多重PCR在质粒拷贝数检测中的应用

聚合酶链式反应(PCR)作为常规分子克隆技术已在分子生物学的各个领域得到广泛应用.然而,多重PCR技术应用于质粒拷贝数检测的研究尚未见报道.为深入探索多重PCR在质粒拷贝数测定中的应用,首先利用构建的多重PCR引物设计及评估体系分别针对细菌基因组DNA和质粒载体DNA序列设计多重PCR引物;然后以转化有不同质粒载体的大...     

Faster, simpler, more-specific methods for improved molecular detection of Phytophthora ramorum in the field

Phytophthora ramorum is the causal agent of sudden oak death. The pathogen also affects a wide range of tree, shrub, and herbaceous species in natural and landscaped environments as well as plants in the nursery industry. A TaqMan real-time PCR method for the detection of this pathogen in the field has been described previously; this paper describes the development of a number of assays based on this method which have various advantages for use in the field. A scorpion real-time PCR assay that is twice as fast as TaqMan was developed, allowing the detection of P. ramorum in less than 30 min. Also designed was a loop-mediated isothermal amplification (LAMP) assay, which allowed sensitive and specific detection of P. ramorum in 45 min using only a heated block. A positive reaction was identified by the detection of the LAMP product by color change visible to the naked eye.     

Estimating the annual number of breeding attempts from breeding dates using mixture models

Well-established statistical methods exist to estimate variation in a number of key demographic rates from field data, including life-history transition probabilities and reproductive success per attempt. However, our understanding of the processes underlying population change remains incomplete without knowing the number of reproductive attempts individuals make annually; this is a key demographic rate for which we have no satisfactory method of estimating. Using census data to estimate this parameter from requires disaggregating the overlying temporal distributions of first and subsequent breeding attempts. We describe a Bayesian mixture method to estimate the annual number of reproductive attempts from field data to provide a new tool for demographic inference. We validate our method using comprehensive data on individually-marked song sparrows Melospiza melodia , and then apply it to more typical nest record data collected over 45 years on yellowhammers Emberiza citrinella . We illustrate the utility of our method by testing, and rejecting, the hypothesis that declines in UK yellowhammer populations have occurred concurrently with declines in annual breeding frequency.     

A one-step method for direct nonsurgical transfer of frozen-thawed bovine embryos

One impediment to the more widespread use of freezing as an adjunct to embryo transfer in cattle has been the method used to remove the protective compound from the thawed embryos. Recently, a method has been developed that permits bovine embryos to be diluted out of the protective solution within the plastic straw in which the embryos were originally rrozen and thawed. The method requires only about 10 minutes to perform and does not require a microscope or other laboratory equipment. Therefore, frozen bovine embryos can be thawed, diluted, and transferred nonsurgically into recipient cattle under conditions quite similar to those used for artificial insemination. A large number of field trials of this method have been performed during the past 2 1 2 years. A total of 327 pregnancies have been established by the nonsurgical transfer of 1259 embryos that had been frozen, thawed, and diluted by this method.     

Effects of field size when using Kerley's histological method for determination of age at death

The field size at which a bone is read affects the results obtained when using Kerley's histological method for age estimation, even after applying the recommended correction factor. Whereas there is no tendency for any one of three field sizes tested to consistently underestimate or overestimate age, a field size closest to that used by Kerley in his original study had significantly lower variances for its age estimates, and thus provides greater reliability. This particular field size yields more precise estimates because it is sampling a pattern and number of structures more similar to that of Kerley. Correction factors cannot equalize the counts of osteons and osteon fragments because of spatial variations in the distributions of these histological structures. A field size similar to that used by Kerley in gathering the data from which he developed his regression equations must be used to assure that the same pattern and number of structures is being sampled. For this reason, we suggest a field size as close to 2.06 mm² as possible be used when employing Kerley's method.     

The two-wavelength method of microspectrophotometry. I. A microspectrophotometer and tests on model systems

In spite of a number of examples of the apparently successful application of the two-wavelength method of microspectrophotometry to biological materials, a demonstration of the effective transition from the theoretical to the practical aspects of the method has never been given. The following tests were carried out on model systems in order to confirm the practical validity of the method: (1) Spherical droplets of Sudan blue or Sudan IV (varying in volume by as much as 400 times) were measured with the surrounding blank field included and were found to have amounts of chromophore which conformed to the estimated droplet volume to within 3 to 6 per cent. (2) Repeated determinations on the same isolated nucleus gave results which did not vary in spite of wide variations in the area of the photometric field. (3) The results of repeated determinations on the same droplet of dye were unaffected by gross changes in the focus of the objective. The microspectrophotometer on which these measurements were made has been briefly described; it was designed specifically for the two-wavelength method. Some of the details of instrumentation have been discussed, in particular, the effects of non-uniform illumination and the contrasting demands of the two integral parts of this type of photometry. Although it has certain limitations, the two-wavelength method conforms to its theoretical promise and is a practical method of measuring light absorption in complex objects.     

Phylogenetic invariants and geometry

The method of invariants is an important approach in biology for determining phylogenetic information which avoids the problems involving long branch lengths that plague some other methods. In this paper, we present a geometric framework underlying the method of invariants. This perspective sheds new lights on problems in the field. It has recently enabled the solution of questions on the number and structure of phylogenetic invariants and suggests possible avenues for future empirical and theoretical research.     

The Two-Wavelength Method of Microspectrophotometry : I. A Microspectrophotometer and Tests on Model Systems

In spite of a number of examples of the apparently successful application of the two-wavelength method of microspectrophotometry to biological materials, a demonstration of the effective transition from the theoretical to the practical aspects of the method has never been given. The following tests were carried out on model systems in order to confirm the practical validity of the method: (1) Spherical droplets of Sudan blue or Sudan IV (varying in volume by as much as 400 times) were measured with the surrounding blank field included and were found to have amounts of chromophore which conformed to the estimated droplet volume to within 3 to 6 per cent. (2) Repeated determinations on the same isolated nucleus gave results which did not vary in spite of wide variations in the area of the photometric field. (3) The results of repeated determinations on the same droplet of dye were unaffected by gross changes in the focus of the objective. The microspectrophotometer on which these measurements were made has been briefly described; it was designed specifically for the two-wavelength method. Some of the details of instrumentation have been discussed, in particular, the effects of non-uniform illumination and the contrasting demands of the two integral parts of this type of photometry. Although it has certain limitations, the two-wavelength method conforms to its theoretical promise and is a practical method of measuring light absorption in complex objects.     

Faster, Simpler, More-Specific Methods for Improved Molecular Detection of Phytophthora ramorum in the Field

Phytophthora ramorum is the causal agent of sudden oak death. The pathogen also affects a wide range of tree, shrub, and herbaceous species in natural and landscaped environments as well as plants in the nursery industry. A TaqMan real-time PCR method for the detection of this pathogen in the field has been described previously; this paper describes the development of a number of assays based on this method which have various advantages for use in the field. A scorpion real-time PCR assay that is twice as fast as TaqMan was developed, allowing the detection of P. ramorum in less than 30 min. Also designed was a loop-mediated isothermal amplification (LAMP) assay, which allowed sensitive and specific detection of P. ramorum in 45 min using only a heated block. A positive reaction was identified by the detection of the LAMP product by color change visible to the naked eye.     

Gene transfer strategies in plants

In recent years, a large number of gene transfer methods have been developed. However, the results of these studies have often been published in such a way that it has been extremely difficult for researchers to assess the reliability and efficiency of the method, and to judge whether or not integrative transformation has occurred. Thus although an abundance of knowledge exists within the area of gene transfer, its documentation remains disjointed.This report summarises the recent progress which has been made in the field of gene transfer systems in plants and discusses the associated advantages, disadvantages and limitations in an attempt to clarify this issue.     

Estimate of mating pattern of a paternal nest brooder goby of <Emphasis Type="Italic">Rhinogobius</Emphasis>, using egg density in the nest

The mating pattern of the paternal nest brooder Rhinogobius sp. CB (cross band type) exhibits both monogamy and polygyny. However, it is difficult to infer the actual conditions of their mating from their egg stages in the clusters, because multiple females spawn simultaneously or sequentially within short intervals. I examined whether the egg density of the egg clusters could be used as an index of the number of females, based on laboratory and field experiments. Egg densities increased with the number of spawning females. Egg densities of egg clusters collected in the field were categorized into three normal distributions which corresponded to those for the number of mating females in the spawning experiment. Egg density in the nest, therefore, seems to be a useful index for clarifying the mating pattern of this species in the field. By adopting the present method, it is suggested that this species frequently mates polygynously in the wild.     

Three-Dimensional Reconstruction from Reduced Sets of Very Noisy Images Acquired Following a Single-Axis Tilt Schema: Application of a New Three-Dimensional Reconstruction Algorithm and Objective Comparison with Weighted Backprojection

In this work we propose a reconstruction algorithm (ART with blobs) that has not been previously used in electron Tomography and we compare it with the standard method in the field (weighted back projection, WBP). We assume that only a limited set of very noisy images, collected around a single axis tilt, is available; which is a typical situation in Electron Tomography. In general, the reconstruction problem is underdetermined (due to the limited number of projections) and the data are inconsistent (due to the high level of noise). The evaluation of the results is performed in a rigorous way by a task-oriented approach which makes use of numerical observers. ART with blobs outperforms WBP for a number of key tasks. Results are presented both for simplified line integral data and for realistic simulations of macromolecular structures embedded in amorphous ice.     

A simple method to estimate insect mortality from field census data: A modification of the Kiritani-Nakasuji-Manly method

Various methods have been proposed to estimate demographic parameters such as mortality from field census data. Simple methods proposed earlier are applicable only for limited situations. For example, the Kiritani-Nakasuji-Manly method is applicable only if individuals are observable until their death. Improved methods proposed later are not subject to such limitations, but are not so widely used in the field of applied entomology, probably because of the complexity of the calculations involved. In this paper, I propose an intermediate method that requires only a pocket calculator, considering the practical convenience for field scientists. This method, which is a modification of the Kiritani-Nakasuji-Manly method, gives an estimate of the number of individuals entering a stage from the frequency of two stages when the stage duration is known.     

Chemoinformatics: A view of the field and current trends in method development

The chemoinformatics field continues to evolve at the interface between computer science and chemistry. Chemical information and computational approaches in pharmaceutical research are major focal points of chemoinformatics. However, the boundaries of this discipline are rather fluid and the chemoinformatics spectrum is difficult to delineate. The field is in flux, which also provides opportunities for further developments. As a lead-in to the Chemoinformatics Symposium-in-Print, we present a brief view of this discipline (with a little anecdotal touch), highlight current trends in method development, and discuss a number of representative examples.     

Estimating three-dimensional temperature fields during hyperthermia: studies of the optimal regularization parameter and time sampling period

During hyperthermia therapy it is desirable to know the entire temperature field in the treatment region. However, accurately inferring this field from the limited number of temperature measurements available is very difficult, and thus state and parameter estimation methods have been used to attempt to solve this inherently ill-posed problem. To compensate for this ill-posedness and to improve the accuracy of this method, Tikhonov regularization of order zero has been used to significantly improve the results of the estimation procedure. It is also shown that the accuracies of the temperature estimates depend upon the value of the regularization parameter, which has an optimal value that is dependent on the perfusion pattern and magnitude. In addition, the transient power-off time sampling period (i.e., the length of time over which transient data is collected and used) influences the accuracy of the estimates, and an optimal sampling period is shown to exist. The effects of additive measurement noise are also investigated, as are the effects of the initial guess of the perfusion values, and the effects of both symmetric and asymmetric blood perfusion patterns. Random perfusion patterns with noisy data are the most difficult cases to evaluate. The cases studied are not a comprehensive set, but continue to show the feasibility of using state and parameter estimation methods to reconstruct the entire temperature field.     

不同邻作作物对玉米田节肢动物多样性的影响

为探明不同邻作对云南普洱地区玉米田节肢动物多样性的影响.采用目测法和粘虫板等多种诱集法对邻作咖啡、水稻、李树及单作的玉米田节肢动物群落进行系统调查,分析不同邻作作物对玉米田节肢动物群落的影响.结果表明:不同邻作玉米田天敌亚群落主要为双翅目和膜翅目,其中玉米单作田赤池信息量准则(AIC)值最低为-16.858,最优模型为...     

MALDI imaging mass spectrometry for direct tissue analysis: technological advancements and recent applications

Matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a method that allows the investigation of the molecular content of tissues within its morphological context. Since it is able to measure the distribution of hundreds of analytes at once, while being label free, this method has great potential which has been increasingly recognized in the field of tissue-based research. In the last few years, MALDI-IMS has been successfully used for the molecular assessment of tissue samples mainly in biomedical research and also in other scientific fields. The present article will give an update on the application of MALDI-IMS in clinical and preclinical research. It will also give an overview of the multitude of technical advancements of this method in recent years. This includes developments in instrumentation, sample preparation, computational data analysis and protein identification. It will also highlight a number of emerging fields for application of MALDI-IMS like drug imaging where MALDI-IMS is used for studying the spatial distribution of drugs in tissues.     

Near-infrared spectroscopy (NIRS) for taxonomic entomology: A brief review

For over two decades, near-infrared spectroscopy (NIRS) has been applied to a wide spectrum of problems in the field of insect taxonomy. It provides a rapid, non-destructive and relatively cheap method of metabolomic profiling, which can often be used to discriminate closely related species in the same genus. Furthermore, very little training or entomological knowledge is required to operate the instrument. However, a taxonomist is still required to ensure accurate identification of samples used for NIRS model creation and validation. To date, most research has focused on species of economic or epidemiological importance, such as mosquitoes, flies or stored product pests. However, an increasing number of studies are applying NIRS for entomological research with a purely “academic” purpose. As research continues in this field, NIRS has the potential to become more widely accepted in entomology, allowing for the rapid metabolomic profiling of thousands of species.     

Pretreatment for enhanced hydrolysis of cellulosic biomass

This review will cover a number of physical and chemical pretreatment methods for cellulosic substrates which enhance their hydrolysis by cellulase or consumption by microorganisms. While the emphasis is on the literature of the last two years, some earlier work is cited which has influenced the work in the pretreatment field. In order to interpret the effects of a pretreatment method, emphasis in the past has been on crystallinity index (CI) and lignin content. Although these parameters happen often to correlate with the rate or extent of hydrolysis, it is suggested that a more basic parameter is the pore size distribution of the wet substrate and the associated surface area available to the cellulase that is the major factor in determining the effectiveness of a pretreatment method.     

Prediction of electromagnetic field distributions inside biological bodies by using an inverse scattering procedure based on a statistical cooling algorithm

This paper presents an inverse scattering procedure based on a statistical cooling algorithm to predict the electromagnetic field inside a biological body. By knowing only the scattered electric field distribution in a set of observation points external to the biological model, this method seems to be able to predict the electromagnetic field distributions in the investigation domain, minimizing a suitable cost function. To this end, a numerical statistical procedure is used, which allows to treat functions with a large number of unknowns in an efficient manner and to exploit the so called a priori knowledge in the reconstruction process. Some preliminary results are reported, concerning simplified biological geometries, which clearly show the capabilities and effectiveness, and also the current limitations of the proposed approach. Finally, further advances for the proposed imaging technique are indicated and discussed.