Fabrication and application of neoglycolipid arrays in a microtiter plate

The work presented herein is a new noncovalent glycoarray assembly method for microplates created by simply mixing together a carbohydrate and a tetradecylamine. Alpha-mannose was utilized in the model study and product formation was detected by lectin binding. The method can be further extended to array complex carbohydrates.     

Enzyme activity deviates due to spatial and temporal temperature profiles in commercial microtiter plate readers

Microtiter plates (MTP) and automatized techniques are increasingly applied in the field of biotechnology. However, the susceptibility of MTPs to edge effects such as thermal gradients can lead to high variation of measured enzyme activities. In an effort to enhance experimental reliability, to quantify, and to minimize instrument‐caused deviations in enzyme kinetics between two MTP‐readers, we comprehensively quantified temperature distribution in 96‐well MTPs. We demonstrated the robust application of the absorbance dye cresol red as easily applicable temperature indicator in cuvettes and MTPs and determined its accuracy to ±0.16°C. We then quantified temperature distributions in 96‐well MTPs revealing temperature deviations over single MTP of up to 2.2°C and different patterns in two commercial devices (BioTek Synergy 4 and Synergy Mx). The obtained liquid temperature was shown to be substantially controlled by evaporation. The temperature‐induced enzyme activity variation within MTPs amounted to about 20 %. Activity deviations between MTPs and to those in cuvettes were determined to 40 % due to deviations from the set temperature in MTPs. In conclusion, we propose a better control of experimental conditions in MTPs or alternative experimental systems for reliable determination of kinetic parameters for bioprocess development.     

Use of precise spatial data for describing spatial patterns and plant interactions in a diverse Great Basin shrub community

Community-structuring processes continue to be of great interest to plant ecologists, and plant spatial patterns have been linked to processes including disturbance, dispersal, environmental heterogeneity, and plant interactions. Under the assumption that the analysis of the spatial structure of plant communities can help to elucidate the type and importance of the predominant community-structuring processes, many studies have analyzed point pattern data on various plant species. A variety of methods have been devised to acquire point pattern data for individual plants, however, the classic tradeoff between the speed of acquisition and the precision of spatial data has meant that large and precise datasets on plant locations are difficult to obtain. The primary goal of this study was to develop a GPS-based methodology for the rapid collection of precise spatial data on plant locations in a semi-arid shrubland in the Great Basin, USA. The secondary goal was to demonstrate a potential application of this approach by using recently developed univariate and bivariate spatial statistics to test for aggregation within the shrub community, as observed in other semi-arid shrublands. We efficiently mapped 2,358 individuals of five shrub species with a spatial error of ≤0.02 m, and found strong statistical evidence of fine-scale aggregation (1) independent of species, (2) within species, and (3) between two species pairs. Our approach is useful for rapidly collecting precise point pattern data in plant communities, and has other applications related to population modeling, GIS analysis, and conservation.     

Immobilization of enzymes in microtiter plate scale

Different immobilization methods were adapted to the 96-well microtiter plate scale using esterases as model enzymes. The methods tested were based on adsorption, coprecipitation, aggregation and covalent bonding. The protein covered microcrystals proved to be the best method in terms of yield and expressed activity for the test reaction, which was the alcoholysis of p-nitrophenyl acetate with 1-propanol under anhydrous conditions.     

Monitoring modulators of platelet aggregation in a microtiter plate assay

Platelets play a central role in maintaining biological hemostasis. Inappropriate platelet activation is responsible for thrombotic diseases such as myocardial infarction and stroke. Therefore, novel agents that can inhibit platelet activation are necessary. However, assays that monitor platelet aggregation are generally time-consuming and require high volumes of blood and specialized equipment. Therefore, a medium- to high-throughput assay that can monitor platelet aggregation would be considered useful. Such an assay should be sensitive, comparable to the "gold standard" assay of platelet aggregometry, and able to monitor multiple samples simultaneously but with low assay volumes. We have developed such a microtiter assay. It can assay an average of 60 independent treatments per 60 ml blood donation and demonstrates greater sensitivity than the current gold standard assay, namely platelet aggregation in stirring conditions in a platelet aggregometer. The microtiter plate (MTP) assay can detect known inhibitors of platelet function such as indomethacin, aspirin, and ReoPro. It is highly reproducible when using standard doses of agonists such as thrombin receptor-activating peptide (20 microM) and collagen (0.19 mg/ml). Finally, the MTP assay is rapid and sensitive and can detect unknown platelet-modulating agents from a library of compounds.     

林带空间配置与布局优化研究

农田防护林(林带)的空间配置与布局是影响防护林结构和防护效益发挥并持续的关键因素．为达到农田防护林防护效益最大并持续的经营目标。在保证林分多样性和稳定性的条件下．林带必须具有空间上布局的合理性和时间上的连续性．经过多年防护林营林研究实践．在对1992年于辽宁省昌图县双井子乡设计营造的试验示范林带调查的基础上．对农田防护林单条林带方向的设置、带内树木的空间搭配方式、树种组成形式,多条林带或林网的带间距离以及大面积或区域防护林体系的空间景观布局等进行了综合研究．结果表明,单条林带和林网走向都应以垂直主害风作为设计的原则;林带内树木的空间搭配以“品”字型为佳,在不同树种混交的同一条林带中,可利用“边行优势”将生长相对缓慢的树种配置于边行．生长相对迅速的树种配置于内行;多条林带或林网空间配置参数——带间距离的设计应以林带达到初始防护成熟龄时的树高作为成林高．以林带结构变化规律和降低害风比例作为林带设计关键参数;区域防护林体系的空间布局应以景观生态学原理为基础．对林网体系进行评价与调控．     

Mobilizing and integrating big data in studies of spatial and phylogenetic patterns of biodiversity

The current global challenges that threaten biodiversity are immense and rapidly growing. These biodiversity challenges demand approaches that meld bioinformatics, large-scale phylogeny reconstruction, use of digitized specimen data, and complex post-tree analyses (e.g. niche modeling, niche diversification, and other ecological analyses). Recent developments in phylogenetics coupled with emerging cyberinfrastructure and new data sources provide unparalleled opportunities for mobilizing and integrating massive amounts of biological data, driving the discovery of complex patterns and new hypotheses for further study. These developments are not trivial in that biodiversity data on the global scale now being collected and analyzed are inherently complex. The ongoing integration and maturation of biodiversity tools discussed here is transforming biodiversity science, enabling what we broadly term “next-generation” investigations in systematics, ecology, and evolution (i.e., “biodiversity science”). New training that integrates domain knowledge in biodiversity and data science skills is also needed to accelerate research in these areas. Integrative biodiversity science is crucial to the future of global biodiversity. We cannot simply react to continued threats to biodiversity, but via the use of an integrative, multifaceted, big data approach, researchers can now make biodiversity projections to provide crucial data not only for scientists, but also for the public, land managers, policy makers, urban planners, and agriculture.     

Novel application of a quantitative spatial comparison tool to species distribution data

Comparing geographically referenced maps has become an important aspect of spatial ecology (e.g. assessing change in distribution over time). Whilst humans are adept at recognising and extracting structure from maps (i.e. identifying spatial patterns), quantifying these structures can be difficult. Here, we show how the Structural Similarity (SSIM) index, a spatial comparison method adapted from techniques developed in computer science to determine the quality of image compression, can be used to extract additional information from spatial ecological data. We enhance the SSIM index to incorporate uncertainty from the underlying spatial models, and provide a software algorithm to correct for internal edge effects so that loss of spatial information from the map comparison is limited. The SSIM index uses a spatially-local window to calculate statistics based on local mean, variance, and covariance between the maps being compared. A number of statistics can be calculated using the SSIM index, ranging from a single summary statistic to quantify similarities between two maps, to maps of similarities in mean, variance, and covariance that can provide additional insight into underlying biological processes. We demonstrate the applicability of the SSIM approach using a case study of sperm whales in the Mediterranean Sea and identify areas where local-scale differences in space-use between groups and singleton whales occur. We show how novel insights into spatial structure can be extracted, which could not be obtained by visual inspection or cell-by-cell subtraction. As an approach, SSIM is applicable to a broad range of spatial ecological data, providing a novel, implementable tool for map comparison.     

Development of a carbon dioxide-capture assay in microtiter plate for aspartyl-beta-hydroxylase.

CO2-capture methods have been used for assaying many decarboxylating enzymes including hydroxylation-coupled decarboxylation reactions. The traditional CO2-capture method involves performing the reaction in capped tubes and radiometric measurement of trapped 14CO2 by scintillation counting. In this report, a 14CO2-capture method in a 96-well microtiter plate format has been developed and a phosphor imaging system has been employed for sample measurement. The new assay method has been used successfully to assay aspartyl-beta-hydroxylase activity in microtiter plate format. The results obtained here compare favorably with those obtained from the traditional tube method. The method is sensitive, suitable for high throughput, and generally applicable to many CO2-releasing enzyme assays.     

Rapid and sensitive detection of b-galactosidase-producing yeasts by using microtiter plate assay

Yeast b-galactosidase activity was detected by a microtiter plate assay using pNPG or Xgal as substrate in 30 minutes. The detection gave a clear result which is well correlated with the specific b-galactosidase activity present in each strain studied. The microtiter plate assay is an effective method to improve the detection and quantify the b-galactosidase gene in recombinant strains of Saccharomyces cerevisiae.     

Modelling spatial patterns in harbour porpoise satellite telemetry data using maximum entropy

The distribution of harbour porpoises in EU waters is poorly understood, and modelled predictions of their distributions could inform the strategic spatial planning of future exploitation of the marine environment to avoid potential conflicts. We analysed satellite telemetry data from 39 harbour porpoises Phocoena phocoena in inner Danish waters using a modelling tool rooted in maximum entropy: Maxent. Maxent does not require absence data and has been shown to be effective for data characterised by small sample size, sampling bias and locational errors. For each season we used an iterative bootstrapping procedure to randomly select among the most precise records from each of the 39 tagged individuals, and ran Maxent on pooled records based on explanatory environmental variables hypothesised to serve as good proxies for harbour porpoise prey abundance. Among our environmental variables, distance to coast and bottom salinity had the most explanatory power, and their response shapes were relatively consistent across most seasons. The predictive power of the models (assessed by ROC‐AUC) ranged from 0.70 to 0.86 within seasons. The southern Kattegat, the Belt Seas, most western part of the Baltic Sea and the Sound were predicted to have relatively high probabilities of occurrence across seasons. In contrast, the central part of Kattegat and the Baltic Sea south and east of Limhamn and Darss Ridge consistently showed low probabilities of occurrence. Areas with the lowest probabilities of occurrence were generally characterised by high predictive uncertainty. Our methods have implications for the analyses of satellite tagged animals in terrestrial and marine environments. By coupling a bootstrapping procedure with Maxent we circumvented some of the statistical challenges presented by satellite telemetry data to generate spatial predictions within the inner Danish waters.     

Rapid and sensitive detection of D-hydantoinase producing microorganisms by using microtiter plate assay

Summary D-Hydantoinase activity of microorganisms was detected by a microtiter plate assay using dihydrouracil as substrate in 24 h. The detection gave a clear result compared to the spot-test in which the colour development of one colony interfered with colour development by adjacent colonies. The sensitivity of the detection was also higher than that of the spot-test.     

Visual detection of peptidase activity using fluorogenic substrates in a microtiter plate assay

A simple, inexpensive, and sensitive assay for peptidase activity has been devised. The assay was performed in a microtiter plate and was based on fluorogenic peptide substrates, many of which are commercially available. 7-Amino-4-methyl coumarin the fluorescent product liberated during an incubation period of between 1 and 16 h, was detected by inspection of the plate under ultraviolet light of wavelength 356 nm. A fluorometer was not required. Using alpha-chymotrypsin as a model enzyme, with succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-methyl-coumaryl-7-amide as substrate, it was shown that as little as 4 fmol of enzyme could be detected. The method was non-quantitative and was particularly suited to location of enzyme activity in fractions during a purification procedure. The validity of the assay was demonstrated by detection of activity of a known enzyme, alpha-chymotrypsin, after its purification by size-exclusion high-performance liquid chromatography. The method was used to locate two forms of aminopeptidase activity, in fractions from size-exclusion chromatography of an extract from reproductive tissue of Helix aspersa, using L-leucine 4-methyl-coumaryl-7-amide as substrate.     

h-Profile plots for the discovery and exploration of patterns in gene expression data with an application to time course data

Background  

An ever increasing number of techniques are being used to find genes with similar profiles from microarray studies. Visualization of gene expression profiles can aid this process, potentially contributing to the identification of co-regulated genes and gene function as well as network development.     

Mitochondrial genome and diverse inheritance patterns in Pleurotus pulmonarius

Journal of Microbiology - Pleurotus pulmonarius, a member of the Pleurotaceae family in Basidiomycota, is an edible, economically important mushroom in most Asian countries. In this study, the...     

An enzymatic fluorescent assay for the quantification of phosphite in a microtiter plate format

A sensitive fluorometric assay for the quantification of phosphite has been developed. The assay uses the enzymatic oxidation of phosphite to phosphate by a recombinant phosphite dehydrogenase with NAD⁺ as cosubstrate to produce the highly fluorescent reaction product resorufin. The optimized assay can be carried out in a 96-well microtiter plate format for high-throughput screening purposes and has a detection limit of 0.25 nmol phosphite. We used the method to quantify phosphite levels in plant tissue extracts and to determine phosphite dehydrogenase activity in transgenic plants. The assay is suitable for other biological or environmental samples. Because phosphite is a widely used fungicide to protect plants from pathogenic oomycetes, the assay provides a cost-effective and easy-to-use method to monitor the fate of phosphite following application.     

A supervised approach for identifying discriminating genotype patterns and its application to breast cancer data

MOTIVATION: Large-scale association studies, investigating the genetic determinants of a phenotype of interest, are producing increasing amounts of genomic variation data on human cohorts. A fundamental challenge in these studies is the detection of genotypic patterns that discriminate individuals exhibiting the phenotype under study from individuals that do not possess it. The difficulty stems from the large number of single nucleotide polymorphism (SNP) combinations that have to be tested. The discrimination problem becomes even more involved when additional high-throughput data, such as gene expression data, are available for the same cohort. RESULTS: We have developed a graph theoretic approach for identifying discriminating patterns (DPs) for a given phenotype in a genotyped population. The method is based on representing the SNP data as a bipartite graph of individuals and their SNP states, and identifying fully connected subgraphs of this graph that relate individuals enriched for a given phenotypic group. The method can handle additional data types such as expression profiles of the genotyped population. It is reminiscent of biclustering approaches with the crucial difference that its search process is guided by the phenotype under consideration in a supervised manner. We tested our approach in simulations and on real data. In simulations, our method was able to retrieve planted patterns with high success rate. We then applied our approach to a dataset of 72 breast cancer patients with available gene expression profiles, genotyped over 695 SNPs. We detected several DPs that were highly significant with respect to various clinical phenotypes, and investigated the groups of patients and the groups of genes they defined. We found the patient groups to be highly enriched for other phenotypes and to display expression coherency among their profiles. The gene groups displayed functional coherency and involved genes with known role in cancer, providing additional support to their involvement. AVAILABILITY: The program is available upon request.