Modification of commerical and custom-built slab gel electrophoresis units for two-dimensional polyacrylamide gel electrophoresis

Many commercial and custom-built slab gel electrophoresis units can be modified to function as two-dimensional polyacrylamide gel electrophoresis units with the insertion of Plexiglas adapters. These adapters can be made for about $50 a pair and can be used for either temporary or permanent modification of the slab gel units. The physical dimensions of the adapters can be varied to permit great flexibility in the diameter of cylinder gels and the thickness of slab gels that can be run together. For example, proteins from 6-mm cylinder gels can be easily separated on 1-mm slab gels, which can then be dried for autoradiography.     

Fiber optic array biosensors

Optical fiber arrays provide a powerful substrate for creating high-density sensing systems that can address a variety of biological problems. The fiber substrate can be used to create femtoliter wells that can be loaded with individual beads to create high-density arrays for multiplexed screening and analysis. In addition, living cells can be loaded into the arrays, and their individual responses monitored over long time periods, enabling functional screening of biologically active compounds. Adherent cells can be attached to the fiber substrate to provide a rapid method for observing cell migration and for screening anti-migratory compounds. Finally, individual enzyme molecules can be loaded into the array wells enabling single molecule detection via enzyme-catalyzed signal amplification.     

The chemoinvasion assay: a method to assess tumor and endothelial cell invasion and its modulation

Invasive and metastatic cells, as well as endothelial cells, must cross basement membranes (BMs) in order to disseminate or to form new blood vessels. The chemoinvasion assay using the reconstituted BM Matrigel in Boyden blind-well chambers is a very rapid, easy, inexpensive and flexible test that can be used to quantify the invasive potential of most cell types; it can be applied to detect the migratory activity associated with matrix degradation and can also be adapted to study the selective degrading activity on different matrix substrates. Transwell inserts can also be used. Once the optimal experimental conditions are empirically determined for specific cellular models, the chemoinvasion assay can be used for the screening of inhibitors of invasiveness and angiogenesis, or to select for invasive cellular populations. This protocol can be completed in 9 h.     

The use of biomarkers in surveillance, medical screening, and intervention

Building on mechanistic information, much of molecular epidemiologic research has focused on validating biomarkers, that is, assessing their ability to accurately indicate exposure, effect, disease, or susceptibility. To be of use in surveillance, medical screening, or interventions, biomarkers must already be validated so that they can be used as outcomes or indicators that can serve a particular function. In surveillance, biomarkers can be used as indicators of hazard, exposure, disease, and population risk. However, to obtain rates for these measures, the population at risk will need to be assessed. In medical screening, biomarkers can serve as early indicators of disease in asymptomatic people. This allows for the identification of those who should receive diagnostic confirmation and early treatment. In intervention (which includes risk assessment and communication, risk management, and various prevention efforts), biomarkers can be used to assess the effectiveness of a prevention or control strategy as well as help determine whether the appropriate individuals are assigned to the correct intervention category. Biomarkers can be used to provide group and individual risk assessments that can be the basis for marshalling resources. Critical for using biomarkers in surveillance, medical screening, and intervention is the justification that the biomarkers can provide information not otherwise accessible by a less expensive and easier-to-obtain source of information, such as medical records, surveys, or vital statistics. The ability to use validated biomarkers in surveillance, medical screening, and intervention will depend on the extent to which a strategy for evidence-based procedures for biomarker knowledge transfer can be developed and implemented. This will require the interaction of researchers and decision-makers to collaborate on public health and medical issues.     

Nanoparticles,molecular biosensors,and multispectral confocal microscopy

Complex, multilayered nanoparticles hold great promise for more sophisticated drug/gene delivery systems to single cells. Outermost layers can include cell targeting and cell-entry facilitating molecules. The next layer can include intracellular targeting molecules for precise delivery of the nanoparticle complex inside the cell of interest. Molecular biosensors can be used to confirm the presence of expected molecules (for example, reactive oxygen species (ROS) as a surrogate molecule for signs of infection, or for activation in radiation damage, etc.) prior to delivery of counter-measure molecules such as drugs or gene therapy. They can also be used as a feedback control mechanism to control the proper amount of drug/gene delivery for each cell. Importantly, the full nanoparticle system can be used to prevent any cells from encountering the drug unless that cell is specifically targeted. Thus, if a cell is initially non-specifically targeted, a secondary check for other molecular targets which must also be present inside the target cell of interest can be used to catch initial targeting mistakes and prevent subsequent delivery of treatment molecules to the wrong cells. The precise intracellular location of nanoparticles within specific regions of a cell can be confirmed by 3D multispectral confocal microscopy. These single cell molecular morphology measurements can be extended from individual cells, to other cells in a tissue in tissue monolayers or tissue sections.     

Pharmacokinetic requirements for successful site-directed targeting of drugs

Targeted drugs can be defined as those in which features of the molecule, additional to those required for receptor interaction, substantially improve the concentration ratio of active substance at the site of action compared to the site where side-effects occur.These requirements for successful targeting of systemically administered drugs can be determined by pharmacokinetic modeling. The requirements depend on the mechanism of targeting and on whether targeting is to be achieved for continuous therapy or for acute treatment. For continuous therapy (lasting several days) the success of targeting using a prodrug which is locally activated and has linear pharmacokinetics is proportional to the clearance of active drug from the body and inversely proportional the rate constant for leaving the site of action and to the volume of the tissue. The relationship can be mapped graphically and typical values for these parameters are considered so that situations where targeting can be successful can be identified. The prodrug must also have properties which result in sufficient concentration of drug being formed at the site of action. These properties can also be described by simple proportionality relationships and the conditions for success illustrated graphically.Kinetic considerations are also important for targeting. For continuous therapy it is desirable that steady drug concentrations should be reached rapidly. This is best achieved by using molecules which rapidly exchange between body compartments and have low tissue binding. For acute therapy the rules for targeting can be quite different and an example is given where binding can be responsible for a form of targeting.The results emphasise the need for careful consideration of the properties of targeted system taking into account transport, binding and clearance of both prodrug and drug. Specific details of the disease are also critical both in terms of local tissue properties and the desired time course of drug action.     

Development of DNA-mediated transformation systems for plants

The genetic engineering of plants by DNA-mediated gene transfer requires that efficient transformation systems be developed. Considerable progress has been made in manipulating the Ti plasmid of Agrobacterium tumefaciens as a vehicle for delivery of foreign genes into protoplasts of dicotyle-donous plants. Part of the Ti plasmid, the T-DNA, can be incorporated into the genome of the host cell; the T-DNA can carry a foreign DNA sequence which co-integrates with it; under normal conditions, the tumorigenic-causing portion of the T-DNA can be inactivated so that transformed protoplasts can be regenerated and T-DNA with an inserted foreign gene can be stably maintained during regeneration, meiosis and gamete formation. A foreign gene has yet to be expressed in regenerated plants although a T-DNA gene for opine synthesis can function in regenerates. Developing a more ubiquitous transformation system for monocotyledons is further from fruition. Based on transformation systems for simple eukaryotic organisms, it is reasonable to expect that a DNA vector which is capable of amplifying a novel plant gene and which contains both a drug resistance marker to facilitate the selection of transformed plant protoplasts and a species-specific autonomously replicating sequence to ensure the stable maintenance of the input gene in the recipient cell can be constructed.     

Cell motility measurements with an automated microscope system

The motility of 3T3 cells has been studied using a newly developed automated microscope system which is capable of recognizing live unstained cells growing in tissue culture. A large number of individual cells can be rapidly identified and characterized and their precise positions recorded. All cells can be revisited automatically every few minutes, and the new cell positions can be determined. Quantitative data from up to 1 000 cells can then be obtained, and cell movement parameters like cell speed, distance travelled, direction of movement, etc., can be measured for individual cells and for the whole cell population. In addition, for any number of chosen cells, high-resolution digitized images can be taken for further morphological studies, including acquisition of images of individual cells.     

Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications

Oil bodies obtained from oilseeds have been exploited for a variety of applications in biotechnology in the recent past. These applications are based on their non-coalescing nature, ease of extraction and presence of unique membrane proteins—oleosins. In suspension, oil bodies exist as separate entities and, hence, they can serve as emulsifying agent for a wide variety of products, ranging from vaccines, food, cosmetics and personal care products. Oil bodies have found significant uses in the production and purification of recombinant proteins with specific applications. The desired protein can be targeted to oil bodies in oilseeds by affinity tag or by fusing it directly to the N or C terminal of oleosins. Upon targeting, the hydrophobic domain of oleosin embeds into the TAG matrix of oil body, whereas the protein fused with N and/or C termini is exposed on the oil body surface, where it acquires correct confirmation spontaneously. Oil bodies with the attached foreign protein can be separated easily from other cellular components. They can be used directly or the protein can be cleaved from the fusion. The desired protein can be a pharmaceutically important polypeptide (e.g. hirudin, insulin and epidermal growth factor), a neutraceutical polypeptide (somatotropin), a commercially important enzyme (e.g. xylanase), a protein important for improvement of crops (e.g. chitinase) or a multimeric protein. These applications can further be widened as oil bodies can also be made artificially and oleosin gene can be expressed in bacterial systems. Thus, a protein fused to oleosin can be expressed in Escherichia coli and after cell lysis it can be incorporated into artificial oil bodies, thereby facilitating the extraction and purification of the desired protein. Artificial oil bodies can also be used for encapsulation of probiotics. The manipulation of oleosin gene for the expression of polyoleosins has further expanded the arena of the applications of oil bodies in biotechnology.     

Causal diagrams for empirical research

The primary aim of this paper is to show how graphical modelscan be used as a mathematical language for integrating statisticaland subject-matter information. In particular, the paper developsa principled, nonparametric framework for causal inference,in which diagrams are queried to determine if the assumptionsavailable are sufficient for identifying causal effects fromnonexperimental data. If so the diagrams can be queried to producemathematical expressions for causal effects in terms of observeddistributions; otherwise, the diagrams can be queried to suggestadditional observations or auxiliary experiments from whichthe desired inferences can be obtained.     

Principles and strategies in breeding for higher salt tolerance

Summary Salinity is an environmental component that usually reduces yield. Recent advances in the understanding of salt effects on plants have not revealed a reliable physiological or biochemical marker that can be used to rapidly screen for salt tolerance. The necessity of measuring salt tolerance based upon growth in saline relative to non-saline environments makes salt tolerance measurements and selection for tolerance difficult. Additionally, high variability in soil salinity and environmental interactions makes it questionable whether breeding should be conducted for tolerance or for high yield. Genetic techniques can be used to identify the components of variation attributable to genotype and environment, and the extent of genetic variation in saline and nonsaline environments can be used to estimate the potential for improving salt tolerance. Absolute salt tolerance can be improved best by increasing both absolute yield and relative salt tolerance.     

Interactive robots in experimental biology

Interactive robots have the potential to revolutionise the study of social behaviour because they provide several methodological advances. In interactions with live animals, the behaviour of robots can be standardised, morphology and behaviour can be decoupled (so that different morphologies and behavioural strategies can be combined), behaviour can be manipulated in complex interaction sequences and models of behaviour can be embodied by the robot and thereby be tested. Furthermore, robots can be used as demonstrators in experiments on social learning. As we discuss here, the opportunities that robots create for new experimental approaches have far-reaching consequences for research in fields such as mate choice, cooperation, social learning, personality studies and collective behaviour.     

黑河流域生态经济带分异协调规律与耦合发展模式

在西北干旱地区建设黑河流域生态经济带,是从根本上高效配置流域水资源,彻底化解流域上,下游利益冲突,实现利益共享,保护流域生态环境,推进流域可持续发展,全面实施国务院黑河流域分水方案的重要途径,通过对黑河流域生态经济带上－中－下游投入产出效益的比较分析和流域上、中、下游生态－生产、生活系统发展分异及互动协调关系的分析,提出了黑河流域生态经济带上－中－下游多维互动的协调耦合发展模式,进而提出了黑河流域生态经济带建设与发展的主要途径,包括推进黑河流域经济发展一体化和集成管理公司化,大力推行全流域水资源的差异化有偿使用制度,实施流动上、中、下游的水权转让贸易,实行规范的流域财政转移支付制度,建立流域资源与生态环境和经济的整合帐户体系,实行跨行政区域河流边界水量水质达标交接制度,等等。     

Hybridization-based karyotyping of mouse chromosomes: hybridization-bands.

We have developed a method, which we have named hybridization-banding, to identify simultaneously all chromosomes in a mouse metaphase spread. The method uses a combination of hybridization probes labeled with a single fluor to yield a simple, unique, readily identifiable hybridization pattern on each chromosome. The method is superior to Giemsa- or fluorescence-based banding methods for chromosome identification because the hybridization patterns are simpler and easier to identify, and unique patterns can be designed at will for each chromosome. Analysis can be performed with a standard fluorescence microscope, and images can be recorded on film with an ordinary 35-mm camera, making the method useful to many investigators. The method can also be applied to any species for which chromosomes and probes can be prepared.     

利用基因诱捕识别哺乳动物发育调控基因

ES细胞是一种来源于胚胎的多潜能细胞,它可在体外培养并进行基因操作,而且通过囊胚注射制作嵌合体的途径,能将外源基因掺入小鼠的基因库中,因此利用ES细胞可筛选出发生基因突变的小概率事件并获得其遗传突变体．利用基因诱捕载体与ES细胞,研究与哺乳动物发育调控有关的未知基因,这一新技术将成为阐明胚胎发育过程中基因表达的时空格式的有效手段．     

Multi-scale keypoints in V1 and beyond: object segregation, scale selection, saliency maps and face detection

End-stopped cells in cortical area V1, which combine outputs of complex cells tuned to different orientations, serve to detect line and edge crossings, singularities and points with large curvature. These cells can be used to construct retinotopic keypoint maps at different spatial scales (level-of-detail). The importance of the multi-scale keypoint representation is studied in this paper. It is shown that this representation provides very important information for object recognition and face detection. Different grouping operators can be used for object segregation and automatic scale selection. Saliency maps for focus-of-attention can be constructed. Such maps can be employed for face detection by grouping facial landmarks at eyes, nose and mouth. Although a face detector can be based on processing within area V1, it is argued that such an operator must be embedded into dorsal and ventral data streams, to and from higher cortical areas, for obtaining translation-, rotation- and scale-invariant detection.     

植被监测及趋势分析——植被数量生态学中几个理论问题的探讨

 系统监测可以对危机发出预警, 是防治灾害的重要手段。生态监测的基础是植被监测。多物种 多样本 多年的植被定位监测数据隐含 着植被变化的信息。该文探索描述植被的数学工具, 提出植被监测数据的趋势分析方法。植被是资源竞争系统, 可以用多维空间的向量来表示 。在向量空间(射影空间), 不是“距离” , 而是“方向”决定区别; 在植被科学, 不是“产量”, 而是“组成”决定区别。新方法用多维空间 的位置向量来表示植被: 向量的方向表示植被的组成、两向量夹角余弦值表示相似、向量长度表示植被总体。在简缩数据时, 用“中心化”滤 去样本噪音、“标准化”滤去系统噪音, 得到状态向量。在趋势分析时, 定义后、前状态向量的比值为变化趋势; 用当年的状态和趋势的乘积 来预报次年的状态。到次年, 再用实测数据修正、更新来自去年的预报, 是为“卡尔曼滤波”。卡尔曼滤波能降低监测成本, 有效地使用历史 数据, 提高分析精度。     

Looking for novel functions of tau

The lack or excess of the protein tau can be deleterious for neurons. The absence of tau can result in retarded neurogenesis and neuronal differentiation, although adult mice deficient in tau are viable, probably because of the compensation of the loss of tau by other MAPs (microtubule-associated proteins). On the contrary, the overexpression of tau can be toxic for the cell. One way to reduce intracellular tau levels can be achieved by its secretion through microvesicles to the extracellular space. Furthermore, tau can be found in the extracellular space because of the neuronal cell death occurring in neurodegenerative disorders such as Alzheimer's disease. The presence of toxic extracellular tau could be the mechanism for the spreading of tau pathology in these neurodegenerative disorders.     

RnaViz, a program for the visualisation of RNA secondary structure.

RnaViz is a user-friendly, portable, windows-type program for producing publication-quality secondary structure drawings of RNA molecules. Drawings can be created starting from DCSE alignment files if they incorporate structure information or from mfold ct files. The layout of a structure can be changed easily. Display of special structural elements such as pseudo-knots or unformatted areas is possible. Sequences can be automatically numbered, and several other types of labels can be used to annotate particular bases or areas. Although the program does not try to produce an initially non-overlapping drawing, the layout of a properly positioned structure drawing can be applied to a newly created drawing using skeleton files. In this way a range of similar structures can be drawn with a minimum of effort. Skeletons for several types of RNA molecule are included with the program.     

Application of logistic regression to case-control association studies involving two causative loci

Models in which two susceptibility loci jointly influence the risk of developing disease can be explored using logistic regression analysis. Comparison of likelihoods of models incorporating different sets of disease model parameters allows inferences to be drawn regarding the nature of the joint effect of the loci. We have simulated case-control samples generated assuming different two-locus models and then analysed them using logistic regression. We show that this method is practicable and that, for the models we have used, it can be expected to allow useful inferences to be drawn from sample sizes consisting of hundreds of subjects. Interactions between loci can be explored, but interactive effects do not exactly correspond with classical definitions of epistasis. We have particularly examined the issue of the extent to which it is helpful to utilise information from a previously identified locus when investigating a second, unknown locus. We show that for some models conditional analysis can have substantially greater power while for others unconditional analysis can be more powerful. Hence we conclude that in general both conditional and unconditional analyses should be performed when searching for additional loci.