Screening of ligands for redox-active europium using magnetic resonance imaging

We report a screening procedure to predict ligand coordination to Eu^II and Eu^III using magnetic resonance imaging in which bright images indicate complexation and dark images indicate no complexation. Here, paramagnetic Gd^III is used as a surrogate for Eu^III in the screening procedure to enable detection with magnetic resonance imaging. The screening procedure was tested using a set of eight ligands with known coordination to Eu^II and Eu^III, and results were found to be consistent with expected binding. Validation of the screening procedure with known coordination chemistry enables use with new ligands in the future.     

Feature screening with latent responses

A novel feature screening method is proposed to examine the correlation between latent responses and potential predictors in ultrahigh-dimensional data analysis. First, a confirmatory factor analysis (CFA) model is used to characterize latent responses through multiple observed variables. The expectation-maximization algorithm is employed to estimate the parameters in the CFA model. Second, R-Vector (RV) correlation is used to measure the dependence between the multivariate latent responses and covariates of interest. Third, a feature screening procedure is proposed on the basis of an unbiased estimator of the RV coefficient. The sure screening property of the proposed screening procedure is established under certain mild conditions. Monte Carlo simulations are conducted to assess the finite-sample performance of the feature screening procedure. The proposed method is applied to an investigation of the relationship between psychological well-being and the human genome.     

Discovery of novel low molecular weight inhibitors of IMPDH via virtual needle screening

Novel, low molecular weight inhibitors of IMPDH have been discovered through the application of a validated virtual screening protocol. A series of 21 IMPDH inhibitors were used to validate the docking procedure. Application of this procedure to the selection of compounds for screening from an in-house database resulted in a 50-fold reduction in the size of the screening set (3425 to 74 compounds) and gave a hit-rate of 10% on biological evaluation.     

噬菌体抗体库筛选技术研究进展

噬菌体抗体库技术是一项新兴的基因工程抗体技术,应用这项技术获得高特异性抗体的关键之一就是筛选环节。根据抗原性质以及筛选目的的不同,筛选方法的选择也不相同,各种筛选策略的优化对中和抗体的获得有至关重要的作用。     

A rapid plate assay for screening l-asparaginase producing micro-organisms

A pH and dye-based fast procedure for screening l -asparaginase-producing micro-organisms is reported. The procedure is suitable for bacterial and fungal screening. The results are obtained within 24 and 48 h for bacteria and fungi, respectively. The results correlate with quantitative estimations in culture broths.     

Selection of Antibodies with Tailored Properties by Application of High-Throughput Multiparameter Fluorescence-Activated Cell Sorting of Yeast-Displayed Immune Libraries

In this study, we present a multiparameter screening procedure for the identification of target-specific antibodies with prescribed properties. Based on B cell receptor gene repertoires from transgenic rats, yeast surface display libraries were generated, and high-affinity human antibodies were readily isolated. We demonstrate that specific desirable features, i.e., species’ cross-reactivity and a broad epitope coverage can be integrated into the screening procedure using high-throughput fluorescence-activated cell sorting. We show that the applied screening stringencies translate directly into binding properties of isolated human antibody variants.     

Controlled search for the producers of ionophore antibiotics among Streptomycetes

A procedure was developed for directed screening of cultures producing ionophore antibiotics among streptomycetes. The procedure is based on measuring the membrane potential generated in the presence of the Men+/nH+ = -expchanger-protonophore couple. It provided isolation of cultures producing ionophore antibiotics at the fermentation broth stage. It was possible to use the procedure in screening both electrogenic and nonelectrogenic ionophores and to rapidly differentiate them. 5 cultures producing ionophore antibiotics were detected with this procedure; 3 of them carry out nonelectrogenic transport of the cations. The cation transport in the other two cultures was electrogenic. Cation selectivity of the antibiotics produced by the cultures was determined with the procedure. An antibiotic identical to indanomycin was isolated from the culture fluid of Streptomyces chromogenes.     

Simple method for extracting plasmid DNA from lactic acid bacteria

Rapid screening and large-scale plasmid DNA isolation procedures are described for lactic acid bacteria, using glass beads to break cells. The rapid screening procedure allows one to obtain plasmid DNA pellets in less than 1 h. This method has been successfully tested on various bacteria from the genera Lactococcus, Leuconostoc, Lactobacillus, Pediococcus, Streptococcus, Enterococcus and Propionibacterium. This procedure yields plasmid DNA with minor chromosomal and plasmid DNA-degraded form contaminations.     

High Throughput Screening of Signal Transduction Mutants With Luciferase Imaging

A detailed procedure for high throughput genetic screening of hormone and environmental stress signal transduction mutants of Arabidopsis thaliana is described. The screen was carried out with mutagenized plants expressing the firefly luciferase reporter under control of a cold, osmotic stress, and absciscic acid responsive promoter. A thermoelectrically cooled CCD camera was used to detect luminescence emitted by the plants in response to stresses or ABA. Advantages of the screening procedure include high throughput, capability to identify low as well as high expression mutants and employment of a highly sensitive but affordable imaging system and software. This procedure can be used to study complex signal transduction networks in higher plants.     

Screening of xylanolytic bacteria using a colour plate method

A simple procedure is described for the screening and isolation of xylanolytic bacteria, able to degrade hemicellulose. Bagasse hemicellulose was purified, cross-linked with green dye for use as indicator, and used in a Petri dish assay for the determination of xylanolytic activity. Hydrolysis of the substrate causes the formation of transparent halos around bacterial colonies. The screening procedure was used to select thermoresistant bacteria. The four best isolates were characterized and classified as Bacillus species.     

A two-phase procedure for QTL mapping with regression models

It is typical in QTL mapping experiments that the number of markers under investigation is large. This poses a challenge to commonly used regression models since the number of feature variables is usually much larger than the sample size, especially, when epistasis effects are to be considered. The greedy nature of the conventional stepwise procedures is well known and is even more conspicuous in such cases. In this article, we propose a two-phase procedure based on penalized likelihood techniques and extended Bayes information criterion (EBIC) for QTL mapping. The procedure consists of a screening phase and a selection phase. In the screening phase, the main and interaction features are alternatively screened by a penalized likelihood mechanism. In the selection phase, a low-dimensional approach using EBIC is applied to the features retained in the screening phase to identify QTL. The two-phase procedure has the asymptotic property that its positive detection rate (PDR) and false discovery rate (FDR) converge to 1 and 0, respectively, as sample size goes to infinity. The two-phase procedure is compared with both traditional and recently developed approaches by simulation studies. A real data analysis is presented to demonstrate the application of the two-phase procedure.     

Convenient method for detecting 14CO2 in multiple samples: application to rapid screening for mutants.

A procedure is presented for the rapid screening of bacterial colonies to detect mutants unable to produce 14CO2 from a labeled precursor. The method is especially useful for mass screening for mutants that cannot be easily detected by their phenotypic characteristics.     

A highly cost effective method of mass screening for thalassaemia.

A simple, fast, and reliable two step procedure for the detection of non-alpha-thalassaemias in mass screening programmes is presented. Step 1 consists of a study of red cell morphology and a one tube red cell osmotic fragility tests. This step eliminates the non-thalassaemic samples; the rest are processed through step 2, consisting of determination of red cell indices and haemoglobin studies. Over the past seven years this procedure has been used at this centre in mass screening secondary school students in Latium. Blood samples from 289 763 students were examined, and 6838 cases of thalassaemia detected. It is estimated that 0.35 +/- 0.25% of subjects with thalassaemia escaped detection by this procedure.     

Detection of Mycotoxins by Thin-Layer Chromatography: Application to Screening of Fungal Extracts

A convenient thin-layer chromatographic screening procedure for the detection of 18 mycotoxins is described.     

噬菌体cDNA文库筛选方法的改良

cDNA文库的构建和简便、快速的筛选是获得全长基因的重要途径,基于PCR的筛库方法具有快捷、灵敏的特点。研究改进了基于PCR的噬菌体cDNA文库筛选方法,用液体分装的方法,替代了文库筛选的关键步骤——涂板分区,省去了噬菌体文库铺平板、浸染、培养、划块洗脱的操作过程,使筛库的工作量减少,进一步提高了筛选速度和获得阳性克隆的效率。     

Pre-docking filter for protein and ligand 3D structures

Virtual drug screening using protein-ligand docking techniques is a time-consuming process, which requires high computational power for binding affinity calculation. There are millions of chemical compounds available for docking. Eliminating compounds that are unlikely to exhibit high binding affinity from the screening set should speed-up the virtual drug screening procedure. We performed docking of 6353 ligands against twenty-one protein X-ray crystal structures. The docked ligands were ranked according to their calculated binding affinities, from which the top five hundred and the bottom five hundred were selected. We found that the volume and number of rotatable bonds of the top five hundred docked ligands are similar to those found in the crystal structures and corresponded with the volume of the binding sites. In contrast, the bottom five hundred set contains ligands that are either too large to enter the binding site, or too small to bind with high specificity and affinity to the binding site. A pre-docking filter that takes into account shapes and volumes of the binding sites as well as ligand volumes and flexibilities can filter out low binding affinity ligands from the screening sets. Thus, the virtual drug screening procedure speed is increased.     

Optimized and automated protocols for high-throughput screening of amylosucrase libraries

This article describes the design and validation of a general procedure for the high-throughput isolation of amylosucrase variants displaying higher thermostability or increased resistance to organic solvents. This procedure consists of 2 successive steps: an in vivo selection that eliminates inactive variants followed by automated screening of active variants to isolate mutants displaying enhanced features. The authors chose an Escherichia coli expression vector, allowing a high production rate of the recombinant enzyme in miniaturized culture conditions. The screening assay was validated by minimizing variability for various parameters of the protocol, especially bacterial growth and protein production in cultures in 96-well microplates. Recombinant amylosucrase production was normalized by decreasing the coefficient of variance from 27% to 12.5%. Selective screening conditions were defined to select variants displaying higher thermostability or increased resistance to organic solvents. A first-generation amylosucrase variant library, constructed by random mutagenesis, was subjected to this procedure, yielding a mutant displaying a 25-fold increased stability at 50 degrees C compared to the parental wild-type enzyme.     

Pilot study of gas chromatographic–mass spectrometric screening of newborn urine for inborn errors of metabolism after treatment with urease

Gas chromatographic–mass spectrometric (GC–MS) techniques for urinary organic acid profiling have been applied to high-risk screening for a wide range of diseases, mainly for inborn errors of metabolism (IEM), rather than to low-risk screening or mass screening. Using a simplified procedure with urease-pretreatment and the GC–MS technique, which allows simultaneous determination of organic acids, amino acids, sugars and sugar acids, we performed a pilot study of the application of this procedure to neonatal urine screening for 22 IEM. Out of 16 246 newborns screened, 11 cases of metabolic disorders were chemically diagnosed: two each of methylmalonic aciduria and glyceroluria, four of cystinuria, and one each of Hartnup disease, citrullinemia and α-aminoadipic aciduria/α-ketoadipic aciduria. The incidence of IEM was thus one per 1477, which was higher than the one per 3000 obtained in the USA in a study targeting amino acids and acylcarnitines in newborn blood spots by tandem mass spectrometry. Also, 227 cases were found to have transient metabolic abnormalities: 108 cases with neonatal tyrosinuria, 99 cases with neonatal galactosuria, and 20 cases with other transient metabolic disorders. Two hundred and thirty-eight cases out of 16 246 neonates (approximately 1/68) were thus diagnosed using this procedure as having either persistent or transient metabolic abnormalities.