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The Sysmex(®) CS-2100i is a fully automated multiparameter hemostasis analyzer equipped with a photo-optical clot detection unit, a cap-piercing system and a pre-analytical check screens for interfering substances such as bilirubin, lipids and haemolysis (HIL system). It is designed to perform coagulation tests as well as chromogenic and immunologic assays. The aim of the present study was to evaluate its performance. The tests performed were routine coagulation (prothrombin time, activated partial thromboplastin time, fibrinogen, factor VIII and factor V), chromogenic (antithrombin) and immunologic assays (D-Dimer). The intra-assay and inter-assay coefficients of variation (CV) were below 5% for most parameters both in the normal and in the pathological range (exceptions: intra-assay CV = 5.65% for the fibrinogen in the low range of concentrations; and inter-assay CV = 6% for clotting factor). The measured lower limits of linearity for factor VIII and factor V were satisfactory. No sample or reagent carryover was detected in the conditions of the study. Our results demonstrated that using the CS-2100i analyzer, routine coagulation testing can be performed with satisfactory precision.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged as the causal agent of an endemic atypical pneumonia, infecting thousands of people worldwide. Although a number of promising potential vaccines and therapeutic agents for SARS-CoV have been described, no effective antiviral drug against SARS-CoV is currently available. The intricate, sequential nature of the viral entry process provides multiple valid targets for drug development. Here, we describe a rapid and safe cell-based high-throughput screening system, dual envelope pseudovirion (DEP) assay, for specifically screening inhibitors of viral entry. The assay system employs a novel dual envelope strategy, using lentiviral pseudovirions as targets whose entry is driven by the SARS-CoV Spike glycoprotein. A second, unrelated viral envelope is used as an internal control to reduce the number of false positives. As an example of the power of this assay a class of inhibitors is reported with the potential to inhibit SARS-CoV at two steps of the replication cycle, viral entry and particle assembly. This assay system can be easily adapted to screen entry inhibitors against other viruses with the careful selection of matching partner virus envelopes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Sulfamethoxydiazine (SMD), which is often used for animal disease treatment, is harmful to human health. No SMD residue should be detected in food in some countries, such as USA and Japan. Therefore, it is significant to develop a high-throughput, high-sensitivity and accurate method for the determination of the content of SMD in food. In this paper, chemiluminescence enzyme immunoassay (CLEIA) was developed for quantification of SMD. For this method, the limit of detection was 3.2 pg/ml, the linear range was from 10 to 2000 pg/ml, the within-day and inter-day precision were below 13% and below 18%, respectively, and the recovery was from 85% to 105%. Milk and egg were selected as samples to be examined with this method, and the result indicated that this CLEIA method was suitable for screening and quality control of food., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The standard (STD) 5 × 5 hybrid median filter (HMF) was previously described as a nonparametric local backestimator of spatially arrayed microtiter plate (MTP) data. As such, the HMF is a useful tool for mitigating global and sporadic systematic error in MTP data arrays. Presented here is the first known HMF correction of a primary screen suffering from systematic error best described as gradient vectors. Application of the STD 5 × 5 HMF to the primary screen raw data reduced background signal deviation, thereby improving the assay dynamic range and hit confirmation rate. While this HMF can correct gradient vectors, it does not properly correct periodic patterns that may present in other screening campaigns. To address this issue, 1 × 7 median and a row/column 5 × 5 hybrid median filter kernels (1 × 7 MF and RC 5 × 5 HMF) were designed ad hoc, to better fit periodic error patterns. The correction data show periodic error in simulated MTP data arrays is reduced by these alternative filter designs and that multiple corrective filters can be combined in serial operations for progressive reduction of complex error patterns in a MTP data array.

Development and progression of colon cancer may be related to cytokines. Cytokines with diagnostic value have been identified individually but have not been implemented into clinical praxis. Using a multiplex protein array, the authors explore a panel of cytokines simultaneously and compared its performance to carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9). Serum concentrations of 12 cytokines were simultaneously determined by multiplex biochip technology in 50 colon cancer patients and 50 healthy controls. Serum levels of interleukin-8 (IL-8) and CEA were significantly higher in cancer patients than in healthy controls. Areas under the receiver operating characteristic curves (AUCs) were largest for IL-8, followed by CEA, vascular endothelial growth factor (VEGF), and CA 19-9. Analyses regarding marker combinations showed an advantage over single marker performance for CEA, VEGF, and CA 19-9 but not for IL-8. Multiplex biochip array technology represents a practical tool in cytokine and cancer research when simultaneous determination of different biomarkers is of interest. The results suggest that the assessment of IL-8, CEA, VEGF, and possibly CA 19-9 serum levels could be useful for colon cancer screening with the potential of also detecting early stage tumors. Further validation studies using these and additional markers on a multiplex array format are encouraged.

Currently, there are no reliable RBC invasion assays to guide the discovery of vaccines against Plasmodium vivax, the most prevalent malaria parasite in Asia and South America. Here we describe a protocol for an ex vivo P vivax invasion assay that can be easily deployed in laboratories located in endemic countries. The assay is based on mixing enriched cord blood reticulocytes with matured, trypsin-treated P vivax schizonts concentrated from clinical isolates. The reliability of this assay was demonstrated using a large panel of P vivax isolates freshly collected from patients in Thailand.

Background: Visceral leishmaniasis is the most severe form of leishmaniasis. Approximately 20% of zoonotic human visceral leishmaniasis worldwide is caused by Leishmania infantum, which is also known as Leishmania chagasi in Latin America, and disease incidence is increasing in urban and peri-urban areas of the tropics. In this form of disease, dogs are the main reservoirs. Diagnostic methods used to identify Leishmania infected animals are not able to detect all of the infected ones, which can compromise the effectiveness of disease control. Therefore, to contribute to the improvement of diagnostic methods for canine visceral leishmaniasis (CVL), we aimed to identify and test novel antigens using high-throughput analysis., Methodology/principal Findings: Immunodominant proteins from L. infantum were mapped in silico to predict B cell epitopes, and the 360 predicted peptides were synthesized on cellulose membranes. Immunoassays were used to select the most reactive peptides, which were then investigated with canine sera. Next, the 10 most reactive peptides were synthesized using solid phase peptide synthesis protocol and tested using ELISA. The sensitivity and specificity of these peptides were also compared to the EIE-LVC Bio-Manguinhos kit, which is recommended by the Brazilian Ministry of Health for use in leishmaniasis control programs. The sensitivity and specificity of the selected synthesized peptides was as high as 88.70% and 95.00%, respectively, whereas the EIE-LVC kit had a sensitivity of 13.08% and 100.00% of specificity. Although the tests based on synthetic peptides were able to diagnose up to 94.80% of asymptomatic dogs with leishmaniasis, the EIE-LVC kit failed to detect the disease in any of the infected asymptomatic dogs., Conclusions/significance: Our study shows that ELISA using synthetic peptides is a technique with great potential for diagnosing CVL; furthermore, the use of these peptides in other diagnostic methodologies, such as immunochromatographic tests, could be beneficial to CVL control programs.

Aqueous solubility is an important physicochemical parameter for any potential drug candidate, and high-throughput kinetic assays are frequently used in drug discovery to give an estimate of a compound's aqueous solubility. However, the aqueous solubility data from an equilibrium (thermodynamic) shake-flask technique is considered more relevant, but is slower and more labor intensive to generate. A highly automated aqueous equilibrium solubility shake-flask technique is described and validated on a set of 15 marketed drugs, whose aqueous solubilities cover four orders of magnitude. The assay uses a Tecan Freedom Evo 200 liquid handling robot (Tecan Group Ltd., Männerdorf, Switzerland) with integrated appliances for the transportation, decapping and recapping, and centrifugation of sample tubes. These bespoke automation solutions help overcome the labor intensive steps associated with performing conventional, gold standard, aqueous equilibrium solubility shake-flask measurements, enabling the assay to be used as a primary-wave drug discovery screen., (Copyright © 2011 Society for Laboratory Automation and Screening. Published by Elsevier Inc. All rights reserved.)

Hit selection is the ultimate goal in many high-throughput screens. Various analytic methods are available for this purpose. Some commonly used ones are z score, z* score, strictly standardized mean difference (SSMD), SSMD*, and t statistic. It is critical to know how to use them correctly because the misusage of them can readily produce misleading results. Here, the author presents basic concepts, elaborates their commonality and difference, describes some common misusage that people should avoid, and uses simulated simple examples to illustrate how to use them correctly.

A set of 25 monoprotic bases is proposed as internal standards for pK(a) determination by capillary electrophoresis. The pK(a) of the bases is determined and compared with available literature data. The capillary electrophoresis internal standard method offers numerous advantages over other typical methods for pK(a) determination, especially of analysis time and buffer preparation. However, it requires disposing of appropriate standards with reference pK(a) value. The set of bases established in this work together with the set of acids previously established provide a reference set of compounds with well-determined acidity constants that facilitate the process of selecting appropriate internal standards for fast pK(a) determination by capillary electrophoresis in high throughput screening of pharmaceutical drugs. In addition, the performance of the method when acidic internal standards are used for the determination of acidity constants of basic internal standards has also been tested. Although higher errors may be expected in this case, good agreement is observed between determined and literature values. These results indicate that in most cases structural similarity between the analyte and the internal standard might not be an essential requirement in the internal standard method., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Methionine aminopeptidase (MAP) (E.C. 3.4.11.18) is a metallopeptidase that cleaves the N-terminal methionine (Met) residue from some proteins. MAP is essential for growth of several bacterial pathogens, making it a target for antibacterial drug discovery. MAP enzymes are also present in eukaryotic cells, and one is a target for antiangiogenic cancer therapy. To screen large compound libraries for MAP inhibitors as the starting point for drug discovery, a high-throughput-compatible assay is valuable. Here the authors describe a novel assay, which detects the Met product of MAP-catalyzed peptide cleavage by coupling it to adenosine triphosphate (ATP)-dependent production of S-adenosyl-L-methionine (SAM) and inorganic phosphate (P(i)) by SAM synthetase (MetK) combined with inorganic pyrophosphatase. The three P(i) ions produced for each Met consumed are detected using Malachite Green/molybdate reagent. This assay can use any unmodified peptide MAP substrate with an N-terminal Met. The assay was used to measure kinetic constants for Escherichia coli MAP using Mn(2+) as the activator and the peptide Met-Gly-Met-Met as the substrate, as well as to measure the potency of a MAP inhibitor. A Mn(2+) buffer is described that can be used to prevent free Mn(2+) depletion by chelating compounds from interfering in screens for MAP inhibitors.

This report presents the high-resolution image acquisition and processing instrument for compound management applications (HIAPI-CM). The HIAPI-CM combines imaging spectroscopy and machine-vision analysis to perform rapid assessment of high-throughput screening (HTS) compound library quality. It has been customized to detect and classify typical artifacts found in HTS compound library microtiter plates (MTPs). These artifacts include (1) insufficient volume of liquid compound sample, (2) compound precipitation, and (3) colored compounds that interfere with HTS assay detection format readout. The HIAPI-CM is also configured to automatically query and compare its analysis results to data stored in a LIMS or corporate database, aiding in the detection of compound registration errors. To demonstrate its capabilities, several compound plates (n=5760 wells total) containing different artifacts were measured via automated HIAPI-CM analysis, and results compared with those obtained by manual (visual) inspection. In all cases, the instrument demonstrated high fidelity (99.8% empty wells; 100.1% filled wells; 94.4% for partially filled wells; 94.0% for wells containing colored compounds), and in the case of precipitate detection, the HIAPI-CM results significantly exceeded the fidelity of visual observations (220.0%). As described, the HIAPI-CM allows for noninvasive, nondestructive MTP assessment with a diagnostic throughput of about 1min per plate, reducing analytical expenses and improving the quality and stewardship of HTS compound libraries., (Copyright © 2011 Society for Laboratory Automation and Screening. Published by Elsevier Inc. All rights reserved.)

Cellular manganese (Mn) uptake and transport dynamics can be measured using a cellular fura-2 manganese extraction assay (CFMEA). The assay described here uses immortalized murine striatal cell line and primary cortical astrocytes, but the method is equally adaptable to other cultured mammalian cells. An ultrasensitive fluorescent nucleic acid stain for quantification of double-stranded DNA (dsDNA) in solution, Quant-iT PicoGreen, has been utilized for normalization of Mn concentration in the cultured cells, following Mn (II) chloride (MnCl(2)) exposure. Depending on the cell type and density, other methods, e.g., protein determination assays or cell counts, may also be used for normalization. Methods are described for rapidly stopping Mn uptake and transport processes at specified times, extraction, and quantification of cellular Mn content, and normalization of Mn levels to dsDNA concentration., (© 2011 by John Wiley & Sons, Inc.)

The nuclear receptor retinoid-related orphan receptor gamma (RORγ) has become an attractive target for drug discovery due to its important role in the development and differentiation of Th17 cells, a subset of T cells that produce interleukin-17 and are involved in the pathogenesis of human inflammatory and autoimmune diseases. To facilitate the drug discovery efforts in this area, we have developed a cellular assay for screening for RORγ inverse agonists. We stably engineered a tetracycline-inducible Gal4 DNA-binding domain/RORγ ligand-binding domain fusion protein into an upstream activation sequence driven-beta-lactamase reporter gene cell line. Due to its constitutive activity, the induced Gal4-RORγ expression leads to increased reporter activity, which can be knocked down using RORγ ligand-binding domain-specific RNA interference oligos. Using this assay, we tested several recently reported ligands for RORγ and observed varying levels of partial inverse agonist activity at μM concentrations. Additionally, we screened a small library of biologically active compounds with this assay and demonstrated its robustness and usefulness in high-throughput screening and follow-up studies for this emerging drug target.

Proteases are industrially important enzymes but often have to be improved for their catalytic efficiency and stabilities to suit applications. Flow cytometry screening technology based on in vitro compartmentalization in double emulsion had been developed and applied on directed evolution of paraoxonase and β-galactosidase. Further advancements of flow cytometry-based screening technologies will enable an ultra-high throughput of variants offering novel opportunities in directed enzyme evolution under high mutational loads. For the industrially important enzyme class of proteases, a first flow cytometry-based screening system for directed protease evolution has been developed based on an extracellular protease-deficient Bacillus subtilis strain (WB800N), a model protease (subtilisin Carlsberg), and a water-in-oil-in-water double-emulsion technology. B. subtilis WB800N cells are encapsulated in double emulsion with a fluorogenic substrate (rhodamine 110-containing peptide), allowing the screening of protease variants in femtoliter compartments at high throughput. The protease screening technology was validated by employing an epPCR mutant library with a high mutational load and screened for increased resistance toward the inhibitor antipain dihydrochloride. A variant (K127R, T237P, M239I, I269V, Y310F, I372V) with an improved relative resistance was isolated from a small population of active variants, validating the reported protease flow cytometry screening technology for increased inhibitor resistance.

Growth rate is an important variable and parameter in biology with a central role in evolutionary, functional genomics, and systems biology studies. In this review the pros and cons of the different technologies presently available for high-throughput measurements of growth rate are discussed. Growth rate can be measured in liquid microcultivation of individual strains, in competition between strains, as growing colonies on agar, as division of individual cells, and estimated from molecular reporters. Irrespective of methodology, statistical issues such as spatial biases and batch effects are crucial to investigate and correct for to ensure low false discovery rates. The rather low correlations between studies indicate that cross-laboratory comparison and standardization are pressing issue to assure high-quality and comparable growth-rate data., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The primary aim of this study was to measure the inherent experimental variability in plant freezing assays involving microtitre plates. Laurus nobilis leaf strips were used as experimental material. Data analysis involved variability measurements among and within microtitre plates. Statistically significant variability (p < 0.05) was observed in both cases. The second aim was to test the effectiveness of five experimental designs, in controlling the experimental error. According to our results the variability in microtitre plate freezing assays can be controlled by the use of blocked experimental designs and single well plots.

Protein-protein interactions are key to many aspects of the cell, including its cytoskeletal structure, the signaling processes in which it is involved, or its metabolism. Failure to form protein complexes or signaling cascades may sometimes translate into pathologic conditions such as cancer or neurodegenerative diseases. The set of all protein interactions between the proteins encoded by an organism constitutes its protein interaction network, representing a scaffold for biological function. Knowing the protein interaction network of an organism, combined with other sources of biological information, can unravel fundamental biological circuits and may help better understand the molecular basics of human diseases. The protein interaction network of an organism can be mapped by combining data obtained from both low-throughput screens, i.e., "one gene at a time" experiments and high-throughput screens, i.e., screens designed to interrogate large sets of proteins at once. In either case, quality controls are required to deal with the inherent imperfect nature of experimental assays. In this chapter, we discuss experimental and statistical methodologies to quantify error rates in high-throughput protein-protein interactions screens.

A fast method for the determination of acidity constants by CZE has been recently developed. This method is based on the use of an internal standard of pK(a) similar to that of the analyte. In this paper we establish the reference pK(a) values of a set of 24 monoprotic neutral acids of varied structure that we propose as internal standards. These compounds cover the most usual working pH range in CZE and facilitate the selection of adequate internal standards for a given determination. The reference pK(a) values of the acids have been established by the own internal standard method, i.e. from the mobility differences between different acids of similar pK(a) in the same pH buffers. The determined pK(a) values have been contrasted to the literature pK(a) values and confirmed by determination of the pK(a) values of some acids of the set by the classical CE method. Some systematic deviations of mobilities have been observed in NaOH buffer in reference to the other used buffers, overcoming the use of NaOH in the classical CE method. However, the deviations affect in a similar degree to the test compounds and internal standards allowing thus, the use of NaOH buffer in the internal standard method. This fact demonstrates the better performance of the internal standard method over the classical method to correct mobility deviations, which together with its fastness makes it an interesting method for the routine determination of accurate pK(a) values of new pharmaceutical drugs and drug precursors., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrP(C) to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD(50)). Brain tissue from 263K scrapie-affected hamsters gave SD(50) values of 10(11)-10(12)/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD(50) values of 10(3.5)-10(5.7)/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD(50) values of 10(2.0)-10(2.9)/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity.

Background: The aim of the present study was to evaluate standard reference material (SRM) 1955 commutability as a reference material for serum folate using automated methods. We also designed so as to reduce the intermethod variability present in different automated methods., Methods: Using a microbiological assay related to the 'information value' of SRM 1955 as a comparison method, we investigated the possibility of standardization for the assay values of serum folate as measured by the automated methods (Access, Centaur and Elecsys). In the assay of 50 patient sera by these automated methods, we corrected observed values by the SRM 1955 and compared with comparison values., Results: The observed values of SRM 1955 Levels I, II and III were within or outside (but near) a 95% prediction interval obtained from patient sera by the automated methods. The normalized residuals obtained from SRM 1955 were within ±3.0 (in SD units), which enabled us to conclude that the SRM 1955 had a physicochemical characterization similar to native serum. Twelve patients were assessed as hypofolataemia (<6.0 ng/mL) and 38 patients as normal (≥6.0 ng/mL). Before correction, folate levels in six of 12 patients were lower than 6.0 ng/mL, and those in seven of 38 patients were higher than 6.0 ng/mL with the automated methods. After correction, low levels were found in four of 12 patients, and normal levels were found in 33 of 38 patients., Conclusions: The use of SRM 1955 would help to reduce the intermethod variability present in different automated methods for serum folate measurement.

Current methods for high-throughput screening (HTS) use a serial process to evaluate compounds as inhibitors toward a single therapeutic target, but as the demand to reduce screening time and cost continues to grow, one solution is the development of multiplex technology. In this communication, the multiplex assay capability of a mass spectrometry (MS)-based readout system is verified using a kinase and esterase reaction simultaneously. Furthermore, the MS-based readout is shown to be compatible with a typical HTS workflow by identifying and validating several new inhibitors for each enzyme from a small library of compounds. These data confirm that it is possible to monitor inhibition of multiple therapeutic targets with one pass through the compound repository, thus demonstrating the potential for MS-based methods to become a method of choice for HTS of isolated enzymes.

Identification of active compounds in high-throughput screening (HTS) contexts can be substantially improved by applying classical experimental design and statistical inference principles to all phases of HTS studies. The authors present both experimental and simulated data to illustrate how true-positive rates can be maximized without increasing false-positive rates by the following analytical process. First, the use of robust data preprocessing methods reduces unwanted variation by removing row, column, and plate biases. Second, replicate measurements allow estimation of the magnitude of the remaining random error and the use of formal statistical models to benchmark putative hits relative to what is expected by chance. Receiver Operating Characteristic (ROC) analyses revealed superior power for data preprocessed by a trimmed-mean polish method combined with the RVM t-test, particularly for small- to moderate-sized biological hits.

Nek2 is a serine/threonine protein kinase that localizes to the centrosome and is implicated in mitotic regulation. Overexpression of Nek2 induces premature centrosome separation and nuclear defects indicative of mitotic errors, whereas depletion of Nek2 interferes with cell growth. As Nek2 expression is upregulated in a range of cancer cell lines and primary human tumors, inhibitors of Nek2 may have therapeutic value in cancer treatment. The authors used a radiometric proximity assay in a high-throughput screen to identify small-molecule inhibitors of Nek2 kinase activity. The assay was based on the measurement of the radiolabeled phosphorylated product of the kinase reaction brought into contact with the surface of wells of solid scintillant-coated microplates. Seventy nonaggregating hits were identified from approximately 73,000 compounds screened and included a number of toxoflavins and a series of viridin/wortmannin-like compounds. The viridin-like compounds were >70-fold selective for Nek2 over Nek6 and Nek7 and inhibited the growth of human tumor cell lines at concentrations consistent with their biochemical potencies. An automated mechanism-based microscopy assay in which centrosomes were visualized using pericentrin antibodies confirmed that 2 of the viridin inhibitors reduced centrosome separation in a human tumor cell line. The data presented show that pharmacological inhibition of Nek2 kinase results in the expected phenotype of disruption to centrosome function associated with growth inhibition and further supports Nek2 as a target for cancer drug discovery.

Identification and characterization of kinase inhibitor potency and selectivity is often an iterative process in which a library of compounds is first screened against a single kinase, and hits from that screen are then profiled against other kinases to determine specificity. By developing kinase assays that employ either a terbium- or a europium-based time-resolved fluorescence resonance energy transfer (TR-FRET) readout, one can take advantage of the distinct emission properties of these labels to develop assays for 2 kinases that can be performed simultaneously in the same well. This not only increases the information content provided per assay well but can immediately provide information on compound specificity. The authors have applied this strategy to the development of multiplexed assays for 2 examples systems: EGFR and IKKbeta, as well as lipid kinase family members mTOR and PIK3C3. They demonstrate the ability of these multiplexed assays to characterize selective kinase inhibitors in a dose-response mode, with no difference in results obtained from traditional single kinase assays performed separately.

The tyrosine kinase Wee1 is part of a key cellular sensing mechanism that signals completion of DNA replication, ensuring proper timing of entry into mitosis. Wee1 acts as an inhibitor of mitotic entry by phosphorylating cyclin-dependent kinase CDK1. Wee1 activity is mainly regulated at the protein level through its phosphorylation and subsequent degradation by the ubiquitin proteasome pathway. To facilitate identification of small molecules preventing Wee1 degradation, a homogeneous cell-based assay was developed using HeLa cells transiently transfected with a Wee1-luciferase fusion protein. To ensure ultra-high-throughput screening (uHTS) compatibility, the assay was scaled to a 1536-well plate format and cells were transfected in bulk and cryopreserved. This miniaturized homogeneous assay demonstrated robust performance, with a calculated Z' factor of 0.65 +/- 0.05. The assay was screened against a publicly available library of approximately 218,000 compounds to identify Wee1 stabilizers. Nonselective, cytotoxic, and promiscuous compounds were rapidly triaged through the use of a similarly formatted counterscreen that measured stabilization of an N-cyclin B-luciferase fusion protein, as well as execution of viability assessment in the parental HeLa cell line. This screening campaign led to the discovery of 4 unrelated cell-permeable small molecules that showed selective Wee1-luciferase stabilization with micromolar potency. One of these compounds, SID4243143 (ML 118), was shown to inhibit cell cycle progression, underscoring the importance of Wee1 degradation to the cell cycle. Results suggest that this uHTS approach is suitable for identifying selective chemical probes that prevent Wee1 degradation and generally applicable to discovering inhibitors of the ubiquitin proteasome pathway.

Drug discovery programmes require accurate in vitro systems for drug screening and testing. Traditional cell culture makes use of 2D (two-dimensional) surfaces for ex vivo cell growth. In such environments, cells are forced to adopt unnatural characteristics, including aberrant flattened morphologies. Therefore there is a strong demand for new cell culture platforms which allow cells to grow and respond to their environment in a more realistic manner. The development of 3D (three-dimensional) alternative substrates for in vitro cell growth has received much attention, and it is widely acknowledged that 3D cell growth is likely to more accurately reflect the in vivo tissue environments from which cultured cells are derived. 3D cell growth techniques promise numerous advantages over 2D culture, including enhanced proliferation and differentiation of stem cells. The present review focuses on the development of scaffold technologies for 3D cell culture.

TRPM7 is a ubiquitously expressed and constitutively active divalent cation channel essential for cell survival and proliferation because it provides a mechanism for Mg2+ entry. This makes the channel an attractive target for proliferative diseases. In keeping with its role in Mg2+ homeostasis, TRPM7 is inhibited by intracellular Mg2+ and Mg-ATP. TRPM7 has been implicated in anoxia-mediated cell death following brain ischemia. Despite its critical role in ischemic cell death and cell proliferation, there are no reports of selective inhibitors of TRPM7. The authors developed and optimized a fluorescent dye-based bioassay measuring the fluorescence quench of fura-2 by TRPM7-mediated Mn2+ influx in HEK293 cells that stably overexpress TRPM7. The following bioassay conditions were evaluated: (a) cell density, (b) dye loading conditions, (c) bioassay temperature, (d) concentration of the fura-2 quenching agent Mn2+, and (e) concentration of vehicle solvent. The bioassay was validated by measuring the effects of the known (nonselective) inhibitor 2-APB and La3+ on Mn2+ influx, and furthermore, the performance of the assay was evaluated by screening a subset of a marine bacteria-derived extract library. The quality of the bioassay window is excellent based on an established statistical parameter used to evaluate high-throughput screening window quality (Z and Z' factors > or =0.5).

An automated system using on-line solid-phase extraction and HPLC with UV detection was developed for the determination of faropenem in human plasma and urine. Analytical process was performed isocratically with two reversed-phase columns connected by a switching valve. After simple pretreatment for plasma and urine with acetonitrile, a volume of 100microl upper layer of the plasma or urine samples was injected for on-line SPE column switching HPLC-UV analysis. The analytes were retained on the self-made trap column (Lichrospher C(18), 4.6mmx37mm, 25microm) with the loading solvent (20mM NaH(2)PO(4) adjusted pH 3.5) at flow rate of 2mlmin(-1), and most matrix materials were removed from the column to waste. After 0.5min washing, the valve was switched to another position so that the target analytes could be eluted from trap column to analytical column in the back-flush mode by the mobile phase (acetonitrile-20mM NaH(2)PO(4) adjusted pH 3.5, 16:84, v/v) at flow rate of 1.5mlmin(-1), and then separated on the analytical column (Ultimate XB-C(18), 4.6mmx50mm, 5microm).The complete cycle of the on-line SPE preconcentration purification and HPLC separation of the analytes was 5min. Calibration curves with good linearities (r=0.9994 for plasma sample and r=0.9988 for urine sample) were obtained in the range 0.02-5microgml(-1) in plasma and 0.05-10microg ml(-1) in urine for faropenem. The optimized method showed good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. The method was successfully utilized to quantify faropenem in human plasma and urine to support the clinical pharmacokinetic studies., (Copyright 2009 Elsevier B.V. All rights reserved.)

High-throughput screening (HTS) is a well-established process for lead discovery in Pharma and Biotech companies and is now also being used for basic and applied research in academia. It comprises the screening of large chemical libraries for activity against biological targets via the use of automation, miniaturized assays and large-scale data analysis. Since its first advent in the early to mid 1990s, the field of HTS has seen not only a continuous change in technology and processes, but also an adaptation to various needs in lead discovery. HTS has now evolved into a mature discipline that is a crucial source of chemical starting points for drug discovery. Whereas in previous years much emphasis has been put on a steady increase in screening capacity ('quantitative increase') via automation and miniaturization, the past years have seen a much greater emphasis on content and quality ('qualitative increase'). Today, many experts in the field see HTS at a crossroad with the need to decide on either higher throughput/more experimentation or a greater focus on assays of greater physiological relevance, both of which may lead to higher productivity in pharmaceutical R&D. In this paper, we describe the development of HTS over the past decade and point out our own ideas for future directions of HTS in biomedical research. We predict that the trend toward further miniaturization will slow down with the balanced implementation of 384 well, 1536 well, and 384 low volume well plates. Furthermore, we envisage that there will be much more emphasis on rigorous assay and chemical characterization, particularly considering that novel and more difficult target classes will be pursued. In recent years we have witnessed a clear trend in the drug discovery community toward rigorous hit validation by the use of orthogonal readout technologies, label free and biophysical methodologies. We also see a trend toward a more flexible use of the various screening approaches in lead discovery, that is, the use of both full deck compound screening as well as the use of focused screening and iterative screening approaches. Moreover, we expect greater usage of target identification strategies downstream of phenotypic screening and the more effective implementation of affinity selection technologies as a result of advances in chemical diversity methodologies. We predict that, ultimately, each hit finding strategy will be much more project-related, tailor-made, and better integrated into the broader drug discovery efforts.

In high-throughput biochemical assays performed in multiwell plates, the effect of test samples on the activity of the biochemical system is usually measured by optical means such as absorbance, fluorescence, luminescence, or scintillation counting. The test sample often causes detection interference when it remains in the well during the measurement. Interference may be due to light absorption, fluorescence quenching, sample fluorescence, chemical interaction of the sample with a detection reagent, or depression of the meniscus. A simple method is described that corrects for such interference well by well. The interference is measured in a separate artifact assay plate. An appropriate arithmetic correction is then applied to the measurement in the corresponding well of the activity assay plate. The correction procedure can be used for single-point screening or potency measurements on serial dilutions of test samples.