Sightseeing Around the Single Cosinor

Misuses of the single cosinor analysis are numerous. We give three rules of thumb, providing guidelines as to whether results obtained from this analysis are dubious. This short note avoids mathematical development so as to be comprehensible to the nonspecialist. However, a few endnotes, give some technical comments.     

Sampling strategies for using female gametophytes to estimate heterozygosity in conifers

Summary Unbiased estimators of genotype and allele frequencies and their respective variances are obtained for loci identified by mendelian segregation in haploid female gametophytes from individual trees. By a minimum sampling variance criterion, the allocation of experimental effort between the number of female gametophytes analysed per tree and the number of trees sampled per population is examined for a fixed total amount of experimental effort. For estimating heterozygosity, the optimum sampling design for many (generally most) cases is three female gametophytes per tree, but may be more than three depending upon the true genotype frequencies in the population. For estimating allele frequencies, the optimum sampling design is one female gametophyte per tree except in cases where a strong negative correlation exists between alleles within genotpyes. Guidelines are discussed for determining a suitable number of female gametophytes to be analysed per tree in order to estimate heterozygosity.     

单分子免疫检测技术研究进展*

早诊断、早发现、早治疗是提升肿瘤患者生存率的主要手段。临床常用的免疫学检测方法如酶联免疫吸附法、化学发光法等,其检测灵敏度多限制在10^-14~10^-12 mol/L,无法满足早期诊断的需求。单分子免疫检测法,可将待检测分子限制在极小空间范围内(nL以下),对检测信号进行绝对计数,从而实现痕量(可达10^-18 mol/L)标志物的检测。这一超高灵敏度技术实现的关键在于将检测范围限制在极小体积内。经过数十年发展,不论是物理隔离还是利用纳米孔,抑或通过改进显微镜性能,均可在极小体积内(10^-21 L)对信号进行检测。目前基于微阵列的SimoA检测系统已成为单分子免疫检测的金标准,Quanterix公司基于此开发的HD-1分析仪已进入市场应用。基于微液滴的单分子免疫检测技术主要限于实验室,但具有床旁检测的优势。重点介绍了基于物理隔离形式如微阵列和微液滴的单分子免疫检测进展,为进一步开发超高灵敏度检测方法并促进未来临床应用提供理论基础。     

Single particle reconstruction of the human apo-transferrin-transferrin receptor complex

Most organisms depend on iron as a co-factor for proteins catalyzing redox reactions. Iron is, however, a difficult element for cells to deal with, as it is insoluble in its ferric (Fe3+) form and potentially toxic in its ferrous (Fe2+) form. Thus, in vertebrates iron is transported through the circulation bound to transferrin (Tf) and delivered to cells through an endocytotic cycle involving the transferrin receptor (TfR). We have previously presented a model for the Tf-TfR complex in its iron-bearing form, the diferric transferrin (dTf)-TfR complex [Cheng, Y., Zak, O., Aisen, P., Harrison, S.C., Walz, T., 2004. Structure of the human transferrin receptor-transferrin complex. Cell 116, 565-576]. We have now calculated a single particle reconstruction for the complex in its iron-free form, the apo-transferrin (apoTf)-TfR complex. The same density map was obtained by aligning raw particle images or class averages of the vitrified apoTf-TfR complex to reference models derived from the structures of the dTf-TfR or apoTf-TfR complex. We were unable to improve the resolution of the apoTf-TfR density map beyond 16A, most likely because of significant structural variability of Tf in its iron-free state. The density map does, however, support the model for the apoTf-TfR we previously proposed based on the dTf-TfR complex structure, and it suggests that receptor-bound apoTf prefers to adopt an open conformation.     

单分子磁体的研究进展

单分子磁体为纳米尺寸,磁特性源自单个分子的内部,可以独立地作为一个磁功能单元,是突破尺寸对传统磁体性能制约的一条连径。已知单分子磁体基本上是含Mn、Fe、V、Cr和一些其他金属元素的簇合物．有望用来制造分子器件、磁存储材料等。本文介绍了一些典型的单分子磁体的研究和发展趋势。     

Rapid Communication: Neuropeptide Expression and Processing as Revealed by Direct Matrix-Assisted Laser Desorption Ionization Mass Spectrometry of Single Neurons

Abstract: Neuropeptides were directly detected in single identified neurons and the neurohemal area of peptidergic (neuroendocrine) systems in the Lymnaea brain by using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The samples were placed in matrix solution and ruptured to allow mixing of cell contents with the matrix solution. After formation of matrix crystals, the analytes were analyzed by MALDI-MS. It was surprising that clean mass spectra were produced, displaying extreme sensitivity of detection. In one of the neuroendocrine systems studied, we could demonstrate for the first time, by comparing the peptide patterns of soma and of neurohemal axon terminals, that processing of the complex prohormone expressed in this system occurs entirely in the soma. In the other system studied, novel peptides could be detected in addition to peptides previously identified by conventional molecular biological and peptide chemical methods. Thus, complex peptide processing and expression patterns could be predicted that were not detected in earlier studies using conventional methods. As the first MALDI- MS study of direct peptide fingerprinting in the single neuron these experients demonstrate that MALDI-MS forms a new and valuable approach to the study of the synthesis and expression of bioactive peptides, with potential application to single-cell studies in vertebrates, including humans.     

Primary production of four dominant salt-marsh angiosperms in the SW Netherlands

Results of a one-year evaluation of aboveground production of four dominant salt-marsh angiosperms show a distinctly higher level of production as compared with reported results for similar studies of salt-marsh vegetations in The Netherlands.Net aboveground production estimates based on production of both living and dead matter varied considerably depending on how the data were computed: Spartina anglica: 1162 to 1649 g m² yr¹; Triglochin maritima: 568 to 783 g m² yr¹; Halimione portulacoides: 790 to 1434 g m² yr¹; Elytrigía pungens: 474 to 878 g m² yr¹. Aboveground production estimates, using a paired plot method, are also strongly dependent on the computation of the data and ranged for Spartina anglica from 2139 to 2659 g m² yr¹ and for Elytrigia pungens from 1416 to 1787 g m² yr¹. Evaluation of the results of the production estimates suggested that the actual aboveground production is best approached using a paired plot method.Nomenclature follows Heukels-van Ooststroom (1977), Flora van Nederland, 19th ed. Wolters-Noordhoff, Groningen.Thanks are due to Dr P. Ketner (Wageningen) for his valuable comments and to Dr A. G. Vlasblom (Yerseke) for providing statistical advice.Communication No. 252.     

焦磷酸测序测定SNP的常见问题与解决方法

目的:探讨焦磷酸测序技术对单核苷酸多态性分型因测序图谱中存在的一些典型问题而导致分型结果不准确的解决方法。方法:以VKORC1基因1639 GA位点、CYP2C19基因636 GA位点及UGT1A1基因TA重复序列(TA)6(TA)7的多态性检测为例,分别采用优化PCR条件、改变测序时dNTP的加入顺序以及设立外标校正的方法来解决上述问题,从而提高焦测序对SNP分型的准确性。结果:通过升高PCR退火温度,可以显著提高VKORC1基因的扩增特异性,降低了测序图谱中非特异性信号峰强度;通过优化测序时dNTP的加入顺序,CYP2C19基因636 GA位点的准确分型结果可通过观察测序图谱中相关信号峰的有无而简单获得,避免了比较信号峰的相对强度;通过比较待测样本与已知基因型的外标样本的测序图谱来确定待测样本的基因型,提高了对UGT1A1基因TA重复序列(TA)6(TA)7多态性的分型准确性。结论:本文针对焦测序在测定SNP时的常见问题所提出的相应解决方法不仅简单、经济有效,而且在临床应用方面具有可靠性。     

用甘蔗渣生产单细胞蛋白的初步试验

本文报道利用微生物混合培养技术,以甘蔗渣为唯一碳源、生产单细胞蛋白的初步研究。将２株纤维单胞菌、３株霉菌、１株酵母进行分别培养和不同组合的培养,结果发现,混合培养较单株培养好。培养基在未经灭菌的情况下,以（ＮＨ４）２ＳＯ４为氮源,ｐＨ６．０,于３２℃振荡培养１０８ｈ,发酵产物的粗蛋白含量是甘蔗渣原材料的１２倍以上。     

大规模单核苷酸多态性分析与肿瘤易感性

单核苷酸多态性(SNPs)是人类基因组中最常见的变异形式。作为第三代遗传标记,SNP在基因定位、克隆、遗传多态性方面具有广泛应用,特别是作为基因诊断标记在预防医学中具有十分重要的作用。近年来,随着人类基因组计划的发展,数以百万计的SNP被陆续发现,并可在公共数据库中免费获得。SNP数量的快速增加和SNP检测方法的发展,为其在肿瘤易感性领城的应用提供了可能。在本综述中,我们介绍了几种高通量检测SNP的分析方法,总结了大规模SNP分析技术在肿瘤易感性中的应用,介绍了目前人们对于不同人群中的SNP分析、肿瘤易感基因、个体肿瘤易感性的理解,以及研究SNP标记与肿瘤易感性关系时存在的难点。     

焦磷酸测序测定SNP的常见问题与解决方法

目的：探讨焦磷酸测序技术对单核苷酸多态性分型因测序图谱中存在的一些典型问题而导致分型结果不准确的解决方法。方法：以VKORC1基因1639 G〉A位点、CYP2C19基因636 G〉A位点及UGT1A1基因TA重复序列（TA）6〉（TA）7的多态性检测为例,分别采用优化PCR条件、改变测序时dNTP的加入顺序以及设立外标校正的方法来解决上述问题,从而提高焦测序对SNP分型的准确性。结果：通过升高PCR退火温度,可以显著提高VKORC1基因的扩增特异性,降低了测序图谱中非特异性信号峰强度;通过优化测序时dNTP的加入顺序,CYP2C19基因636 G〉A位点的准确分型结果可通过观察测序图谱中相关信号峰的有无而简单获得,避免了比较信号峰的相对强度;通过比较待测样本与已知基因型的外标样本的测序图谱来确定待测样本的基因型,提高了对UGT1A1基因TA重复序列（TA）6〉（TA）7多态性的分型准确性。结论：本文针对焦测序在测定SNP时的常见问题所提出的相应解决方法不仅简单、经济有效,而且在临床应用方面具有可靠性。     

Microdissection and PCR Amplification of Single Soybean Chromosome

Glass needles were successfully used to dissect the soybean (Glycine max L. ) single chromosome under the micromanipulator in this research. Two dissected soybean chromosomes were digested by Sau3A in two 0.5 mL Eppendorf tubes respectively. The two ends of chromosomal fragments were ligated with Sau3A linker adoptor. After two rounds of PCR amplification, smear DNA fragments ranged from 0.3 to 3 kb were acquired. Southern hybridization result showed the PCR products from the two single soybean chromosomes were homogeneous with the soybean genomic DNA, indicating that DNAs from the two single chromosomes have been successfully amplified. At the same time, the amplified products from the two of the distinguished single chromosome appeared somewhat different. The authors dissected the small chromosomes only by a traditional inverted microscope. Therefore, this research provides a plausible chance for amplification and microcloning of single small chromosomes.     

单分子磁体的研究进展

单分子磁体为纳米尺寸,磁特性源自单个分子的内部,可以独立地作为一个磁功能单元,是突破尺寸对传统磁体性能制约的一条途径。已知单分子磁体基本上是含Mn、Fe、V、Cr和一些其他金属元素的簇合物,有望用来制造分子器件、磁存储材料等。本文介绍了一些典型的单分子磁体的研究和发展趋势。     

Glutamate Decarboxylase Activities in Single Vertebrate Neurons

An enzymatic microassay method for glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) was improved to a degree yielding high sensitivity and low blank. Single cell bodies of anterior horn cells and dorsal root ganglion cells were dissected out from the freeze-dried sections of rabbit and chicken spinal cords and Purkinje cell bodies from those of rabbit cerebellum. A minute amount of GABA, present in single neurons or synthesized by GAD in single neurons, was enzymatically converted to NADPH. The NADPH was amplified 10,000-350,000-fold and measured, using an enzymatic amplification reaction (NADP cycling). GAD was contained in all Purkinje cell bodies and its average activity was four- to fivefold higher than those of the molecular and granular layers of rabbit cerebellum. The GABA concentration was threefold higher in Purkinje cell bodies than in these layers. GAD activity, at a level similar to that in the cerebellar layers, was found in almost all the cell bodies of anterior horn cells from rabbit and chicken. GABA was detected in 40% of rabbit neurons and not in chicken neurons. Dorsal root ganglion cells from both species contained no measurable GAD or GABA.     

具脉冲扩散效应的单种群动力学模型

讨论了两斑块间脉冲扩散的单种群动力学模型,利用离散动力系统频闪映射理论,得到了种群持续生存的充分条件．结论31,1~了现实的生物种群动力学性质,也丰富了脉冲微分方程理论．     

Single chain antibodies that recognize the N-glycosylation site

We aimed to identify antibodies that can recognize the Asn-Xaa-Ser/Thr(NXS/T) N-glycosylation site that guides oligosaccharyltransferase (OT) activity. We used synthetic Asn-Cys-Ser/Thr(NCS/T) tripeptides conjugated to bovine serum albumin to isolate single chain antibody fragments of a variable region (scFv) from the Griffin 1 phage antibody library. Although Ser and Thr have different side chains, the scFv proteins thus isolated bound to both NCS and NCT with Kd values of the order of 10(-6) M and accepted the substitution of the Cys residue with various amino acids, including Ala, Gly, and Val. However, these proteins recognized neither Asn-Pro-Ser/Thr nor non-NXS/T tripeptides. The scFv proteins recognized NCS/T and N-glycosylation site of mutant yeast protein disulfide isomerase when they were in their native but not denatured state. These results indicate that antibody recognition of the NXS/T motif is conformation dependent and suggest that NXS/T spontaneously adopts a specific conformation that is necessary for antibody recognition. These features are likely to correlate with the known binding specificity of OT.     

A comprehensive system of cosinor treatment programs written for the Apple II microcomputer

A comprehensive set of cosinor treatment programs has been written for an Apple II microcomputer. The system includes Single Cosinor, Mean (population) Cosinor and Serial Sections analyses as well as extensive graphics and file management. The package is integrated and used through a hierarchically ordered system of menus and choices. 48k memory and two disk drives are required, and both EPSON and SILENTYPE printers are supported.     

imzML--a common data format for the flexible exchange and processing of mass spectrometry imaging data

The application of mass spectrometry imaging (MS imaging) is rapidly growing with a constantly increasing number of different instrumental systems and software tools. The data format imzML was developed to allow the flexible and efficient exchange of MS imaging data between different instruments and data analysis software. imzML data is divided in two files which are linked by a universally unique identifier (UUID). Experimental details are stored in an XML file which is based on the HUPO-PSI format mzML. Information is provided in the form of a 'controlled vocabulary' (CV) in order to unequivocally describe the parameters and to avoid redundancy in nomenclature. Mass spectral data are stored in a binary file in order to allow efficient storage. imzML is supported by a growing number of software tools. Users will be no longer limited to proprietary software, but are able to use the processing software best suited for a specific question or application. MS imaging data from different instruments can be converted to imzML and displayed with identical parameters in one software package for easier comparison. All technical details necessary to implement imzML and additional background information is available at www.imzml.org.     

Single molecule fluorescence study of the Bacillus thuringiensis toxin Cry1Aa reveals tetramerization

Pore-forming toxins constitute a class of potent virulence factors that attack their host membrane in a two- or three-step mechanism. After binding to the membrane, often aided by specific receptors, they form pores in the membrane. Pore formation either unfolds a cytolytic activity in itself or provides a pathway to introduce enzymes into the cells that act upon intracellular proteins. The elucidation of the pore-forming mechanism of many of these toxins represents a major research challenge. As the toxins often refold after entering the membrane, their structure in the membrane is unknown, and key questions such as the stoichiometry of individual pores and their mechanism of oligomerization remain unanswered. In this study, we used single subunit counting based on fluorescence spectroscopy to explore the oligomerization process of the Cry1Aa toxin of Bacillus thuringiensis. Purified Cry1Aa toxin molecules labeled at different positions in the pore-forming domain were inserted into supported lipid bilayers, and the photobleaching steps of single fluorophores in the fluorescence time traces were counted to determine the number of subunits of each oligomer. We found that toxin oligomerization is a highly dynamic process that occurs in the membrane and that tetramers represent the final form of the toxins in a lipid bilayer environment.     

Single molecule measurements and biological motors

Recent technological advances in lasers and optical detectors have enabled a variety of new, single molecule technologies to be developed. Using intense and highly collimated laser light sources in addition to super-sensitive cameras, the fluorescence of single fluorophores can now be imaged in aqueous solution. Also, laser optical tweezers have enabled the piconewton forces produced by pair of interacting biomolecules to be measured directly. However, for a researcher new to the field to begin to use such techniques in their own research might seem a daunting prospect. Most of the equipment that is in use is custom-built. However, most of the equipment is essence fairly simple and the aim of this article is to provide an entry point to the field for a newcomer. It focuses mainly on those practical aspects which are not particularly well covered in the literature, and aims to provide an overview of the field as a whole with references and web links to more detailed sources elsewhere. Indeed, the opportunity to publish an article such as this on the Internet affords many new opportunities (and more space!) for presenting scientific ideas and information. For example, we have illustrated the nature of optical trap data with an interactive Java simulation; provided links to relevant web sites and technical documents, and included a large number of colour figures and plots. Our group’s research focuses on molecular motors, and the bias of this article reflects this. It turns out that molecular motors have been a paradigm (or prototype) for single molecule research and the field has seen a rapid development in the techniques. It is hoped that the methods described here will be broadly applicable to other biological systems.This is an interactive contribution, which can be accessed at: