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PubMed2004

High-throughput imaging of bacterial colonies grown on filter plates with application to serum bactericidal assays.

The ability to accurately enumerate viable bacteria has applications in antibiotic screening assays, toxicology testing, and serological assays for functional antibodies. An impediment to high-throughput bacterial assays is the requirement to grow bacteria as individual colonies on semisolid media containing agar. We have now developed a method for growth, staining, and counting of bacterial colonies in 96-well filter plates. A unique feature of the method is that colony size is inversely proportional to the number of colonies in each well, presumably due to nutrient depletion. As a result, as many as 300 colony-forming units (cfu) can be detected as discrete colonies within a single assay well. The resulting colonies can be counted automatically using an imaging system originally developed for ELISPOT assays. The method has been applied to the measurement of serum bactericidal activity (SBA) in human sera.

Adult↗
PubMed2005

Novel approaches to plant drug discovery based on high throughput pharmacological screening and genetic manipulation.

Plants are potentially important for novel therapeutic drug leads, but the slowness of conventional methods for investigation of plants limits enthusiasm in the pharmaceutical industry. To overcome some of the drawbacks, we have applied high throughput pharmacological screening (HTPS) to crude plant extracts. Using a "differential smart screen", (DSS) the spectrum of activity contained in a crude extract is measured at several closely related receptor subtypes. This spectrum is then compared to that of known compounds. A unique spectrum suggests that the extract merits further investigation. Evaluation of species and environmental libraries of whole plants has demonstrated the value of this approach for rapid prioritization of plants for investigation. In addition, genomic and genetic manipulation of plants and plant cell cultures can increase the value of DSS. For example, the whole genomic potential of a plant species for biodiversity can be accessed by using gain of function mutations to generate a "functional genomics library" of mutant clonal cultures, and the bioactivity of these cultures tested by DSS. Clones that overproduce activity differing from the wild-type plant can be identified in this way. This "Natural Products Genomics" (NPG) strategy is limited by the massive numbers of clonal cultures that are required to cover all possible gain-of-function mutations. The rapidity and efficiency of this process can be improved by using transgenic plants expressing appropriate mammalian proteins. These may be designed to make the plant cell resemble a human cell for a specific form of toxicity. Now, "unnatural selection" of resistant mutant clones can be used to provide a sub-population potentially enriched in useful compounds. Alternatively, transgenic plant cells can be used for "in situ screening" in which a mammalian receptor protein, linked to a reporter construct, such as green fluorescent protein, is expressed. Clonal cultures that produce ligands for this receptor can now be rapidly identified visually in an ultra-HTPS. Overall, our aim is to use pharmacological screening, together with functional genomic approaches, to make plant drug discovery competitive with combinatorial chemistry.

Drug Evaluation, Preclinical↗
PubMed2001

High-throughput peptide identification from protein digests using data-dependent multiplexed tandem FTICR mass spectrometry coupled with capillary liquid chromatography.

Tandem mass spectrometry (MS/MS) plays an important role in the unambiguous identification and structural elucidation of biomolecules. In contrast to conventional MS/MS approaches for protein identification where an individual polypeptide is sequentially selected and dissociated, a multiplexed-MS/MS approach increases throughput by selecting several peptides for simultaneous dissociation using either infrared multiphoton dissociation (IRMPD) or multiple frequency sustained off-resonance irradiation (SORI) collisionally induced dissociation (CID). The high mass measurement accuracy and resolution of FTICR combined with knowledge of peptide dissociation pathways allows the fragments arising from several different parent ions to be assigned. Herein we report the application of multiplexed-MS/MS coupled with on-line separations for the identification of peptides present in complex mixtures (i.e., whole cell lysate digests). Software was developed to enable "on-the-fly" data-dependent peak selection of a subset of polypeptides from each FTICR MS acquisition. In the subsequent MS/MS acquisitions, several coeluting peptides were fragmented simultaneously using either IRMPD or SORI-CID techniques. The utility of this approach has been demonstrated using a bovine serum albumin tryptic digest separated by capillary LC where multiple peptides were readily identified in single MS/MS acquisitions. We also present initial results from multiplexed-MS/MS analysis of a D. radiodurans whole cell digest to illustrate the utility of this approach for high-throughput analysis of a bacterial proteome.

Amino Acid Sequence↗
PubMed2026

Identifying critical lysines in mammalian histone H3 with high-throughput CRISPR prime editing.

Histone post-translational modifications are fundamental to genome regulation, yet dissecting the functions of individual histone marks in mammals remains challenging due to the presence of multiple histone gene copies. Here we develop a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) prime editing platform enabling precise, reversible and combinatorial mutagenesis of canonical and noncanonical histone H3 genes within their native genomic context. Using systematic lysine-to-arginine substitutions benchmarked against synonymous controls, we identify key residues, including H3K4, H3K9, H3K14, H3K18 and H3K79, whose mutation compromises fitness in mouse embryonic stem cells. We further show that H3K56, linked to genome stability in yeast and Drosophila, has a conserved role in mammalian cells. Through analysis of selected double mutants, we uncover functional crosstalk across residues, with combinations such as H3K27R + H3K36R impairing stem cell self-renewal and altering transcription. Altogether, this study establishes a functional map of histone H3 lysines in mammals and provides a broadly applicable platform for systematic dissection of chromatin regulation.

Histones↗
PubMed2002

Enzyme fragment complementation: a flexible high throughput screening assay technology.

High-affinity complementation of a small fragment of beta-galactosidase to an inactive deletion mutant of the enzyme forms a stable heteromeric enzyme complex capable of hydrolyzing substrates to produce either chemiluminescent or fluorescent signals. This review describes a series of screening assays in which the small beta-galactosidase fragment, Enzyme Donor or ProLabel, is either chemically conjugated or recombinantly fused to small molecules or proteins, respectively. Chemical conjugation forms the basis of several HitHunter HTS assays in which competitive displacement of the ProLabel conjugate from either a binding protein (receptor or antibody) is induced by the analyte in question. In this manner, a calibration curve is generated, to measure cellular analytes including 3',5'-cyclic AMP. Changes in this second messenger, occurring due to G protein-coupled receptor (GPCR) activation, can thus be easily measured in a homogeneous assay. Similar assays have been developed for tyrosine kinases, serine threonine kinases, nuclear hormone receptors, and proteases. A second form of assay technology involves measurement of cellular protein expression, in which the protein is fused to ProLabel. Analysis can be undertaken in crude cell lysates, or with intact cells, using beta-galactosidase complementation in a microtiter plate. This homogeneous technology is highly sensitive and has been developed to measure protein expression changes occurring in response to pathway activation by targets such as GPCRs, tyrosine kinase receptors, and proteases. In summary, the DiscoveRx technology using beta-galactosidase complementation provides a robust and flexible assay technology for use in cell-free and cell-based HTS.

Animals↗
PubMed1999

High-throughput plasmid DNA purification for 3 cents per sample.

To accommodate the increasingly rapid rates of DNA sequencing we have developed and implemented an inexpensive, expeditious method for the purification of double-stranded plasmid DNA clones. The robust nature, high throughput, low degree of technical difficulty and extremely low cost have made it the plasmid DNA preparation method of choice in both our expressed sequence tag (EST) and genome sequencing projects. Here we report the details of the method and describe its application in the generation of more than 700 000 ESTs at a rate exceeding 16 000 per week.

DNA, Bacterial↗
PubMed2002

Large-scale, high-throughput screening for coagulation and hematologic phenotypes in mice.

The Mouse Phenome Project is an international effort to systematically gather phenotypic data for a defined set of inbred mouse strains. For such large-scale projects the development of high-throughput screening protocols that allow multiple tests to be performed on a single mouse is essential. Here we report hematologic and coagulation data for more than 30 inbred strains. Complete blood counts were performed using an Advia 120 analyzer. For coagulation testing, we successfully adapted the Dade Behring BCS automated coagulation analyzer for use in mice by lowering sample and reagent volume requirements. Seven automated assay procedures were developed. Small sample volume requirements make it possible to perform multiple tests on a single animal without euthanasia, while reductions in reagent volume requirements reduce costs. The data show that considerable variation in many basic hematological and coagulation parameters exists among the inbred strains. These data, freely available on the World Wide Web, allow investigators to knowledgeably select the most appropriate strain(s) to meet their individual study designs and goals.

Animals↗
PubMed2006

Novel method for high-throughput phenotyping of sleep in mice.

Assessment of sleep in mice currently requires initial implantation of chronic electrodes for assessment of electroencephalogram (EEG) and electromyogram (EMG) followed by time to recover from surgery. Hence, it is not ideal for high-throughput screening. To address this deficiency, a method of assessment of sleep and wakefulness in mice has been developed based on assessment of activity/inactivity either by digital video analysis or by breaking infrared beams in the mouse cage. It is based on the algorithm that any episode of continuous inactivity of > or =40 s is predicted to be sleep. The method gives excellent agreement in C57BL/6J male mice with simultaneous assessment of sleep by EEG/EMG recording. The average agreement over 8,640 10-s epochs in 24 h is 92% (n = 7 mice) with agreement in individual mice being 88-94%. Average EEG/EMG determined sleep per 2-h interval across the day was 59.4 min. The estimated mean difference (bias) per 2-h interval between inactivity-defined sleep and EEG/EMG-defined sleep was only 1.0 min (95% confidence interval for mean bias -0.06 to +2.6 min). The standard deviation of differences (precision) was 7.5 min per 2-h interval with 95% limits of agreement ranging from -13.7 to +15.7 min. Although bias significantly varied by time of day (P = 0.0007), the magnitude of time-of-day differences was not large (average bias during lights on and lights off was +5.0 and -3.0 min per 2-h interval, respectively). This method has applications in chemical mutagenesis and for studies of molecular changes in brain with sleep/wakefulness.

Algorithms↗
PubMed2003

Development of sensitive, high-throughput one-tube RT-PCR-enzyme hybridisation assay to detect selected bacterial fish pathogens.

Bacterial monitoring and surveillance is critical for the early detection of pathogens to avoid the spread of disease. To facilitate this, an efficient, high-performance and high-throughput method to detect the presence of femotgram amounts of ribosomal RNA from 4 bacterial fish pathogens: Aeromonas salmonicida; Tenacibaculum maritimum (formerly Flexibacter maritimus); Lactococcus garvieae; and Yersinia ruckeri was developed. The system uses NucleoLink strips for liquid- and solid-phase PCR in 1 tube, to perform RT-PCR-enzyme hybridisation assays (RT-PCR-EHA) detecting 4 fg or less of rRNA from pure cultures and between 1 and 9 CFU per 200 microl sample volume from selective-enrichment culture media. The liquid-phase amplicons were visualised by gel electrophoresis and the solid-phase amplicons detected using internal probes and visualised using colorimetric detection and p-nitrophenylphosphate.

Aeromonas↗
PubMed1986

Improved screening for beta-lactam antibiotics. A sensitive, high-throughput assay using DD-carboxypeptidase and a novel chromophore-labeled substrate.

The very sensitive and specific method for the detection of beta-lactam antibiotics using DD-carboxypeptidase (DDCase) from Actinomadura strain R39 has been improved to meet the requirements of a high-throughput beta-lactam screening from culture broths of microorganisms. The method is based on a novel chromophor-labeled substrate N alpha-acetyl-N epsilon-4-(7-nitrobenzofurazanyl)-L-lysyl-D-al anyl-D-alanine (ANLA2) which is converted by DDCase into ANLA1 with only one D-alanine residue left. Both compounds are intensely yellow as well as highly fluorescent and can be separated by thin-layer chromatography. This allows easy determination of residual DDCase activity after reaction with beta-lactams by simple visual inspection of chromatograms. Also, many assays can be run at a time without sophisticated instrumentation. Details of the method as well as some results of a beta-lactam screening performed with this type of assay are described.

4-Chloro-7-nitrobenzofurazan↗
PubMed2005

Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling.

A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.

Animals↗
PubMed2005

High-throughput screening of inhibitory potential of nine cytochrome P450 enzymes in vitro using liquid chromatography/tandem mass spectrometry.

The early detection of potential drug-drug interactions is an important issue of drug discovery that has led to the development of high-throughput screening (HTS) methods for potential drug interactions. We developed a HTS method for potential interactions of inhibitory drugs for nine human P450 enzymes using cocktail incubation and tandem mass spectrometry in vitro. This new method involves incubation of two cocktail doses and single cassette analysis. The two cocktail doses in vitro were developed to minimize solvent effects and mutual drug interactions among substrates: cocktail A was composed of phenacetin for CYP1A2, coumarin for CYP2A6, paclitaxel for CYP2C8, S-mephenytoin for CYP2C19, dextromethorphan for CYP2D6, and midazolam for CYP3A4; and cocktail B was composed of three substrates including bupropion for CYP2B6, tolbutamide for CYP2C9, and chlorzoxazone for CYP2E1. In the incubation study of these cocktails, the reaction mixtures were pooled and simultaneously analyzed using liquid chromatography/tandem mass spectrometry employing a fast gradient. The method was validated by comparing the inhibition data obtained from the incubation of each individual probe substrate alone with data from the new method. The IC50 value of each inhibitor in the cocktail agreed well with that of the individual probe drug as well as with values previously reported in the literature. As a HTS method for potential interactions of the inhibition of these nine P450 enzymes, this new method will be useful in the drug discovery process and for the mechanistic understanding of drug interactions.

Chromatography, Liquid↗
PubMed2003

A fluorescence-based high-throughput screening assay for inhibitors of human immunodeficiency virus-1 reverse transcriptase-associated ribonuclease H activity.

A fluorescence resonance energy transfer assay readily applicable to 96-well and 384-well microplate formats with robotic operation was developed to enable high-throughput screening for inhibitors of human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT)-associated RNase H activity, an underexplored target for antiretroviral development. The assay substrate is an 18-nucleotide 3'-fluorescein-labeled RNA annealed to a complementary 18-nucleotide 5'-Dabcyl-modified DNA. The intact duplex has an extremely low background fluorescent signal and provides up to 50-fold fluorescent signal enhancement following hydrolysis. The size and sequence of the duplex are such that HIV-1 RT-RNase H cuts the RNA strand close to the 3' end. The fluorescein-labeled ribonucleotide fragment readily dissociates from the complementary DNA at room temperature with immediate generation of a fluorescent signal. This assay is rapid, inexpensive, and robust, providing Z' factors of 0.8 and coefficients of variation of about 5%. The assay can be carried out both in real-time (continuous) and in "quench" modes; the latter requires only two addition steps with no washing and is thus suitable for robotic operation. Several chemical libraries totaling more than 106,000 compounds were screened with this assay in approximately 1 month.

Base Sequence↗
PubMed2004

High-throughput methods for measuring heparanase activity and screening potential antimetastatic and anti-inflammatory agents.

Heparanase plays an important role in the degradation of the extracellular matrix. It is implicated in inflammation, tumor angiogenesis and metastasis. We have developed two high-throughput methods for measuring heparanase activity and screening potential inhibitors. The first method involves coating fibroblast growth factor (FGF) on microtiter plates and capturing fluorescein isothiocyanate (FITC)-labeled heparin sulfate (HS), which is used as a substrate for heparanase digestion. Labeled HS fragments are released into the medium and quantitated by fluorescence intensity measurement. We have implemented this assay method into a Zeiss uHTS system and screened compound libraries for heparanase inhibitors. The second method involves labeling HS with biotin followed by FITC to generate a dual-labeled HS. The labeled material is bound to streptavidin-coated plates and used as a substrate for heparanase digestion. Both methods are sensitive and easily applicable to robotic systems. In addition, we have labeled both HS and biotin-HS with Eu-chelate, a fluorophore that exhibits long decay fluorescence. Assays using Eu-labeled HS and Eu-labeled biotin-HS have been developed and show higher sensitivity than those using FITC-labeled material. Furthermore, assays using Eu-chelate HS (or biotin-HS) should eliminate the interference of fluorescence compounds.

Animals↗
PubMed2007

High-throughput mapping of the chromatin structure of human promoters.

Our understanding of how chromatin structure influences cellular processes such as transcription and replication has been limited by a lack of nucleosome-positioning data in human cells. We describe a high-resolution microarray approach combined with an analysis algorithm to examine nucleosome positioning in 3,692 promoters within seven human cell lines. Unlike unexpressed genes without transcription-preinitiation complexes at their promoters, expressed genes or genes containing preinitiation complexes exhibit characteristic nucleosome-free regions at their transcription start sites. The combination of these nucleosome data with chromatin immunoprecipitation-chip analyses reveals that the melanocyte master regulator microphthalmia-associated transcription factor (MITF) predominantly binds nucleosome-free regions, supporting the model that nucleosomes limit sequence accessibility. This study presents a global view of human nucleosome positioning and provides a high-throughput tool for analyzing chromatin structure in development and disease.

Algorithms↗
PubMed2001

A DNA fingerprinting procedure for ultra high-throughput genetic analysis of insects.

Existing procedures for the generation of polymorphic DNA markers are not optimal for insect studies in which the organisms are often tiny and background molecular information is often non-existent. We have used a new high throughput DNA marker generation protocol called randomly amplified DNA fingerprints (RAF) to analyse the genetic variability in three separate strains of the stored grain pest, Rhyzopertha dominica. This protocol is quick, robust and reliable even though it requires minimal sample preparation, minute amounts of DNA and no prior molecular analysis of the organism. Arbitrarily selected oligonucleotide primers routinely produced approximately 50 scoreable polymorphic DNA markers, between individuals of three independent field isolates of R. dominica. Multivariate cluster analysis using forty-nine arbitrarily selected polymorphisms generated from a single primer reliably separated individuals into three clades corresponding to their geographical origin. The resulting clades were quite distinct, with an average genetic difference of 37.5 +/- 6.0% between clades and of 21.0 +/- 7.1% between individuals within clades. As a prelude to future gene mapping efforts, we have also assessed the performance of RAF under conditions commonly used in gene mapping. In this analysis, fingerprints from pooled DNA samples accurately and reproducibly reflected RAF profiles obtained from individual DNA samples that had been combined to create the bulked samples.

Animals↗
PubMed2004

High-throughput generation of P. falciparum functional molecules by recombinational cloning.

Large-scale functional genomics studies for malaria vaccine and drug development will depend on the generation of molecular tools to study protein expression. We examined the feasibility of a high-throughput cloning approach using the Gateway system to create a large set of expression clones encoding Plasmodium falciparum single-exon genes. Master clones and their ORFs were transferred en masse to multiple expression vectors. Target genes (n = 303) were selected using specific sets of criteria, including stage expression and secondary structure. Upon screening four colonies per capture reaction, we achieved 84% cloning efficiency. The genes were subcloned in parallel into three expression vectors: a DNA vaccine vector and two protein expression vectors. These transfers yielded a 100% success rate without any observed recombination based on single colony screening. The functional expression of 95 genes was evaluated in mice with DNA vaccine constructs to generate antibody against various stages of the parasite. From these, 19 induced antibody titers against the erythrocytic stages and three against sporozoite stages. We have overcome the potential limitation of producing large P. falciparum clone sets in multiple expression vectors. This approach represents a powerful technique for the production of molecular reagents for genome-wide functional analysis of the P. falciparum genome and will provide for a resource for the malaria resource community distributed through public repositories.

Animals↗
PubMed2004

Identification of retinoid-modulated proteins in squamous carcinoma cells using high-throughput immunoblotting.

Retinoids have shown clinical efficacy in cancer chemoprevention and therapy presumably by modulating the growth, differentiation, and apoptosis of normal, premalignant, and malignant cells. To better understand the mechanisms by which retinoids exert their effects, we used a high-throughput Western blotting method (Becton-Dickinson PowerBlot) to evaluate changes in the levels of cellular signaling proteins in head and neck squamous cell carcinoma cells treated with the cytostatic all-trans-retinoic acid or with the proapoptotic retinoids 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid or N-(4-hydroxyphenyl)retinamide. Treatments of the head and neck squamous cell carcinoma cells with these retinoids for 24 h resulted in increased levels of 14, 22, and 22 proteins and decreased levels of 5, 10, and 7 proteins, respectively. The changes in the levels of the following proteins were confirmed by conventional western immunoblotting: all-trans-retinoic acid increased ELF3, topoisomerase II alpha, RB2/p130, RIG-G, and EMAPII and decreased MEF2D and cathepsin L. N-(4-Hydroxyphenyl)retinamide up-regulated ELF3, c-Jun, Rb2/p130, JAK1, p67phox, Grb2, O(6)-methylguanine-DNA methyltransferase, and Ercc-1. 6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid increased Rb2/p130, c-Jun, Sp1, Sin, and tomosyn and decreased cathepsin L, Mre11, and topoisomerase II alpha. Some of these proteins were also modulated by these retinoids in other human cancer cell lines. A subset of the proteins were modulated similarly by the different retinoids, whereas changes in other proteins were unique for each retinoid. These results suggest that the mechanisms by which these retinoids modulate proteins are distinct but may overlap. Some of the retinoid-modulated proteins identified in this study may be novel candidates for mediating different responses to retinoids.

Antineoplastic Agents↗