieresii and Actinobacillus suis by sequence analysis of the ribosomal operon and to find a species-specific area for in situ detection of A. pleuropneumoniae. Amplification and sequence analysis of the 16S-23S rDNA ribosomal intergenic sequence (RIS) from the three species showed the existence of two RIS's, differing by about 100 bp. Both sequences contained a region resembling the ribonuclease III cleavage site found in Escherichia coli. The smaller RIS contained a Glu-tRNA gene, and the larger one contained genes encoding Ile-tRNA and Ala-tRNA. These tRNA's showed a high sequence homology to the respective tRNA genes found in E. coli. Sequence analysis of the RIS's showed a high degree of genetic similarity of 24 strains of A. pleuropneumoniae. The larger RIS's were different between the 3 species tested. The sequence of the 16S ribosomal gene was determined for 8 serotypes of A. pleuropneumoniae. These sequences showed only minor base differences, indicating a close genetic relatedness of these serotypes within the species. An oligonucleotide DNA probe designed from the 16S rRNA gene sequence of A. pleuropneumoniae was specific for all strains of the target species and did not cross react with A. lignieresii, the closest known relative of A. pleuropneumoniae. This species-specific DNA probe labeled with fluorescein was used for in situ hybridization experiments to detect A. pleuropneumoniae in biopsies of diseased porcine lungs.     

Volumetric analysis of epilepsy surgery resections using high resolution magnetic imaging: technical report

A method is described for accurately measuring the volume and site of epilepsy surgery resections utilizing magnetic resonance imaging. Accuracy has been assessed using post-mortem studies, and both the intra- and interobserver variability is consistently less than 5%. The technique has so far been applied to 25 patients following a variety of operations for medically intractable epilepsy. It provides the crucially accurate baseline required for meaningful follow-up outcome studies of epilepsy surgery. Consequently, it should allow the development of more precise prognostic indices for such operations.     

利用高分辨率卫星影像分析工业城市的土地利用

The spatial differentiation of land use changes of Tuticorin is studied using high resolution LISS Ⅲ satellite imagery and Maximum Likelihood algorithms. The classification accuracy of 95.2% was obtained. In this study, the land use of Tuticorin is classified as settlement, salt pan, agricultural land, wasteland, water bodies and shrubs. The settlement area is increased to 4.6 km2 during the year 2001 and 2006. The settlement area change is mainly driven by growth of industries and migration of people from peripheral villages. Shrub is increased to 3.63 km2 in the six year period. Water logging due to growth of shrubs in Tuticorin leads to several environmental and health hazard. This study warrants proper urban planning for Tuticorin for sustainable use of resource and environment.     

Transient liquid phase sintering of high density Fe3Al using Fe and Fe2Al5/FeAl2 powders Part 1: Experimentation and results

Abstract

High density Fe₃Al was produced through transient liquid phase sintering, using rapid heating rates of greater than 150 K min^-1 and a mixture of prealloyed and elemental powders. Prealloyed Fe₂Al₅/FeAl₂ (50Fe/50Al, wt-%) powder was added to elemental iron powder in a ratio appropriate for producing an overall Fe₃Al (13·87 wt-%) ratio. The heating rate, sintering time, sintering temperature, green density and powder particle size were controlled during the study. Heating rate, sintering time and powder particle size had the most significant influence upon the sintered density of the compacts. The highest sintered density of 6·12 Mg m^-3 (92% of the theoretical density for Fe3Al) was achieved after 15 minutes of sintering at 1350°C, using a 250 K min^{- 1} heating rate, 1-6 μm Fe powders and 5·66 μm alloy powders.

SEM microscopy suggests that agglomerated Fe₂Al₅/ FeAl₂ particles, which form a liquid during sintering, are responsible for a significant portion of the remaining porosity in high sintered density compacts, creating stable pores, larger than 100 μm diameter, after melting. High density was achieved by minimising the Kirkendall porosity formed during heating by unbalanced diffusion and solubility between the iron and Fe₂Al₅/FeAl₂ components. The lower diffusion rate of aluminium in the prealloyed powder into the iron compared with elemental aluminium in iron, coupled with a fast heating rate, is expected to permit minimal iron-aluminium interdiffusion during heating so that when a liquid forms the aluminium dissolves in the iron to promote solidification at a lower aluminium content. This leads to a further reduction in porosity.