False labelling due to quenching failure of N‐hydroxy‐succinimide–ester‐coupled dyes

In comparative fluorescence gel electrophoresis experiments, cross‐talk was detected. It was traced back to a failure in the quenching process in typical labelling protocols. Despite a huge excess of potential reaction sites for the N‐hydroxy‐succinimide–ester‐coupled dye, sufficient active dye molecules were available after the quenching step to label protein molecules un‐specifically. It could be shown that only a 100‐fold increase in the amount of quencher will silence residual dye to such an extent that no artificial signals are detected.     

A method for finding optimal RNA secondary structures using a new entropy model (vsfold)

We are developing a program to calculate optimal RNA secondary structures. The model uses di-nucleotide pairing energies as with most traditional approaches. However, for long-range entropy interactions, the approach uses an entropy-loss model based on the accumulated sum of the entropy of bonding between each base-pair weighted inversely by the correlation of the RNA sequence (the Kuhn length). Stiff RNA forms very different structures from flexible RNA. The results demonstrate that the long-range folding is largely governed by this entropy and the Kuhn length.     

基于SVM和平均影响值的人肿瘤信息基因提取

基于基因表达谱的肿瘤分类信息基因选取是发现肿瘤特异表达基因、探索肿瘤基因表达模式的重要手段。借助由基因表达谱获得的分类信息进行肿瘤诊断是当今生物信息学领域中的一个重要研究方向,有望成为临床医学上一种快速而有效的肿瘤分子诊断方法。鉴于肿瘤基因表达谱样本数据维数高、样本量小以及噪音大等特点,提出一种结合支持向量机应用平均影响值来寻找肿瘤信息基因的算法,其优点是能够搜索到基因数量尽可能少而分类能力尽可能强的多个信息基因子集。采用二分类肿瘤数据集验证算法的可行性和有效性,对于结肠癌样本集,只需3个基因就能获得100%的留一法交叉验证识别准确率。为避免样本集的不同划分对分类性能的影响,进一步采用全折交叉验证方法来评估各信息基因子集的分类性能,优选出更可靠的信息基因子集。与基它肿瘤分类方法相比,实验结果在信息基因数量以及分类性能方面具有明显的优势。     

A/H6N1亚型禽流感病毒致病性评估及与2009 A/H1N1亚型流感病毒在哺乳动物体内的遗传兼容性

目的探讨人、禽流感病毒在哺乳动物体内的遗传兼容性,为下一步研究H6亚型禽流感病毒重配和致病性变异的分子机制奠定基础。方法野鸭源A/H6N1亚型禽流感病毒A/Mallard/SanJiang/275/2007以101EID50～106EID50的攻毒剂量经鼻内途径感染小鼠,通过临床症状观察、病毒滴定和病理切片观察进行病毒学和组织学两方面检测对小鼠的致病性;同时,将此病毒与2009年A/H1N1流感病毒A/Changchun/01/2009(H1N1)混合感染豚鼠,分析两株病毒在哺乳动物体内的遗传兼容性。每天采集豚鼠鼻洗液并用噬斑纯化技术获得重配病毒,对获得的重配病毒进行全基因组序列的测定。结果 H6N1亚型禽流感病毒能直接感染小鼠,但对小鼠不致死。106EID50的攻毒剂量可有效感染小鼠,攻毒后第5天,小鼠表现出被毛较粗乱、活动减少、体重下降、呼吸急促的临床症状,但至攻毒后第10天开始康复,而对照组(MOCK)小鼠在14 d的观察期内无明显临床症状。病毒滴定结果表明,该病毒主要在小鼠肺脏和鼻甲骨中复制,病毒滴度可达104.5EID50/mL。病理学观察发现感染小鼠肺泡壁增厚,有大量炎性细胞浸润,纤维蛋白渗出并伴有轻微出血;在A/H6N1和A/H1N1混合感染豚鼠的重配实验中,经过三轮噬斑纯化从豚鼠鼻洗液中分离到6株重配病毒,说明A/H6N1亚型禽流感病毒与A/H1N1亚型流感病毒具有很好的遗传兼容性,能在豚鼠体内能发生重配。结论野鸭源A/H6N1亚型流感病毒无需适应就能够感染哺乳动物;该病毒与A/H1N1流感病毒具有很好的遗传兼容性,在哺乳动物体内能够发生基因重配,产生新的重配病毒,其公共卫生意义应引起高度关注。     

Tetrapod Track Assemblage in the Hettangian of Sołtyków,Poland, and its Paleoenvironmental Background

Large theropod and sauropod footprints predominated in the continental Zagaje Formation (early Hettangian) exposed in the So?tyków outcrop. Geological and paleobotanical data indicate an inland seasonal habitat with low and high growing vegetation on the flood plain. The theropod-sauropod ichnofauna also contains occasional footprints of small ornithischians and mammals. The So?tyków track assemblage, together with other similar assemblages, suggests a wide range of habitats occupied by sauropods.     

Compatibility among entomopathogenic hyphocreales and two beneficial insects used to control Trialeurodes vaporariorum (Hemiptera: Aleurodidae) in Mediterranean greenhouses

The effect of the combined use of Encarsia formosa or Macrolophus caliginosus and one of three marketed mycoinsecticides, Mycotal® (Leucanicillium muscarium-based), Naturalis-L™ (Beauveria bassiana-based) and PreFeRal® (Isaria fumosorosea-based), on the control of the whitefly, Trialeurodes vaporariorum, was studied under laboratory and greenhouse conditions. The results of both types of tests, the bioassays and the greenhouse trials, for all combinations of E. Formosa with each of the three mycoinsecticides showed that the total mortality of larval populations of T. vaporariorum was not affected. The mortality of T. vaporariorum larvae treated in the second instar revealed the capacity for both B. bassiana- and L. muscarium-based formulations and E. formosa to kill the host either separately or in association. Because of its higher pathogenic activity (under our test conditions), L. muscarium provoked a large proportion of mycoses in larvae exposed to parasitization. In contrast, the efficacy of parasitization was higher in larvae treated with B. bassiana and exposed to E. formosa because of a lower pathogenic activity of the fungus. Bioassays carried out with third-instar larvae of T. vaporariorum showed a low susceptibility to both tested fungi. Consequently, mortalities recorded in larvae subjected to the combined treatments by consecutive exposures or at 2-4 days post-parasitization were mainly caused by the development of the parasitoid. Greenhouse trials showed that fungus-induced mortality of T. vaporariorum in plants treated with L. muscarium, I. fumosorosea, and B. Bassiana was significant compare to control. L. muscarium, B. bassiana and I. fumosorosea killed young whitefly larvae and limited parasitization to 10% or less. Second-instar larvae of M. caliginosus were not susceptible to L. muscarium and B. bassiana formulations with any mode of contamination: direct spraying of larvae, spraying on the foliar substrate or by contaminated T. vaporariorum prey. In greenhouse trials, M. caliginosus populations treated with fungi were not significantly affected compared to controls.     

Asparagine 175 of connexin32 is a critical residue for docking and forming functional heterotypic gap junction channels with connexin26

The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn(175) of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels.     

Confidence scores for prediction models

In medical statistics, many alternative strategies are available for building a prediction model based on training data. Prediction models are routinely compared by means of their prediction performance in independent validation data. If only one data set is available for training and validation, then rival strategies can still be compared based on repeated bootstraps of the same data. Often, however, the overall performance of rival strategies is similar and it is thus difficult to decide for one model. Here, we investigate the variability of the prediction models that results when the same modelling strategy is applied to different training sets. For each modelling strategy we estimate a confidence score based on the same repeated bootstraps. A new decomposition of the expected Brier score is obtained, as well as the estimates of population average confidence scores. The latter can be used to distinguish rival prediction models with similar prediction performances. Furthermore, on the subject level a confidence score may provide useful supplementary information for new patients who want to base a medical decision on predicted risk. The ideas are illustrated and discussed using data from cancer studies, also with high-dimensional predictor space.     

Formation of lamellar cross bridges in the annulus fibrosus of the intervertebral disc is a consequence of vascular regression

Cross bridges are radial structures within the highly organized lamellar structure of the annulus fibrosus of the intervertebral disc that connect two or more non-consecutive lamellae. Their origin and function are unknown. During fetal development, blood vessels penetrate deep within the AF and recede during postnatal growth. We hypothesized that cross bridges are the pathways left by these receding blood vessels.Initially, the presence of cross bridges was confirmed in cadaveric human discs aged 25 and 53 years. Next, L1-L2 intervertebral discs (n = 4) from sheep ranging in age from 75 days fetal gestation to adult were processed for paraffin histology. Mid-sagittal sections were immunostained for endothelial cell marker PECAM-1. The anterior and posterior AF were imaged using differential interference contrast microscopy, and the following parameters were quantified: total number of distinct lamellae, total number of cross bridges, percentage of cross bridges staining positive for PECAM-1, cross bridge penetration depth (% total lamellae), and PECAM-1 positive cross bridge penetration depth.Cross bridges were first observed at 100 days fetal gestation. The overall number peaked in neonates then remained relatively unchanged. The percentage of PECAM-1 positive cross bridges declined progressively from almost 100% at 100 days gestation to less than 10% in adults. Cross bridge penetration depth peaked in neonates then remained unchanged at subsequent ages. Depth of PECAM-1 positive cross bridges decreased progressively after birth. Findings were similar for both the anterior and posterior.The AF lamellar architecture is established early in development. It later becomes disrupted as a consequence of vascularization. Blood vessels then recede, perhaps due to increasing mechanical stresses in the surrounding matrix. In this study we present evidence that the pathways left by receding blood vessels remain as lamellar cross bridges. It is unclear whether the presence of cross bridges in the aging and degenerating intervertebral disc would be advantageous or detrimental, and this question should be addressed by future studies.