Detectability of radiation damage to melanoma cells using ESR spectroscopy. A review

Comparison of indirect and direct methods applicable for the examination of radiation damage to melanoma cells leads to the conclusion that the only method useful for its detection appears to be the indirect method. The paper describes the principle, examples of radiobiological applications, and perspectives of future development of a new indirect method of measuring oxygen consumption by neoplastic cells under in vitro conditions. The method is based on the use of a spin label technique.     

Indirect emissions of forest bioenergy: detailed modeling of stump‐root systems

Indirect carbon dioxide emissions from producing bioenergy from tree stumps and roots depend critically on the decomposition rate of these harvest residues if they were left in forest to decay. We developed a method to improve the current estimates of these emissions. First, the 3D structure of uprooted stump‐root systems was modeled based on terrestrial laser‐scanning data. Second, information obtained on the size distribution of the stumps and the roots was used to simulate their decomposition and to estimate the indirect emissions. The method was able to describe the structure of stump‐root systems at a clear‐cut boreal Norway spruce test site. Compared with earlier results based on the diameter of stumps alone, the new estimates of the decomposition rate were slightly higher and, consequently, those of the indirect emissions slightly lower. The method is useful to collect information on the indirect emissions of stump‐root bioenergy quickly in different forests.     

A new method for evaluating the specificity of indirect readout in protein-DNA recognition

Proteins recognize a specific DNA sequence not only through direct contact (direct readout) with base pairs but also through sequence-dependent conformation and/or flexibility of DNA (indirect readout). However, it is difficult to assess the contribution of indirect readout to the sequence specificity. What is needed is a straightforward method for quantifying its contributions to specificity. Using Bayesian statistics, we derived the probability of a particular sequence for a given DNA structure from the trajectories of molecular dynamics (MD) simulations of DNAs containing all possible tetramer sequences. Then, we quantified the specificity of indirect readout based on the information entropy associated with the probability. We tested this method with known structures of protein-DNA complexes. This method enabled us to correctly predict those regions where experiments suggested the involvement of indirect readout. The results also indicated new regions where the indirect readout mechanism makes major contributions to the recognition. The present method can be used to estimate the contribution of indirect readout without approximations to the distributions in the conformational ensembles of DNA, and would serve as a powerful tool to study the mechanism of protein-DNA recognition.     

METHODS FOR FASTER PARSIMONY ANALYSIS

Abstract — Several algorithms to speed up branch swapping searches for most parsimonious trees are described. The method for indirect tree length calculation when moving a clipped clade, based on final states for the divided tree, is expanded to take into account polymorphic characters, and to include the possibility of rejecting several locations as suboptimal by checking just one node. Three different algorithms for faster estimation of final state assignments for the divided tree based on calculations for the whole tree are presented. The first of these is approximate; it uses information from the final state sets for the whole tree. The second is exact, but it is slower than the first, and requires more memory; it is based on the union of the state sets of the descendants for each node. The third is also exact; it requires more memory and programming effort than the other two but it is faster, it is based on final and preliminary state sets for the whole tree ("incremental two-pass optimization"). Efficient ways to derive state assignments for collapsing trees, based on final states for the divided tree, are described. The recently proposed method of "incremental optimization" is discussed. It is likely that searches using that method will be no faster than searches using indirect calculation as originally described, and will be quite slower than the modified indirect calculation described here. Searches using that method will probably be significantly slowed down when zero-length branches are to be collapsed, since shortcuts for faster collapsing are not directly applicable.     

Efficiency of DNA extraction methods on the evaluation of soil microeukaryotic diversity

The evaluation of microbial molecular diversity has been mainly based on the extraction of total DNA from environmental samples. The indirect extraction methods, which have been used for prokaryotes, have never been used to recover soil microeukaryotic DNA. We evaluated the efficiency of an improved indirect DNA extraction protocol developed herein and the direct lysis (the sodium dodecyl sulfate (SDS)-based method and commercial DNA extraction kit) on estimating the molecular diversity of soil microbial eukaryotes. DNA quality and quantity as well as denaturing gradient gel electrophoresis (DGGE) profiles were determined using three soil samples from different stations. The indirect method detected the highest DGGE bands in spite of the low DNA yield. The commercial kit detected a lower number of DGGE bands than the indirect method. The SDS-based method produced the lowest DGGE bands and DNA purity but the highest yield. Using the indirect method, we further evaluated the effect of freezing and air-dried preservations on estimating the microeukaryotic diversity. In spite of the low DNA yield obtained from the air-dried preservation, no significant differences were found in either the number of DGGE bands or the DNA purity between two manners. Our results indicate that the improved indirect method could obtain a high purity of intracellular DNA and high efficiency in the estimation of molecular diversity of soil microbial eukaryotes.     

Sensitive detection of hybridocytochemical results by means of reflection-contrast microscopy

A new sensitive method for visualization of nonautoradiographic hybridization results in microscopic preparations is described. The method is based on the reflection of the incident light by diaminobenzidine precipitates deposited at the site of hybridization during an indirect hybridocytochemical procedure. The reflected light is detected by means of reflection-contrast microscopy. The applicability of the procedure is demonstrated with nucleic acid probes modified with 2-acetylaminofluorene groups. These in turn are localized in situ by an indirect immunoperoxidase reaction. Besides its sensitivity, this simple visualization technique possesses the additional advantages, over absorption and fluorescence microscopy, that it provides a total DNA counterstain and a chromosomal banding pattern.     

A new method of estimating the pollen dispersal curve independently of effective density

We introduce a novel indirect method of estimating the pollen dispersal curve from mother-offspring genotypic data. Unlike an earlier indirect approach (TwoGener), this method is based on a normalized measure of correlated paternity between female pairs whose expectation does not explicitly depend on the unknown effective male population density (d(e)). We investigate the statistical properties of the new method, by comparison with those of TwoGener, considering the sensitivity to reductions of d(e), relative to census density, resulting from unequal male fecundity and asynchronous flowering. Our main results are: (i) it is possible to obtain reliable estimates of the average distance of pollen dispersal, delta, from indirect methods, even under nonuniform male fecundity and variable flowering phenology; (ii) the new method yields more accurate and more precise delta-estimates than TwoGener under a wide range of sampling and flowering scenarios; and (iii) TwoGener can be used to obtain approximate d(e) estimates, if needed for other purposes. Our results also show that accurately estimating the shape of the tail of the pollen dispersal function by means of indirect methods remains a very difficult challenge.     

Estimation of pK(a) values using microchip capillary electrophoresis and indirect fluorescence detection

Microchip capillary electrophoresis (CE), coupled with indirect fluorescence detection was investigated for estimating the pK(a) values of non-fluorescent compounds. The CE method is based on the differences in electrophoretic mobility of the analyte as a function of the pH of the running buffer. Nine compounds were tested, including several of pharmaceutical importance, with pK(a) values from 10.3 to 4.6. All buffers contained 5-TAMRA as the fluorescent probe for indirect detection. Calculated pK(a) values agreed well with literature values obtained by traditional methods, differing not more than 0.2 from the literature value. The current work on single lane chips demonstrates the principle of microchip CE with indirect detection as a viable method for estimating pK(a) values. However, increased throughput will be required using a multilane chip to enable the approach to be used practically.     

Estimation of deciduous forest leaf area index using direct and indirect methods

This study evaluated one semi-direct and three indirect methods for estimating leaf area index (LAI) by comparing these estimates with direct estimates derived from litter collection. The semi-direct method uses a thin metallic needle to count a number of contacts across fresh litter layers. One indirect method is based on the penetration of diffuse global radiation measured over the course of a day. The second indirect method uses the LAI-2000 plant canopy analyser (PCA) which measures diffuse light penetration from five different sky sectors simultaneously. The third indirect method uses the Demon portable light sensor to measure the penetration of direct beam sunlight at different zenith angles over the course of half a day. The Poisson model of gap frequency was applied to estimate plant area index (PAI) from observed transmittances using the second and third methods. Litter collection from 11 temperate decidous forests gave values of LAI ranging from 1.7 to 7.5. Estimates based on the needle method showed a significant linear relationship with LAI values obtained from litter collections but were systematically lower (by 6–37%). PAI estimates using all three indirect techniques (fixed light sensor system, LAI-2000 and Demon) showed a strong linear relationship with LAI derived from litter collection. Differences, averaged over all forest stands, between PAI estimates from each of the three indirect methods and LAI from litter collections were below 2%. If we consider that LAI=PAI–WAI (wood area index) then, all three indirect methods underestimated LAI by an additional factor close to the value of WAI. One reason could be a local clumping of architectural canopy components: in particular, the spatial dispositions of branchlets and leaves are not independent, leading to a non-random relationship between the distributions of these two canopy components.     

牛分枝杆菌Mb0950c的免疫特性及其血清学检测方法的建立

【目的】利用原核表达系统对牛分枝杆菌Mb0950c蛋白进行表达和纯化,通过小鼠模型评价其免疫原性,建立血清学间接ELISA方法用于牛结核病的临床检测。【方法】构建pET32a-Mb0950c原核表达质粒,并转化至BL21(DE3)中诱导蛋白的表达,对蛋白进行纯化。使用流式细胞术(flow cytometry,FCM)、ELISA等对该蛋白在小鼠中的免疫原性进行分析。建立基于Mb0950c的间接ELISA方法,评价该方法的临床检测潜力。【结果】SDS-PAGE和Western blotting结果显示,成功获得了可溶性Mb0950c蛋白,且具有良好免疫反应性;FCM结果显示,Mb0950c蛋白上调了T细胞表面CD69分子的表达。细胞因子和抗体结果表明,该蛋白能够诱导特异性的IFN-γ和IL-4的分泌,同时能诱导机体分泌特异性的抗体,且以IgG1型为主。建立了ELISA检测方法应用于牛结核临床检测,结果显示,该方法与牛结核外周血IFN-γ外释放试验和皮试试验结果的阳性符合率、阴性符合率和总符合率分别为65.7%、97.9%和72.4%。【结论】在原核表达系统中可溶性表达Mb0950c蛋白,...     

茶尺蠖核型多角体病毒多角体蛋白单克隆抗体的制备

为了建立一种基于免疫反应检测茶尺蠖核型多角体病毒的方法,以纯化后的茶尺蠖核型多角体病毒作为抗原,免疫BALB/c小鼠,将小鼠脾脏细胞与小鼠骨髓瘤细胞Sp2/0融合,经间接ELISA筛选及克隆得到了一株稳定分泌单克隆抗体的杂交瘤细胞株,命名为7D3。同时克隆并在大肠杆菌中表达了EoNPV多角体蛋白基因,获得重组多角体蛋白。经Western blotting鉴定,该抗体可与EoNPV的多角体蛋白特异性结合。利用制备EoNPV多角体蛋白的单克隆抗体,建立了间接ELISA测定EoNPV的方法。     

Determination of standardized transpiration used as an indirect method for considering stomatal aperture under field conditions

An indirect method for determination of stomata apertures is described. It is based on comparison of two simultaneous curves: the one obtained under natural conditions and the second obtained under standard conditions with minimum air humidity which can be considered as a criterion of stomata apertures.     

Determination of standardized transpiration used as an indirect method for considering stomatal aperture under field conditions

An indirect method for determination of stomata apertures is described. It is based on comparison of two simultaneous curves : the one obtained under natural conditions and the second obtained under standard conditions with minimum air humidity which can be considered as a criterion of stomata apertures.     

基于不同需求层次的中国城镇居民消费隐含碳排放时空演变机制

正确认识不同需求水平下人均隐含碳排放量的变化,对实现低碳经济及低碳生活具有重要的参考价值。基于居民消费需求层次建立不同需求层次与隐含碳排放的对应关系,将人均隐含碳排放分解为生存型、发展型、奢侈型三类,并运用投入产出法进行核算,在对不同需求层次人均隐含碳排放的空间格局演变分析的基础上采用空间面板方法对其驱动机制进行甄别。结果显示,在全国层面,各需求层次人均隐含碳排放均呈现上升趋势,空间分布不均衡性主要体现在南北差异上,北部地区始终是各需求层次人均隐含碳排放的主要空间载体,其中多数省分生存型人均隐含碳排放上升势头较强,发展型和奢侈型的高值区在省份数量上分别呈现先减后增与逐渐增加的变化趋势;不同需求层次人均隐含碳排放水平相似的地区在空间上呈集聚分布,具有较强的"马太效应";空间面板模型结果显示技术减排是降低不同需求层次人均隐含碳排放的重要举措,而人口规模在各需求层次上的负向减排作用远小于正向的人口结构效应,宏观经济因素主要表现为增排效应,而居民消费因素的作用通道存在差异。此外,部分因素在各需求层次上存在显著空间外溢效应,应重视区域间的横向联动减排效应,做好隐含碳减排的统筹协调工作。     

Choice of Smoothing Parameters for Direct Kernels in Discrimination

Direct kernels, due to LAUDER (1983), as an alternative to the indirect kernel method in discriminant analysis are considered. It is shown that direct kernels may be based on any kernel function known in discrete density estimation. The choice of smoothing parameters is based on general loss functions and a family of loss functions which are specific for the discrimination problem is introduced. Examples with distance dependent and distance independent smoothing parameters are given to illustrate the applicability.     

Calculation of the"Net Additions to Stock" Indicator for the Czech Republic Using a Direct Method

Abstract: Net additions to stock (NAS) are an indicator based on economy-wide material flow accounting and analysis. NAS, a measure of the physical growth rate of an economy, can be used for estimates of future waste flows. It is calculated using two methods: The indirect method of calculation is a simple difference between all input and output flows, whereas the direct method involves measuring the amounts of materials added to particular categories of physical stock and the amounts of waste flows from these stocks.
The study described in this article had one leading objective: to make available direct NAS data for the Czech Republic, which could later be used for predicting future waste flows. Two additional objectives emerged from the first: (1) to develop a method for direct NAS calculation from data availability in the Czech Republic; (2) to calculate NAS directly, compare the results with those achieved in indirect NAS calculation, and discuss the identified differences.
The NAS for the Czech Republic calculated by the direct method is equal to approximately 65 million tonnes on average in 2000–2002 and is approximately 27% lower than the NAS acquired by the indirect method of calculation. The actual values of directly calculated NAS and its uncertainties suggest that the indirect NAS is more likely to be an overestimation than an underestimation. Durables account for about 2% of the total direct NAS, whereas the rest is attributed to infrastructure and buildings. The direct NAS is dominated by nonmetal construction commodities such as building stone and bricks, which equal approximately 89% of the total direct NAS.
Calculation of NAS by the direct method has been proved to be feasible in the Czech Republic. Moreover, uncertainties related to direct NAS are lower than those related to indirectly acquired NAS.     

大鼠垂体远侧部内分泌细胞的四重免疫组织化学染色方法

本文介绍一种显示大鼠垂体远侧部内分泌细胞的四重免疫组织化学染色方法。此法以PAP 法为基础,结合 DAB 银加强法和间接免疫金标记技术。方法比较简单,所需试剂容易得到。     

藻胆蛋白荧光探针及其标记

藻胆蛋白是一系列新型的荧光标记探针,具有优良的荧光特性,以藻胆蛋白荧光探针标记抗体还可用于血清可溶性抗原（或抗体）的荧光免疫检测,其标记方法可分为直接法和间接法。结合藻胆蛋白的特点,研究藻胆蛋白的标记方法有助于提高荧光免疫检测的灵敏度。     

A simple technique for the direct determination of maintenance energy coefficient: An example with Zymomonas mobilis

Summary Maintenance energy plays an important role both in basic kinetic studies as well as in process development. However the determination of the maintenance energy coefficient is based so far on indirect time consuming measurements assuming linearity between growth rate and substrate uptake rate. This paper presents a simple and fast method using cell recycling techniques for the direct determination of maintenance energy coefficient.     

Method for rapid DNA extraction from bacterial communities of different soils

A method for indirect DNA extraction from various soils significantly differing in their physicochemical properties has been developed. The proposed method is based on cell desorption from soil particles using a Tris-EDTA (TE) buffer supplemented with polyvinylpolypyrrolydone (PVPP) and sodium dodecylsulfate (SDS). Methods for subsequent cell lysis and purification of DNA preparations based on alkaline lysis followed by chromatography on ion-exchange resins were described by us earlier. The purity of the DNA preparations obtained did not depend on the type of soil. It was shown that the DNA preparations can be used for the amplification of rather large fragments, e.g., sequences spanning the complete 16S rRNA gene.