Analysis of normal and modified nucleosides in urine by capillary electrophoresisa)

Summary Modified nucleosides excreted in urine have been studied as potential diagnostic markers for cancer and AIDS, and as indicators for the whole-body turnover of RNA. Until now, reversed-phase (RP) HPLC and, to some extent, immunoassays are the preferred analytical methods for urinary nucleosides. A new capillary electrophoretic method for the analysis of normal and modified nucleosides in urine has been developed and optimized in our laboratory. The separation of nucleosides extracted from normal human urine on phenyl boronic acid affinity chromatography columns was performed in uncoated 565 mm (500 mm to detection window) × 50 μm i.d. capillary tubing using a 300 mM SDS—25 mM borate—50 mM phosphate buffer (pH 6.7), a 45-s load, a voltage of 7.5 kV (41 μA) and UV detection at 260 and 210 nm. The average recovery of the nucleosides was 91 %. The calibration curves were linear over all physiological and pathophysiological concentration ranges and the limits of detection were at micromolar levels. Reproducibility of migration times were better than 1 % (coefficient of variation,CV), and the reproducibilities of the determined concentrations were better than 5 % for standards and 6–15 % for extracted urine. The developed method was used to quantify 15 normal and modified nucleosides in 25 normal urines to establish reference ranges. The analysis time was less than 45 min. Dedicated to Professor E. Bayer on the occasion of his 70th birthday. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.     

CT血流灌注结合血脑屏障标志蛋白对中毒性休克患者神经元损伤的评价价值

选择2017年12月~2018年11月我院收治的100例经临床确认的中毒性休克患者的临床资料,所有患者均进行CT血流灌注成像检查,原始图像经工作站自带的脑灌注软件处理后产生灌注参数伪彩图像,测量病变侧及对侧大脑半球多个解剖部位的脑血流量(CBF)、脑血容量(CBV)、平均通过时间(MTT)、达峰时间(TTP)等灌注参数,计算病变侧/对侧的对比值(rCBF、rCBV、rMTT、rTTP)。并使用免疫组学方法检测不同患者外周血及脑脊液中血脑屏障通透性与紧密连接蛋白claudin-5、occludin、ZO-1、JAM-1、NOS和NSE的表达水平变化,并比较灌注参数与血脑屏障标志蛋白水平的相关性。结果显示,观察组患者外周血及脑脊液rCBF显著下降,rTTP降低,rPS升高(P<0.05),但两组rMTT、rCBV比较,差异无统计学意义(P>0.05)。观察组患者外周血及脑脊液claudin-5、occludin、ZO-1、NOS显著下降,JAM-1、NSE明显升高(P<0.05)。观察组claudin-5、occludin、ZO-1和NOS与rCBF、rTTP呈正相关,与rPS呈负相关;JAM-1和NSE与rCBF、rTTP呈负相关,与rPS呈正相关。rCBF、rTTP、JAM-1、NSE与神经元损伤程度呈正相关,claudin-5、occludin、ZO-1和NOS与神经元损伤程度呈负相关。上述结果表明CT血流灌注参数结合血脑屏障标志蛋白可有效评估中毒性休克患者神经元损伤程度,值得推广和应用。     

Evaluation of the microhaplotype markers in kinship analysis

Microhaplotype markers are emerging forensic genetic markers, which may supplement existing markers. Consisting of two to four SNPs with an extent of <200 bp, microhaplotype can be genotyped through massively parallel sequencing technology. Articles that have been published suggested that microhaplotype markers have good application prospect in forensics. Multiallelic haplotype loci are potentially important in certain forensic works, as the stutter and high mutation rate of short tandem repeats and the low polymorphism of single nucleotide polymorphisms may limit the power of these two kind of regular markers. In this study, we explored the potential of 11 new microhaplotype loci in kinship analysis. The results suggested that these loci have relatively high polymorphic information in different populations worldwide and relatively high system effectiveness in the kinship analysis. Microhaplotypes have potential for forensic kinshipg analysis.     

A high-performance SNP panel developed by machine-learning approaches for characterizing genetic differences of Southern and Northern Han Chinese,Korean, and Japanese individuals

Population stratification analyses targeting genetically closely related East Asians have revealed that distinguishable differentiation exists between Han Chinese, Korean, and Japanese individuals, as well as between southern (S-) and northern (N-) Han Chinese. Previous studies offer a number of choices for ancestry informative single nucleotide polymorphisms (AISNPs) to discriminate East-Asian populations. In this study, we collected and examined the efficiency of 1185 AISNPs using frequency and genotype data from various publicly available databases. With the aim to perform fine-scale classification of S-Han, N-Han, Korean, and Japanese subjects, machine-learning methods (Softmax and Random Forest) were used to screen a panel of highly informative AISNPs and to develop a superior classification model. Stepwise classification was implemented to increase and balance the discrimination in the process of AISNP selection, first discriminating Han, Korean, and Japanese individuals, and then characterizing stratification between S-Han and N-Han. The final 272-AISNP panel is an alternative optimization of various previous works, which promises reliable and >90% accuracy in classification of the four East-Asian groups. This AISNP panel and the machine-learning model could be a useful and superior choice in medical genome-wide association studies and in forensic investigations for unknown suspect identity.     

A new weakly basic amino‐reactive fluorescent label for use in isoelectric focusing and chip electrophoresis

In this study, we present a novel amino‐reactive fluorescence marker (referred to as UR‐431), which is well suited for electrophoretic techniques. A main feature of this marker is its weakly basic behavior when conjugated to analytes. Labeled primary amines exhibit a positive net charge and accordingly a cathodic mobility below a pH of 2.4. The label features a pH‐independent fluorescence emission and is thus very interesting for electrophoretic applications such as IEF. The absorption maximum of this yellow daylight chromophore is at 431 nm, whereas fluorescence emission peaks at 537 nm (quantum yield≈0.1). The label was successfully conjugated to amines, peptides and proteins and separated via CE and MCE. The on‐chip detection limit of labeled lysine using a mercury‐lamp‐based fluorescence microscope was determined as 12 nM. An important feature of the new label is that it effects only a subtle change of the pI of proteins compared with common anionic labels, e.g. FITC. pI values of proteins were investigated by comparing native proteins and labeled proteins in CIEF. UR‐431 was also applied to sensitive detection of amines and peptides in MCE.     

High-performance counter-current chromatography purification and off-line mass spectrometry monitoring and identification of pyrrolizidine alkaloid markers of tropical Ghanaian honey

The nutritional and medicinal properties of honey have been well-documented. However, honey has occasionally been contaminated with hepatotoxic pyrrolizidine alkaloids as a result of bees foraging on the flowers of pyrrolizidine alkaloid plants. This study establishes a simple and rapid method to determine the marker pyrrolizidine alkaloids in honey using high-performance counter-current chromatography and an off-line electrospray ionization-tandem mass spectrometry, in order to identify the botanical sources responsible for the contamination. The honey sample was initially liquid-liquid extracted (sulfuric acid/hexane, 2:3, v/v) to enrich the pyrrolizidine alkaloids and subsequently purified by a semi-preparative high-performance counter-current chromatography using a solvent system, hexane/butanol/1% aqueous ammonia, 1:1:2, v/v, based on partition coefficient measurements of the target alkaloids. The recovered fractions were profiled by injecting them sequentially into an off-line electrospray ionization-tandem mass spectrometry device to monitor the preparative molecular weight based on elution and extrusion modes. The monitored lycopsamine-type pyrrolizidine alkaloids and their N-oxides (m/z 300, 316; lycopsamine, intermedine, rinderine, and echinatine) were used as the phytochemical markers to identify plants like Chromolaena odorata, Ageratum spp., or Heliotropium spp. to be responsible for the pyrrolizidine alkaloid contamination. Identification of these pyrrolizidine alkaloid plants could guide beekeepers in locating their beehives in order to minimize their potential liver damaging effects.     

Wall slip of mayonnaises in viscometers

The shear flow of mayonnaise is generally characterized by an apparent yield stress, shear thinning in steady flow, stress overshoots upon inception of flow and other time-dependent effects. These observations are usually understood to be the result of structural rearrangement within the material. Additionally and separately, the possibility that emulsions may exhibit apparent wall slip on a microscopic scale at a solid-liquid boundary has been reported by some researchers. Thus, observed rheological behavior is likely to be the result of the interplay between these two phenomena. In the present work, it is demonstrated that when measurements are sought to be made on mayonnaise using rotational viscometers visible wall slip occurs, rendering such instruments ineffective for the purpose of making viscosity measurements even at shear rates as low as 10^–3s^–1. The factors that influence the onset and extent of slip are investigated with the help of parallel plate viscometers, and it is concluded that the observed “yielding” of mayonnaise is actually an artifact of the onset of macroscopic slip. Slip effects are also found in capillary flow but are ameliorated with increasing shear rate. To circumvent these problems, it is proposed that extensional viscometry be employed for determining the flow behavior of mayonnaises. Received: 18 August 1997 Accepted: 1 April 1998     

A secretome-based methodology may provide a better characterization of the virulence of Listeria monocytogenes: preliminary results

Four strains of Listeria monocytogenes with different levels of virulence were studied. Two strains were consistently evaluated as virulent (strain 3077) and of low virulence (strain 3993), whereas the other two strains (3006 and 3049) originated conflicting results in what the evaluation tests were concerned: both were shown to exhibit low virulence when evaluated by in vitro assays, but virulent when the analyses were performed under in vivo conditions.To clarify the virulence potential of the selected strains, a proteomic approach was used after incubating L. monocytogenes cultures under conditions favoring the expression of virulence factors (minimal medium, at 37 °C). Bacterial proteins present in the liquid culture media were precipitated from late exponential phase cultures, fractionated by SDS-PAGE and identified by MALDI-TOF-MS.Three virulence factors differentially expressed were detected: protein p60, listeriolysin O (LLO) and internalin C (InlC). Clustering analysis of the four L. monocytogenes strains based on their secretome profiles allowed their categorization in two groups: the virulent group, composed by strains 3077 and 3049, and the low virulence group, containing strains 3993 and 3006. The results presented in this work suggest that the virulent potential of a particular L. monocytogenes strain may be predicted from the levels of both listeriolysin O (LLO) and internalin C (InlC) present in its secretome when the bacterium is grown under conditions favoring the expression of virulence factors. Following validation of this proposal through the analysis of a large array of strains, this methodology exhibits a great potential to be developed into an accurate and rapid method to characterize L. monocytogenes strain virulence.     

改进示波计时电位法

本文克服了dE/dt～E曲线线性范围窄、分辨率差等缺点,把dE/dt～E曲线的切口变成峰形,具有再现性好、线性范围宽、灵敏度高、分辨率好等优点.采用内标法和频谱分析进行了定量测试工作,取得了满意的结果.     

5种体色瓯江彩鲤线粒体COII基因的序列差异和遗传标记研究

利用PCR方法扩增获得瓯江彩鲤（Cyprinus carpio var．color）线粒体DNA的COⅡ基因,测定该基因片段序列．分析了瓯江彩鲤“全红”、“粉玉”、“粉花”、“麻花”和“大花”共20只个体的序列核苷酸位点差异和遗传多态性．结果表明,“大花”和“全红”遗传多样性最为丰富,“粉花”的遗传多样性相对贫乏．在长度为604bp的基因片段中,共检测到9个多态性核苷酸位点（占1．49%）,20只个体具有4种基因型,5种体色各自的平均核苷酸位点差异分别为0．38％、0．2％、0、0．13％和0．47％．UPGMA分子系统聚类树显示,“粉花”、“麻花”和“粉玉”的遗传关系接近;“全红”相对独立成一支,与其他4种体色瓯江彩鲤的遗传关系较远．COⅡ基因可以作为区分三分支群体的遗传标记．