Repeatability of and Relationship between Potential COPD Biomarkers in Bronchoalveolar Lavage,Bronchial Biopsies,Serum, and Induced Sputum

Chronic Obstructive Pulmonary Disease (COPD) is a chronic inflammatory disease, primarily affecting the airways. Stable biomarkers characterizing the inflammatory phenotype of the disease, relevant for disease activity and suited to predict disease progression are needed to monitor the efficacy and safety of drug interventions. We therefore analyzed a large panel of markers in bronchoalveolar lavage, bronchial biopsies, serum and induced sputum of 23 healthy smokers and 24 smoking COPD patients (GOLD II) matched for age and gender. Sample collection was performed twice within a period of 6 weeks. Assays for over 100 different markers were validated for the respective matrices prior to analysis. In our study, we found 51 markers with a sufficient repeatability (intraclass correlation coefficient >0.6), most of these in serum. Differences between groups were observed for markers from all compartments, which extends (von-Willebrand-factor) and confirms (e.g. C-reactive-protein, interleukin-6) previous findings. No correlations between lung and serum markers were observed, including A1AT. Airway inflammation defined by sputum neutrophils showed only a moderate repeatability. This could be improved, when a combination of neutrophils and four sputum fluid phase markers was used to define the inflammatory phenotype.In summary, our study provides comprehensive information on the repeatability and interrelationship of pulmonary and systemic COPD-related markers. These results are relevant for ongoing large clinical trials and future COPD research. While serum markers can discriminate between smokers with and without COPD, they do not seem to sufficiently reflect the disease-associated inflammatory processes within the airways.     

Spatial consistency of circle on the pedagogic pommel horse: influence of expertise

Gymnastics is a closed-skill sport, and the repeatability of a specific technique is accepted as a valuable indicator of a performer's level of expertise. The circle movement, defined as a gyrating movement in the horizontal plane, is a key component of all contemporary pommel horse exercises. The aims of this study were to determine the spatial consistency of the circle movement and to assess the influence of expertise on the repeatability of this skill performed on the pedagogic pommel horse. Six expert gymnasts and six nonexperts performed 10 circles on this apparatus. A 3-dimensional analysis system recorded the trajectories of 6 markers fixed on the right and left ankles, hips, and shoulders. The spatiotemporal consistency was assessed by the SD of the marker trajectories during the circle sequence. The results showed that the shoulder and trajectories were more consistent than the ankle trajectory (P < 0.05); the marker trajectories were less consistent in the sagittal plane (P < 0.05); and the expert gymnasts showed better repeatability of the ankle trajectory than the nonexpert gymnasts did (P < 0.05). In this context, the use of the SD of the ankle trajectory during the circle sequence could be an interesting tool for trainers to quantify objectively the positional errors of the legs during circular swings and to measure the improvement in movements after specific training.     

In vivo estimation of the glenohumeral joint centre by functional methods: Accuracy and repeatability assessment

Several algorithms have been proposed for determining the centre of rotation of ball joints. These algorithms are used rather to locate the hip joint centre. Few studies have focused on the determination of the glenohumeral joint centre. However, no studies have assessed the accuracy and repeatability of functional methods for glenohumeral joint centre.This paper aims at evaluating the accuracy and the repeatability with which the glenohumeral joint rotation centre (GHRC) can be estimated in vivo by functional methods. The reference joint centre is the glenohumeral anatomical centre obtained by medical imaging. Five functional methods were tested: the algorithm of Gamage and Lasenby (2002), bias compensated (Halvorsen, 2003), symmetrical centre of rotation estimation (Ehrig et al., 2006), normalization method (Chang and Pollard, 2007), helical axis (Woltring et al., 1985). The glenohumeral anatomical centre (GHAC) was deduced from the fitting of the humeral head.Four subjects performed three cycles of three different movements (flexion/extension, abduction/adduction and circumduction). For each test, the location of the glenohumeral joint centre was estimated by the five methods. Analyses focused on the 3D location, on the repeatability of location and on the accuracy by computing the Euclidian distance between the estimated GHRC and the GHAC. For all the methods, the error repeatability was inferior to 8.25 mm. This study showed that there are significant differences between the five functional methods. The smallest distance between the estimated joint centre and the centre of the humeral head was obtained with the method of Gamage and Lasenby (2002).     

SELDI-TOF-MS技术寻找肿瘤转移相关的标志蛋白研究进展

由于SELDI-TOF-MS技术简便、快速、灵敏度高、重复性好等优点,目前大量应用于肿瘤相关的研究,不但为肿瘤早期诊断相关标志蛋白的寻找提供了有效的手段,而且它在寻找肿瘤转移相关的标志蛋白中的作用越来越受到人们的关注。因为对于肿瘤转移相关标志蛋白的检测有利于人们更清楚地认识肿瘤转移的机制,更重要的是为临床医生提供对病人情况的更全面的判断,制定更适合病人自身的个体化的治疗方案。本文对国内外发表的利用SELDI-TOF-MS技术寻找在大肠癌、胃癌、肺癌、乳腺癌、前列腺癌、卵巢癌、肝细胞肝癌、头颈部癌等转移相关标志蛋白的研究文献作一综述。     

SSR分子标记在梨种质资源研究中的应用

SSR（simple sequence repeat）标记,又称微卫星标记,具有多态性高、重复性好、共显性、检测简单等优点,在果树树种的多样性、品种和亲缘关系鉴定等方面得到了广泛应用。就近年来SSR分子标记在梨种质资源研究上的应用,综述了SSR分子标记的筛选与开发研究、种质资源的分类与多样性研究、品种鉴别与亲子鉴定研究以及重要性状的SSR标记与定位研究进展,并对存在问题进行分析和展望,旨在为相关研究工作提供参考。     

全柱成像毛细管等电聚焦电泳测定艾塞那肽等电点

目的:采用新一代全柱成像毛细管等电聚焦电泳技术(CIEF-WCID)测定艾塞那肽等电点。方法:采用互补性金属氧化物半导体成像技术对样品等电聚焦过程进行实时记录,根据适宜的marker计算得到艾塞那肽的等电点,并对方法的准确度与重复性进行考察。结果:测得艾塞那肽等电点为5.46,与凝胶电泳结果基本一致,相对标准偏差为0.11%。CIEF-WCID方法快速准确,相对误差小于2.5%,重复性良好。结论:CIEF-WCID作为一种新的技术手段可用于艾塞那肽等电点的分析,方法快速、准确、重复性好,可为多肽的质量控制提供一种可靠的分析方法。     

基于RAPD改进的分子标记技术

随机扩增多态DNA(RAPD)由于经济、快速简便、不需预知模板DNA信息、对模板DNA要求较低,因此被大量应用于昆虫学的研究中,如昆虫的鉴定分类、构建连锁图谱、系统发育分析等。但RAPD也有许多缺点,主要是一种显性标记而不能区分杂合体和纯合体,结果的重复性易受多因素的影响等。针对这些缺点,人们通过将RAPD与其它检测技术如聚丙烯酰胺凝胶电泳、单链构象多态(SSCP)、变性梯度凝胶电泳(DGGE)等相结合,提高了其分析的灵敏度和稳定性;再者,还将RAPD标记发展成为其它更稳定的或共显性的标记,如SCAR、微卫星等,为RAPD进一步的应用开阔了前景。文章对这些检测和转化的改进技术进行综述。     

Low-dose CT imaging of radio-opaque markers for assessing human rotator cuff repair: accuracy, repeatability and the effect of arm position

Previous studies have used radiostereometric analysis (RSA) to assess the integrity and mechanical properties of repaired tendons and ligament grafts. A conceptually similar approach is to use CT imaging to measure the 3D position and distance between implanted markers. The purpose of this study was to quantify the accuracy and repeatability of measuring the position and distance between metallic markers placed in the rotator cuff using low-dose CT imaging. We also investigated the effect of repeated or variable positions of the arm on position and distance measures. Six human patients had undergone rotator cuff repair and placement of tantalum beads in the rotator cuff at least one year prior to participating in this study. On a single day each patient underwent nine low-dose CT scans in seven unique arm positions. CT scans were analyzed to assess bias, precision and RMS error of the measurement technique. The effect of repeated or variable positions of the arm on the 3D position of the beads and the distance between these beads and suture anchors in the humeral head were also assessed. Results showed the CT imaging method is accurate and repeatable to within 0.7 mm. Further, measures of bead position and anchor-to-bead distance are influenced by arm position and location of the bead within the rotator cuff. Beads located in the posterior rotator cuff moved medially as much as 20 mm in abduction or external rotation. When clinically relevant CT arm positions such as the hand on umbilicus or at side were repeated, bead position varied less than 4 mm in any anatomic direction and anchor-to-bead distance varied +2.8 to -1.6 mm (RMS 1.3 mm). We conclude that a range of ± 3 mm is a conservative estimate of the uncertainty in anchor-to-bead distance for patients repeatedly scanned in clinically-relevant arm positions.     

Genetic and Physical Mapping of Telomeres in the Rice Blast Fungus, Magnaporthe Grisea

Telomeric restriction fragments were genetically mapped to a previously described linkage map of Magnaporthe grisea, using RFLPs identified by a synthetic probe, (TTAGGG)(3). Frequent rearrangement of telomeric sequences was observed in progeny isolates creating a potential for misinterpretation of data. Therefore a consensus segregation data set was used to minimize mapping errors. Twelve of the 14 telomeres were found to be genetically linked to existing RFLP markers. Second-dimensional electrophoresis of restricted chromosomes confirmed these linkage assignments and revealed the chromosomal location of the two unlinked telomeres. We were thus able to assign all 14 M. grisea telomeres to their respective chromosome ends. The Achilles' cleavage (AC) technique was employed to determine that chromosome 1 markers 11 and CH5-120H were ~1.8 Mb and 1.28 Mb, respectively, from their nearest telomeres. RecA-AC was also used to determine that unlinked telomere 6 was ~530 kb from marker CH5-176H in strain 2539 and 580 kb in Guy11. These experiments indicated that large portions of some chromosome ends are unrepresented by genetic markers and provided estimates of the relationship of genetic to physical distance in these regions of the genome.     

Point and interval estimates of marker location in radiation hybrid mapping.

Radiation hybrid (RH) mapping is a powerful method for ordering loci on chromosomes and for estimating the distances between them. RH mapping is currently used to construct both framework maps, in which all markers are ordered with high confidence (e.g., 1,000:1 relative maximum likelihood), and comprehensive maps, which include markers with less-confident placement. To deal with uncertainty in the order and location of markers, marker positions may be estimated conditional on the most likely marker order, plausible intervals for nonframework markers may be indicated on a framework map, or bins of markers may be constructed. We propose a statistical method for estimating marker position that combines information from all plausible marker orders, gives a measure of uncertainty in location for each marker, and provides an alternative to the current practice of binning. Assuming that the prior distribution for the retention probabilities is uniform and that the marker loci are distributed independently and uniformly on an interval of specified length, we calculate the posterior distribution of marker position for each marker. The median or mean of this distribution provides a point estimate of marker location. An interval estimate of marker location may be constructed either by using the 100(alpha/2) and 100(1-alpha)/2 percentiles of the distribution to form a 100(1-alpha) % posterior credible interval or by calculating the shortest 100(1-alpha) % posterior credible interval. These point and interval estimates take into account ordering uncertainty and do not depend on the assumption of a particular marker order. We evaluate the performance of the estimates on the basis of results from simulated data and illustrate the method with two examples.     

In vivo tests of an improved method for functional location of the subtalar joint axis

The subtalar joint is important in frontal plane movement and posture of the hindfoot. Abnormal subtalar joint moments caused by muscle forces and the ground reaction force acting on the foot are thought to play a role in various foot deformities. Calculating joint moments typically requires knowledge of the location of the joint axis; however, location of the subtalar axis from measured movement is difficult because the talus cannot be tracked using skin-mounted markers. The accuracy of a novel technique for locating the subtalar axis was assessed in vivo using magnetic resonance imaging. The method was also tested with skin-mounted markers and video motion analysis. The technique involves applying forces to the foot that cause pure subtalar joint motion (with negligible talocrural joint motion), and then using helical axis decomposition of the resulting tibiocalcaneal motion. The resulting subtalar axis estimates differed by 6° on average from the true best-fit subtalar axes in the MRI tests. Motion was found to have been applied primarily about the subtalar joint with an average of only 3° of talocrural joint motion. The proposed method provides a potential means for obtaining subject-specific subtalar axis estimates which can then be used in inverse dynamic analyses and subject-specific musculoskeletal models.     

Repeatability assessment of functional methods to estimate the glenohumeral joint centre

Functional methods can be used to determine the centre of rotation (CoR) of a ball-and-socket joint. The algorithms are used to locate rather the hip joint centre than the glenohumeral joint centre. The choice of the most suitable method depends especially on the intra- and inter-session repeatability of these methods. This paper aims at evaluating the intra- and inter-session repeatability of functional methods with which the glenohumeral joint rotation centre (GHRC) can be estimated in vivo. It also estimates the most suitable amplitude of functional movements. Five functional methods were tested: the algorithms of Gamage and Lasenby, bias compensation, symmetrical CoR estimation, normalisation method and helical axis. Ten subjects performed three cycles of three different movements (flexion–extension, abduction–adduction and circumduction). These movements were repeated three times with three different ranges of motion. Six subjects came back in order to evaluate the inter-session repeatability. For each test, the location of the GHRC was estimated by the five methods. The method to solve the functional problem and the range of functional movement affected the GHRC location. The results showed a good to excellent intra-session repeatability. The lowest repeatability error was found for the high amplitude whatever the methods used. The inter-session reliability was moderate. Finally, we suggest the use of functional methods with high amplitude movement in order to locate the GHRC with the best reliability.     

Constrained least-squares optimization for robust estimation of center of rotation

This paper presents a new direct method for estimating the average center of rotation (CoR). An existing least-squares (LS) solution has been shown by previous works to have reduced accuracy for data with small range of motion (RoM). Alternative methods proposed to improve the CoR estimation use iterative algorithms. However, in this paper we show that with a carefully chosen normalization scheme, constrained least-squares solutions can perform as well as iterative approaches, even for challenging problems with significant noise and small RoM. In particular, enforcing the normalization constraint avoids poor fits near plane singularities that can affect the existing LS method. Our formulation has an exact solution, accounts for multiple markers simultaneously, and does not depend on manually-adjusted parameters. Simulation tests compare the method to four published CoR estimation techniques. The results show that the new approach has the accuracy of the iterative methods as well as the short computation time and repeatability of a least-squares solution. In addition, application of the new method to experimental motion capture data of the thumb carpometacarpal (CMC) joint yielded a more plausible CoR location compared to the previously reported LS solution and required less time than all four alternative techniques.     

Locations of genetic markers on the physical map of the chromosome of Neisseria gonorrhoeae FA1090.

To increase the utility of the previously constructed physical map of the chromosome of Neisseria gonorrhoeae FA1090, 28 additional genetic markers were localized on the map. Cloned gonococcal genes were used to probe Southern blots of restriction enzyme-digested DNA separated on pulsed-field gels, thus identifying the fragment in each of several digests to which the probe hybridized and the map location of each gene. The addition of the new markers brings the total number of mapped loci for this strain to 68; the locations of all of those markers on the updated map are shown.     

Chromosomal anchoring of linkage groups and identification of wing size QTL using markers and FISH probes derived from microdissected chromosomes in Nasonia (Pteromalidae: Hymenoptera)

Nasonia vitripennis is a small parasitic hymenopteran with a 50-year history of genetic work including linkage mapping with mutant and molecular markers. For the first time we are now able to anchor linkage groups to specific chromosomes. Two linkage maps based on a hybrid cross (N. vitripennis x N. longicornis) were constructed using STS, RAPD and microsatellite markers, where 17 of the linked STS markers were developed from single microdissected banded chromosomes. Based on these microdissections we anchored all linkage groups to the five chromosomes of N. vitripennis. We also verified the chromosomal specificity of the microdissection through in situ hybridization and linkage analyses. This information and technique will allow us in the future to locate genes or QTL detected in different mapping populations efficiently and fast on homologous chromosomes or even chromosomal regions. To test this approach we asked whether QTL responsible for the wing size in two different hybrid crosses (N. vitripennis x N. longicornis and N. vitripennis x N.giraulti) map to the same location. One QTL with a major effect was found to map to the centromere region of chromosome 3 in both crosses. This could indicate that indeed the same gene/s is involved in the reduction of wing in N. vitripennis and N. longicornis.     

Shear wave elastography of the supraspinatus muscle and tendon: Repeatability and preliminary findings

Shear wave elastography (SWE) is a promising tool for estimating musculoskeletal tissue properties, but few studies have rigorously assessed its repeatability and sources of error. The objectives of this study were to assess: (1) the extent to which probe positioning error and human user error influence measurement accuracy, (2) intra-user, inter-user, and day-to-day repeatability, and (3) the extent to which active and passive conditions affect shear wave speed (SWS) repeatability. Probe positioning and human usage errors were assessed by acquiring SWE images from custom ultrasound phantoms. Intra- and inter-user repeatability were assessed by two users acquiring five trials of supraspinatus muscle and tendon SWE images from ten human subjects. To assess day-to-day repeatability, five of the subjects were tested a second time, approximately 24 h later. Imaging of the phantoms indicated high inter-user repeatability, with intraclass correlation coefficient (ICC) values of 0.68–0.85, and RMS errors of no more than 4.1%. SWE imaging of the supraspinatus muscle and tendon had high repeatability, with intra- and inter-user ICC values of greater than 0.87 and 0.73, respectively. Day-to-day repeatability demonstrated ICC values greater than 0.33 for passive muscle, 0.48 for passive tendon, 0.65 for active muscle, and 0.94 for active tendon. This study indicates the technique has good to very good intra- and inter-user repeatability, and day-to-day repeatability is appreciably higher when SWE images are acquired under a low level of muscle activation. The findings from this study establish the feasibility and repeatability of SWE for acquiring data longitudinally in human subjects.     

Integration of Retrotransposons-Based Markers in a Linkage Map of Barley

A deeper understanding of random markers is important if they are to be employed for a range of objectives. The sequence specific amplified polymorphism (S-SAP) technique is a powerful genetic analysis tool which exploits the high copy number of retrotransposon long terminal repeats (LTRs) in the plant genome. The distribution and inheritance of S-SAP bands in the barley genome was studied using the Steptoe × Morex (S × M) double haploid (DH) population. Six S-SAP primer combinations generated 98 polymorphic bands, and map positions were assigned to all but one band. Eight putative co-dominant loci were detected, representing 16 of the mapped markers. Thus at least 81 of the mapped S-SAP loci were dominant. The markers were distributed along all of the seven chromosomes and a tendency to cluster was observed. The distribution of S-SAP markers over the barley genome concurred with the knowledge of the high copy number of retrotransposons in plants. This experiment has demonstrated the potential for the S-SAP technique to be applied in a range of analyses such as genetic fingerprinting, marker assisted breeding, biodiversity assessment and phylogenetic analyses.     

Meta-analysis of genetic-linkage analysis of quantitative-trait loci

Meta-analysis is an important tool in linkage analysis. The pooling of results across primary linkage studies allows greater statistical power to detect quantitative-trait loci (QTLs) and more-precise estimation of their genetic effects and, hence, yields conclusions that are stronger relative to those of individual studies. Previous methods for the meta-analysis of linkage studies have been proposed, and, although some methods address the problem of between-study heterogeneity, most methods still require linkage analysis at the same marker or set of markers across studies, whereas others do not result in an estimate of genetic variance. In this study, we present a meta-analytic procedure to evaluate evidence from several studies that report Haseman-Elston statistics for linkage to a QTL at multiple, possibly distinct, markers on a chromosome. This technique accounts for between-study heterogeneity and estimates both the location of the QTL and the magnitude of the genetic effect more precisely than does an individual study. We also provide standard errors for the genetic effect and for the location (in cM) of the QTL, using a resampling method. The approach can be applied under other conditions, provided that the various studies use the same linkage statistic.     

Construction of a mitotic linkage map of Fusarium oxysporum based on Foxy -AFLPs

Construction of the first mitotic linkage map of the asexual fungus Fusarium oxysporum, based on a population of 32 parasexual fusion products, is reported. Molecular markers were developed using a modified AFLP technique which combines a Foxy-specific primer with standard adapter primers. The retroposon Foxy is abundantly present and highly variable in location in F. oxysporum isolates: 43% of the Foxy-AFLP markers tested appeared to be polymorphic between the strains Fol004 and Fol029. Of the 102 Foxy markers obtained, 83 segregated in a 1:1 ratio. The remaining fragments showed a skewed segregation pattern in which the Fol004 derived Foxy fragments were overrepresented. Foxy markers were observed to be clustered, suggesting that active Foxy elements may not transpose very far from their initial insertion sites, or that hotspots for insertion may exist. Linkage analysis revealed 23 linkage groups. Physical linkage between segregating markers predicted to be 20 cM apart was confirmed, indicating that the mitotic linkage map is reliable.