Development and validation of a cell-based high-throughput screening assay for TRPM2 channel modulators

TRPM2 is a member of the transient receptor potential melastatin (TRPM)-related ion channel family. The activation of TRPM2 induced by oxidative/nitrosative stress leads to an increase in intracellular free Ca(2+). Although further progress in understanding TRPM2's role in cell and organism physiology would be facilitated by isolation of compounds able to specifically modulate its function in primary cells or animal models, no cell-based assays for TRPM2 function well suited for high-throughput screening have yet been described. Here, a novel suspension B lymphocyte cell line stably expressing TRPM2 was used to develop a cell-based assay. The assay uses the Ca(2+)-sensitive fluorescence dye, Fluo-4 NW (no wash), to measure TRPM2-dependent Ca(2+) transients induced by H(2)O(2) and N-methyl-N'-nitrosoguanidine in a 96-well plate format. Assay performance was evaluated by statistical analysis of the Z' factor value and was consistently greater than 0.5 under optimal conditions, suggesting that the assay is very robust. For assay validation, the effects of known inhibitors of TRPM2 and TRPM2 gating secondary messenger production were determined. Overall, the authors have developed a cell-based assay that may be used to identify TRPM2 ion channel modulators from large compound libraries.     

Allelopathy: a tool for weed management in forest restoration

Forest restoration uses active management to re-establish natural forest habitat after disturbance. However, competition from early successional species, often aggressively invasive exotic plant species, can inhibit tree establishment and forest regeneration. Ideally, restoration ecologists can plant native tree species that not only establish and grow rapidly, but also suppress exotic competitors. Allelopathy may be a key mechanism by which some native trees could reduce the abundance and impact of exotic species. Allelopathy is a recognized tool for weed management in agriculture and agroforestry, but few studies have considered how allelopathic interactions may aid restoration. Here we introduce the “Homeland Security” hypothesis, which posits that some naïve exotic species may be particularly sensitive to allelochemicals produced by native species, providing a tool to reduce the growth and impacts of invasive exotic species on reforestation. This article explores how exploiting allelopathy in native species could improve restoration success and the re-establishment of natural successional dynamics. We review the evidence for allelopathy in agroforestry systems, and consider its relevance for reforestation. We then illustrate the potential for this approach with a case study of tropical forest restoration in Panama. C₄ grasses heavily invade deforested areas in the Panama Canal watershed, especially Saccharum spontaneum L. We measured the effect of leaf litter from 17 potential restoration tree species on the growth of invasive C₄ grasses. We found that leaf litter from legume trees had a greater inhibitory effect on performance of S. spontaneum than did litter from non-legume trees. However, allelopathic effects varied greatly among species within tree functional groups. Further evaluation of intra- and inter-specific interactions will help to improve our selection of restoration species.     

Development and validation of a high-throughput screening assay for the hepatitis C virus p7 viroporin

The HCV p7 protein is not involved in viral RNA replication but is essential for production of infectious virus. Based on its putative ion channel activity, p7 belongs to a family of viral proteins known as viroporins that oligomerize after insertion into a lipid membrane. To screen for compounds capable of interfering with p7 channel function, a low-throughput liposome-based fluorescent dye permeability assay was modified and converted to a robust high-throughput screening assay. Escherichia coli expressing recombinant p7 were grown in high-density fed-batch fermentation followed by a detergent-free purification using a combination of affinity and reversed-phase chromatography. The phospholipid composition of the liposomes was optimized for both p7 recognition and long-term stability. A counterscreen was developed using the melittin channel-forming peptide to eliminate nonspecific screening hits. The p7 liposome-based assay displayed robust statistics (Z' > 0.75), and sensitivity to inhibition was confirmed using known inhibitors.     

Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase

Human estrogen sulfotransferase (SULT1E1) is involved in the regulation of 17beta-estradiol responsiveness and is believed to protect peripheral tissues from excessive estrogenic effects. Several assays already have been developed to investigate the inhibitory effect of endocrine-disrupting compounds (EDCs) on SULT1E1. However, most of these assays make use of the radiolabeled cofactor [(35)S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) or radiolabeled substrate [(3)H]estradiol. In this article, we describe the development and validation of an assay for the inhibition of human SULT1E1 that is rapid and simple and that uses the nonradioactive and noncarcinogenic 1-hydroxypyrene. A gradient HPLC separation of 15 min using a C18-RP column was developed to detect 1-hydroxypyrene and its metabolite pyrene 1-sulfate fluorescently. Time- and protein-dependent formation of pyrene 1-sulfate was investigated, and enzyme kinetics was determined (K(m)=6.4+/-0.8 nM and V(max)=158+/-19 pmol/min/microg SULT1E1). At higher 1-hydroxypyrene concentrations, the assay displayed non-Michaelis-Menten kinetics involving substrate inhibition. IC(50) values have been determined for eight known SULT1E1 inhibitors or competing substrates (17beta-estradiol, 17alpha-estradiol, genistein, 17alpha-ethynylestradiol, estrone, diethylstilbestrol, estriol, and hexestrol) and two previously unknown SULT1E1 inhibitors (zearalenone and dienestrol). The method was demonstrated to be easy, feasible, and highly reproducible for SULT1E1 screening assay inhibition studies.     

Development and validation of a plate technique for screening of microorganisms that produce volatile sulfur compounds

Volatile sulfur compounds (VSCs) are of major importance for flavor development in foodstuffs such as cheeses. Such compounds originate from the amino acid l-methionine, which can be degraded to methanethiol (MTL), a common precursor to a variety of other VSCs. A plate assay based on double-layer petri dishes containing 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB), a chemical used for the estimation of free thiols, in the upper layer provides an easy and reliable detection method for thiol-producing, cheese-ripening microorganisms. MTL production was quantitated by measuring the yellow-orange color intensity resulting from reaction with DTNB. Using this method, 18 Geotrichum candidum strains isolated from cheeses were compared, and the color intensity was found to be correlated with the production of microbial VSCs as measured by gas chromatographic analysis.     

Clark Wissler and the Development of Anthropology in the United States

Wissler receives scant notice today although he was a major figure in American anthropology. During the decades when the historical particularism of Franz Boas dominated American cultural anthropology, Wissler's theories provided a nomothetic alternative. His theories are in current use in various guises. The importance of Wissler has been obscured to some extent because he is often misclassified as a Boasian. However, he consciously worked outside of Boas's influence.     

Development and validation of a time-resolved fluorometric immunoassay for screening of antichlamydial activity using a genus-specific europium-conjugated antibody

The lack of high-throughput assays has limited the screening of new antimicrobials against obligate intracellular bacteria, including chlamydia, which cause a variety of diseases. In this study, a novel technological approach was developed to detect intracellular bacteria using time-resolved fluorometric immunoassay (TR-FIA), and the method was validated for susceptibility testing of Chlamydia pneumoniae. In this cell-based, 96-well plate assay, chlamydial inclusions are labeled with europium-conjugated antibody and quantified as time-resolved fluorometric signals by means of a multilabel counter. To confirm the reliability of the TR-FIA, susceptibilities of C. pneumoniae reference strain Kajaani 7 to a set of antimicrobial agents were determined by the TR-FIA, conventional immunofluorescence staining, and real-time polymerase chain reaction. Minimum inhibitory concentrations measured using the different methods demonstrated good to excellent correlation. Data relating to reproducibility (day-to-day variation 9.0%), as well as to the signal-to-background, signal-to-noise, and Z′ values (6.5, 6.9, and 0.4, respectively), showed the suitability of the TR-FIA for screening. By means of dual labeling with sulfornodamine B the cytotoxicity of test compounds could be detected simultaneously with the susceptibility testing. In summary, the TR-FIA is a convenient, reliable, and objective alternative for detecting chlamydia in vitro. By being considerably less labor intensive and offering significantly higher throughput, the TR-FIA is especially suitable for screening of new antichlamydial compounds.     

Species screening and biomass trials of woody plants in the semi-arid southwest United States

Species screening and selection trials are being conducted to identify appropriate woody species and technology for biofuel farming in the southwest United States. During the initial phase of the program, 26 native and exotic species have been tested at sites in Texas, New Mexico and Arizona. Second season biomass estimates indicate that initial yields of 3·2–8·5 dry Mg ha⁻¹ year⁻¹ are possible without post-establishment irrigation. The species Atriplex canescens (Pursh) Nutt., Prosopis alba Griseb. and Leucaena leucocephala (Lam.) de Wit reached canopy closure within two seasons of growth and are identified as rapid initial biomass producers under the semi-arid conditions of the study area.     

Vermicomposting--An effective tool for the management of invasive weed Parthenium hysterophorus

This study reports the results of vermicomposting with Eisenia fetida of Parthenium hysterophorus mixed with cow dung in different ratios (25%, 50% and 75%) in a 18 weeks experiment. In all the treatments, a decrease in pH, OC_total and C:N ratio, but increase in EC, N_total, P_aval, Ca_total, K_total and heavy metals was recorded. The cocoons production and growth rate (biomass gain worm⁻¹ day⁻¹) were maximum in 100% cow dung. The results indicated that parthenium can be a raw material for vermicomposting if mix with cow dung in appropriate quantity.     

The United States of the United Races: a rejoinder

I respond to a review by C. Matthew Snipp, revisiting how my book connects abolitionist leanings to acceptance of racial mixing in the Early Republic. I reiterate that, contrary to the reviewer's claims, the book does not suggest that the defence of interracial marriage has been a thriving social movement. I correct his reading of my chapter on the Civil War era, referring to both the variety of voices present, and the claims of reformers' opponents, who were the only ones who claimed racial mixing was an aim of the abolitionist movement. Lastly, I defend The United States of the United Races against Professor Snipp's characterization of it as a work anticipating a ‘post-racial’ ideal, embodied by racially mixed people, who would be the end point of the obsolescence of race as a relevant analytic tool.     

Development and status of dedicated energy crops in the United States

The biofuel industry is rapidly growing because of increasing energy demand and diminishing petroleum reserves on a global scale. A multitude of biomass resources have been investigated, with high-yielding, perennial feedstocks showing the greatest potential for utilization as advanced biofuels. Government policy and economic drivers have promoted the development and commercialization of biofuel feedstocks, conversion technologies, and supply chain logistics. Research and regulations have focused on the environmental consequences of biofuels, greatly promoting systems that reduce greenhouse gas emissions and life-cycle impacts. Numerous biofuel refineries using lignocellulosic feedstocks and biomass-based triglycerides are either in production or pre-commercial development phases. Leading candidate energy crops have been identified, yet require additional efforts to realize their full potential. Advanced biofuels, complementing conventional biofuels and other renewable energy sources such as wind and solar, provide the means to substantially displace humanity’s reliance on petroleum-based energy.     

Biological control of the wetlands weed purple loosestrife (Lythrum salicaria) in the Pacific northwestern United States

Purple loosestrife (Lythrum salicaria) is an Eurasian perennial hydrophyte that has become naturalized in wetlands and in and along waterways throughout temperate North America. The ecological integrity of such areas is threatened by rapidly forming monotypic infestations that displace valued flora and diminish critical fish and wildlife habitat. The inability of physical, cultural, and chemical methods to provide adequate control of the weed has led to the development of an insect-based biological control program. The first field releases of the bud and leaf feeding beetles, Galerucella calmariensis and G. pusilla, and a root-mining weevil, Hylobius transversovittatus, were made in the United States and Canada in 1992. A total of 4740 Galerucella spp. adults were released in central Washington during 1992 and 1993 at eight sites and 471 H. transversovittatus egg inoculations were made in 1993 at three locations. Establishment of both Galerucella spp. was confirmed and Hylobius colonization was achieved.     

Proteomics as a tool for clinically relevant biomarker discovery and validation

The excitement associated with clinical applications of proteomics was initially focused on its potential to serve as a vehicle for both biomarker discovery and drug discovery and routine clinical sample analysis. Some approaches were thought to be able to "identify" mass spectral characteristics that distinguished between control and disease samples, and thereafter it was believed that the same tool could be employed to screen samples in a high-throughput clinical setting. However, this has been difficult to achieve, and the early promise is yet to be fully realized. While we see an important place for mass spectrometry in drug and biomarker discovery, we believe that alternative strategies will prove more fruitful for routine analysis. Here we discuss the power and versatility of 2D gels and mass spectrometry in the discovery phase of biomarker work but argue that it is better to rely on immunochemical methods for high-throughput validation and routine assay applications.     

Single-item screening for agoraphobic symptoms: validation of a web-based audiovisual screening instrument

The advent of web-based treatments for anxiety disorders creates a need for quick and valid online screening instruments, suitable for a range of social groups. This study validates a single-item multimedia screening instrument for agoraphobia, part of the Visual Screener for Common Mental Disorders (VS-CMD), and compares it with the text-based agoraphobia items of the PDSS-SR. The study concerned 85 subjects in an RCT of the effects of web-based therapy for panic symptoms. The VS-CMD item and items 4 and 5 of the PDSS-SR were validated by comparing scores to the outcomes of the CIDI diagnostic interview. Screening for agoraphobia was found moderately valid for both the multimedia item (sensitivity.81, specificity.66, AUC.734) and the text-based items (AUC.607-.697). Single-item multimedia screening for anxiety disorders should be further developed and tested in the general population and in patient, illiterate and immigrant samples.     

Development of a novel,high-throughput screening tool for efficient perfusion-based cell culture process development

Perfusion technology has been successfully used for the commercial production of biotherapeutics, in particular unstable recombinant proteins, for more than a decade. However, there has been a general lack of high-throughput cell culture tools specifically for perfusion-based cell culture processes. Here, we have developed a high-throughput cell retention operation for use with the ambr® 15 bioreactor system. Experiments were run in both 24 and 48 reactor configurations for comparing perfusion mimic models, media development, and clone screening. Employing offline centrifugation for cell retention and a variable volume model developed with MATLAB computational software, the established screening model has demonstrated cell culture performance, productivity, and product quality were comparable to bench scale bioreactors. The automated, single use, high-throughput perfusion mimic is a powerful tool that enables us to have rapid and efficient process development of perfusion-based cell culture processes.     

AutoDimer: a screening tool for primer-dimer and hairpin structures

The ability to select short DNA oligonucleotide sequences capable of binding solely to their intended target is of great importance in developing nucleic acid based detection technologies. Applications such as multiplex PCR rely on primers binding to unique regions in a genome. Competing side reactions with other primer pairs or template DNA decrease PCR efficiency: Freely available primer design software such as Primer3 screens for potential hairpin and primer-dimer interactions while selecting a single primer pair. The development of multiplex PCR assays (in the range of 5 to 20 loci) requires the screening of all primer pairs for potential cross-reactivity. However, a logistical problem results due to the number of total number of comparisons required. Comparing the primer set for a 10-plex assay (20 total primer sequences) results in 210 primer-primer combinations that must be screened. The ability to screen sets of candidate oligomers rapidly for potential cross-reactivity reduces overall assay devlelopment time. Here we report the application of a familiar sliding algorithm for comparing two strands of DNA in an overlapping fashion. The algorithm has been employed in a software package wherein the user can compare multiple sequences in a single computational run. After the screening is completed, a score is assigned to potential duplex interactions exceeding a user-defined threshold. Additional criteria of predicted melting temperature (Tm) and free energy of melting (deltaG) are included for further ranking. Sodium counterion and total stand concentrations can be adjusted for the Tm and deltaG calculations. The predicted interactions are saved in a text file for further evaluation.     

美国流域生态健康评价体系的发展和实践

为了达到《清洁水法》规定的水质目标,美国环境部门在过去20多年的实践中逐步发展形成了涵盖整个流域,包括水文、化学、生物等多重指标在内的全国性流域综合生态健康评价体系.该体系已经成为美国流域水质管理系统的重要组成部分,为水环境保护和水生生态系统恢复提供了有力支持.本文从法律行政框架、生态功能分析、生态健康指标、综合评价体系和流域监测系统等方面系统总结了美国环境管理部门,特别是联邦环境保护局在流域生态健康评价方面的发展和实践;并介绍了美国水生资源调查采用的河流、湖泊、河口、海湾、湿地流域健康评价系统.在此基础上,根据我国水环境保护和治理的实际情况,提出了建立流域生态健康科学评价和决策支持体系建议.