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Shareholders and regulators have increasingly been calling upon firms to reign in executive compensation. Most of this discussion has focused on bonuses and stock options, the more observable portions of an executive compensation package. More difficult for investors to assess, however, is long-term incentive pay, such as supplemental executive retirement plans (SERPs), which has become a significant portion of executive compensation. Our study examines whether managers use accelerated share repurchases (ASRs) to increase total compensation through this under-emphasized form of pay. Managers wishing to increase their total compensation through an increase in earnings per share (EPS) should prefer ASRs, which generally have a more immediate and significant impact on EPS relative to open market repurchases (OMRs). For a sample of repurchase firms, we find evidence that managers who have performance-contingent SERPs in place (i.e., a SERP based on salary and bonus or on average earnings), together with EPS-based bonuses and a share repurchase that results in a higher EPS number, are significantly more likely to choose an ASR versus an OMR. JEL Classifications: G34; G35; M4; M41; M52. Data Availability: The data are available from public sources identified in this study.

SYNOPSIS “Little r” restatements occur when a firm's immaterial errors accumulate to a material error in a given year. Unlike “Big R” restatements that must be reported through an SEC 8-K material event filing, little r restatements do not require an 8-K form or a withdrawal of the auditor opinion. This paper documents this previously unexamined form of restatement and analyzes the characteristics of the firms that have used this method of correcting accounting errors over the period 2009 through 2012. Contrary to concerns voiced by regulators and research agencies we find, in multivariate tests, that little r firms are generally more profitable, less complex, and show some evidence of stronger corporate governance and higher audit quality than Big R firms. Compared to non-revising or restating firms, little r firms have lower free cash flows, higher board expertise, higher CFO tenure, are less likely to use a specialist auditor, and less likely to have material weaknesses in their internal controls. We also find that the majority of little r firms do not include any discussion of why these little r's occurred. We discuss policy implications related to disclosure of little r revisions. JEL Classifications: M41; M48; G38. Data Availability: All data used in this study are publicly available from the sources indicated.

Financial analysts are important players in the marketplace. Analysts’ reports, which include forecasts of earnings and stock recommendations, move market prices. Investors, both large and small, rely on the information in reports when forming their investment decisions. Given the relevance of financial analysts’ research, understanding whether their reports are biased is important. Despite an increase in market regulation, evidence suggests that analysts’ reports are biased. Research also finds that analysts’ bias increases when information uncertainty is high. Thus, investors should understand the possible dangers in blindly relying on research by financial analysts.

ATP binding cassette (ABC) transmembrane efflux pumps such as P-glycoprotein (ABCB1), multidrug resistance protein 1 (ABCC1), and breast cancer resistance protein (ABCG2) play an important role in anti-cancer drug resistance. A large number of structurally and functionally diverse compounds act as substrates or modulators of these pumps. In vitro assessment of the affinity of drug candidates for multidrug resistance proteins is central to predict in vivo pharmacokinetics and drug–drug interactions. The objective of this study was to identify and characterize new substrates for these transporters. As part of a collaborative project with Life Technologies, 102 fluorescent probes were investigated in a flow cytometric screen of ABC transporters. The primary screen compared substrate efflux activity in parental cell lines with their corresponding highly expressing resistant counterparts. The fluorescent compound library included a range of excitation/emission profiles and required dual laser excitation as well as multiple fluorescence detection channels. A total of 31 substrates with active efflux in one or more pumps and practical fluorescence response ranges were identified and tested for interaction with eight known inhibitors. This screening approach provides an efficient tool for identification and characterization of new fluorescent substrates for ABCB1, ABCC1, and ABCG2.

This paper provides a comprehensive exploration of the types of accounting fraud committed by firms over the period 1995–2009. Using detailed data from US SEC Accounting and Auditing Enforcement Releases (AAER), we examine the likelihood and timing of analyst coverage decisions and recommendation revisions related to fraud firms versus firms without accounting fraud. We find that analysts have a higher probability of taking the more severe action of dropping coverage rather than only revising down recommendations for firms with any type of accounting fraud and also for specific egregious types of accounting fraud. Through the use of competing hazards models, we also find that accounting frauds and their egregiousness are positively (negatively) associated with the timeliness of the analysts’ action to drop coverage (revise only). Overall, we find that analysts’ actions may be useful in determining the occurrence of accounting fraud prior to the public announcement of the fraud.

The human Bcl-2 family includes six antiapoptotic members (Bcl-2, Bcl-B, Bcl-W, Bcl-X(L), Bfl-1, and Mcl-1) and many proapoptotic members, wherein a balance between the two determines cell life or death in many physiological and disease contexts. Elevated expression of various antiapoptotic Bcl-2 members is commonly observed in cancers, and chemical inhibitors of these proteins have been shown to promote apoptosis of malignant cells in culture, in animal models, and in human clinical trials. All six antiapoptotic members bind a helix from the proapoptotic family member Bim, thus quenching Bim's apoptotic signal. Here, we describe the use of a multiplex, high-throughput flow cytometry assay for the discovery of small molecule modulators that disrupt the interaction between the antiapoptotic members of the Bcl-2 family and Bim. The six antiapoptotic Bcl-2 family members were expressed as glutathione-S-transferase fusion proteins and bound individually to six glutathione bead sets, with each set having a different intensity of red fluorescence. A fluorescein-conjugated Bcl-2 homology region 3 (BH3) peptide from Bim was employed as a universal ligand. Flow cytometry measured the amount of green peptide bound to each bead set in a given well, with inhibitory compounds resulting in a decrease of green fluorescence on one or more bead set(s). Hits and cheminformatically selected analogs were retested in a dose-response series, resulting in three "active" compounds for Bcl-B. These three compounds were validated by fluorescence polarization and isothermal titration calorimetry. We discuss some of the lessons learned about screening a chemical library provided by the National Institutes of Health Small Molecule Repository (∼195,000 compounds) using high-throughput flow cytometry.

After controlling for economic performance (i.e., stock returns), we find that several proxies for analyst incentives as well as accounting-based fundamentals are related to an analyst's decision to drop coverage of a firm. When we separately consider the drop decisions of analysts with High vs. Low Experience, we find that Low Experience analysts place more weight on firm risk and decreases in liquidity when making their drop decision, while High Experience analysts place higher weights on accounting losses and decreasing SG&A margins. Lastly, when High (Low) Experience analysts initiate dropped coverage, their Low (High) Experience peers mimic the dropped coverage fairly quickly (fairly slowly).

The Bcl-2 family contains six anti-apoptotic members, each with a hydrophobic pocket where BH3 helixes (Bcl-2 homology region 3) bind. This binding quenches apoptotic signals from activated BH3 family members. Many tumor cells either have increased expression of one of these six proteins, or become overexpressed under treatment. Herein we provide a protocol for the simultaneous, in vitro screening of a small molecule library against all six anti-apoptotic Bcl-2 family members using a fluoresceinated BH3 peptide. Six fusion proteins made up of glutathione-S-transferase and each of the Bcl-2 members are bound individually to six glutathione bead sets, each set being easily distinguished by their different intensity of red fluorescence. The coated bead sets are washed, combined, and incubated with green fluorescent Bim BH3 peptide and a small molecule in 10 microliter wells for 1 hour. Flow cytometry measures the peptide bound to each bead set without wash steps. The green fluorescence signal for each bed set is resolved, and selective inhibitors are expected to reduce the signal for individual bead sets, with pan-inhibitors affecting all bead sets. Each 384 well plate is analyzed in 12 minutes, typically measuring 200 of 2,000 beads (~10%) of each type per well.

Fluorescence intensity of the pH-sensitive carboxyfluorescein derivative 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was monitored by high-throughput flow cytometry in living yeast cells. We measured fluorescence intensity of BCECF trapped in yeast vacuoles, acidic compartments equivalent to lysosomes where vacuolar proton-translocating ATPases (V-ATPases) are abundant. Because V-ATPases maintain a low pH in the vacuolar lumen, V-ATPase inhibition by concanamycin A alkalinized the vacuole and increased BCECF fluorescence. Likewise, V-ATPase-deficient mutant cells had greater fluorescence intensity than wild-type cells. Thus, we detected an increase of fluorescence intensity after short- and long-term inhibition of V-ATPase function. We used yeast cells loaded with BCECF to screen a small chemical library of structurally diverse compounds to identify V-ATPase inhibitors. One compound, disulfiram, enhanced BCECF fluorescence intensity (although to a degree beyond that anticipated for pH changes alone in the mutant cells). Once confirmed by dose-response assays (EC(50)=26 microM), we verified V-ATPase inhibition by disulfiram in secondary assays that measured ATP hydrolysis in vacuolar membranes. The inhibitory action of disulfiram against V-ATPase pumps revealed a novel effect previously unknown for this compound. Because V-ATPases are highly conserved, new inhibitors identified could be used as research and therapeutic tools in cancer, viral infections, and other diseases where V-ATPases are involved.

Prior literature has found that as uncertainty in a firms information environment increases, optimism increases in equity analysts’ earnings forecasts. The studies suggest an economic incentive explanation, commonly called the management‐relations hypothesis. However, there is conflicting evidence that managers would prefer pessimistic forecasts and encourage analysts to “walk‐down” their forecasts to prevent negative earnings surprises. To test these contradictory findings, this study uses an experimental setting to remove economic incentives from the analyst’s decision process and isolate the cause of observed bias in analysts’ reports. The results of the experiment show that an increase in the perceived uncertainty of the forecasting task results in significantly lower relative optimism in analysts’ earnings forecasts. This finding is consistent with a negativity hypothesis and the managementrelations hypothesis extolled in the empirical research. The findings also show that relative forecast optimism bias is positively related to the level of analysts’ buy/sell recommendations consistent with more recent findings that suggest that analysts use motivated reasoning (the tendency to process information in a manner that supports one’s goal) in their judgments of forecasted earnings and recommendations. Together, these results suggest that analysts consider and use financial information differently depending on their decision goal.

Formylpeptide receptors (FPRs) are implicated in a variety of immunological and inflammatory response cascades. Further understanding of FPR-family ligand interactions could play an integral role in biological and therapeutic discovery. Fluorescent reporter ligands for the family are desirable experimental tools for increased understanding of ligand/receptor interactions. The ligand binding affinity and fluorescent reporting activity of the peptide WK(FL)YMVm was explored though use of the high throughput HyperCyt flow cytometric platform. Relative binding affinities of several known FPR and FPRL1 peptide ligands were compared in a duplex assay format. The fluorescent W-peptide ligand, WK(FL)YMVm, proved to be a high-affinity, cross-reactive reporter ligand for the FPR/FPRL1 duplex assay. Ligand specificity was demonstrated for each receptor, with known, selective peptide ligands. The binding site specificity of the reporter ligand was further verified by a fluorescent confocal microscopy internalization experiment. The fluorescent peptide ligand WK(FL)YMVm binds with high affinity to both FPR and FPRL1. The differential affinities of known peptide ligands were observed with the use of this fluorescent probe in high throughput screening flow cytometry.

Of recent, clinical interest have been two related human G-protein coupled receptors: formylpeptide receptor (FPR), linked to antibacterial inflammation and malignant glioma cell metastasis; and FPR like-1 (FPRL1), linked to chronic inflammation in systemic amyloidosis, Alzheimer's disease, and prion diseases. In association with the National Institutes of Health (NIH) Molecular Library Screening Network, we implemented a flow-cytometry-based high-throughput screening (HTS) approach for identifying selective small molecule FPR and FPRL1 ligands. The screening assay measured the ability of test compounds to competitively displace a high-affinity, fluorescein- labeled peptide ligand from FPR, FPRL1, or both. U937 cells expressing FPR and rat basophil leukemia (RBL) cells expressing FPRL1 were tested together in a “duplex” format. The U937 cells were color coded with red-fluorescent dye allowing their distinction during analysis. Compounds, cells, and fluorescent ligand were sequentially combined (no wash) in 15 μl assay volumes in 384-well plates. Throughput averaged ∼11 min per plate to analyze ∼4,000 cells (∼2,000/receptor) in a 2 μl aspirate from each well. In primary single concentration HTS of 24,304 NIH Small Molecule Repository compounds, 253 resulted in inhibition >30% (181 for FPR, 72 for FPRL1) of which 40 had selective binding inhibition constants (Ki) ≤ 4 μM (34 for FPR and 6 for FPRL1). An additional 1,446 candidate compounds were selected by structure–activity-relationship analysis of the hits and screened to identify novel ligands for FPR (3570-0208, Ki = 95 ± 10 nM) and FPRL1 (BB-V-115, Ki = 270 ± 51 nM). Each was a selective antagonist in calcium response assays and the most potent small molecule antagonist reported for its respective receptor to date. The duplex assay format reduced assay time, minimized reagent requirements, and provided selectivity information at every screening stage, thus proving to be an efficient means to screen for selective receptor ligand probes. © 2008 International Society for Advancement of Cytometry

The overexpression of P-glycoprotein, encoded by the ATP Binding Cassette B1 (ABCB1) gene, contributes to multidrug resistance (MDR) and is considered one of the major obstacles to successful cancer chemotherapy. The authors previously developed a T-lineage acute lymphoblastic leukemia (T-ALL) cell line that overexpresses ABCB1 and exhibits MDR to daunorubicin (DNR), prednisolone, and vincristine. Using this cell line and the fluorescent probe JC-1, they developed a flow cytometry-based, high-throughput screening (HTS) assay that quantifies ABCB1 efflux. They screened a library of 880 off-patent drugs for their ability to inhibit ABCB1 efflux and then measured the ability of 11 lead compounds to reverse in vitro DNR-mediated drug resistance and the toxic doses for each agent. Seven of the 11 drugs were able to reverse drug resistance at a concentration significantly below its toxic dose. Of the remaining 7, only 1 compound, mometasone furoate, has not been previously described as an ABCB1 antagonist to DNR-mediated drug resistance. On the basis of its high ABC modulator activity and relatively large in vitro therapeutic window, this drug warrants further investigation. In addition, the approach used in this study is useful for identifying off-patent drugs that may be repurposed for novel clinical indications.

Estrogen is a hormone critical in the development, normal physiology and pathophysiology of numerous human tissues. The effects of estrogen have traditionally been solely ascribed to estrogen receptor alpha (ERalpha) and more recently ERbeta, members of the soluble, nuclear ligand-activated family of transcription factors. We have recently shown that the seven-transmembrane G protein-coupled receptor GPR30 binds estrogen with high affinity and resides in the endoplasmic reticulum, where it activates multiple intracellular signaling pathways. To differentiate between the functions of ERalpha or ERbeta and GPR30, we used a combination of virtual and biomolecular screening to isolate compounds that selectively bind to GPR30. Here we describe the identification of the first GPR30-specific agonist, G-1 (1), capable of activating GPR30 in a complex environment of classical and new estrogen receptors. The development of compounds specific to estrogen receptor family members provides the opportunity to increase our understanding of these receptors and their contribution to estrogen biology.

Research has shown that high-involvement work practices are positively related to corporate financial performance. However, it is unknown if investors are able to use information on high-involvement practices to predict the performance of specific companies. In this study, we examine earnings forecasts for a sample of Fortune 1000 firms and find professional stock analysts consistently underestimated the earnings of firms that made greater use of high-involvement practices during the 1990s. Based on data collected from newspaper articles and annual reports, we argue that these lower estimates resulted from a lack of information on innovative HR practices. Recommendations to managers for disseminating information on and leveraging highinvolvement HR practices are discussed. © 2006 Wiley Periodicals, Inc.

The accuracy of sell-side analysts' forecast revisions is related to a number of factors, including characteristics of the analyst and the age of the forecast. In this study we examine whether there are differences in how sophisticated and unsophisticated investors use these factors to predict the relative accuracy of forecast revisions. We adapt the lens model methodological approach from the judgment and decision-making literature to investigate these differences in an archival setting. Our results suggest that sophisticated investors have greater knowledge overall about the relation of the factors to forecast accuracy. Further, our evidence is consistent with sophisticated investors relying more on the specific factors that provide the most benefits (relative to their costs) for predicting relative forecast accuracy.

Although three major classes of systemic antifungal agents are clinically available, each is characterized by important limitations. Thus, there has been considerable ongoing effort to develop novel and repurposed agents for the therapy of invasive fungal infections. In an effort to address these needs, we developed a novel high-throughput, multiplexed screening method that utilizes small molecules to probe candidate drug targets in the opportunistic fungal pathogen Candida albicans. This method is amenable to high-throughput automated screening and is based upon detection of changes in GFP levels of individually tagged target proteins. We first selected four GFP-tagged membrane-bound proteins associated with virulence or antifungal drug resistance in C. albicans. We demonstrated proof-of-principle that modulation of fluorescence intensity can be used to assay the expression of specific GFP-tagged target proteins to inhibitors (and inducers), and this change is measurable within the HyperCyt automated flow cytometry sampling system. Next, we generated a multiplex of differentially color-coded C. albicans strains bearing C-terminal GFP-tags of each gene encoding candidate drug targets incubated in the presence of small molecules from the Prestwick Chemical Library in 384-well microtiter plate format. Following incubation, cells were sampled through the HyperCyt system and modulation of protein levels, as indicated by changes in GFP-levels of each strain, was used to identify compounds of interest. The hit rate for both inducers and inhibitors identified in the primary screen did not exceed 1% of the total number of compounds in the small-molecule library that was probed, as would be expected from a robust target-specific, high-throughput screening campaign. Secondary assays for virulence characteristics based on null mutant strains were then used to further validate specificity. In all, this study presents a method for the identification and verification of new antifungal drugs targeted to fungal virulence proteins using C. albicans as a model fungal pathogen.

The formylpeptide receptor (FPR1) and formylpeptide-like 1 receptor (FPR2) are G protein-coupled receptors that are linked to acute inflammatory responses, malignant glioma stem cell metastasis, and chronic inflammation. Although several N-formyl peptides are known to bind to these receptors, more selective small-molecule, high-affinity ligands are needed for a better understanding of the physiologic roles played by these receptors. High-throughput assays using mixture-based combinatorial libraries represent a unique, highly efficient approach for rapid data acquisition and ligand identification. We report the superiority of this approach in the context of the simultaneous screening of a diverse set of mixture-based small-molecule libraries. We used a single cross-reactive peptide ligand for a duplex flow cytometric screen of FPR1 and FPR2 in color-coded cell lines. Screening 37 different mixture-based combinatorial libraries totaling more than five million small molecules (contained in 5,261 mixture samples) resulted in seven libraries that significantly inhibited activity at the receptors. Using positional scanning deconvolution, selective high-affinity (low nM K(i)) individual compounds were identified from two separate libraries, namely, pyrrolidine bis-diketopiperazine and polyphenyl urea. The most active individual compounds were characterized for their functional activities as agonists or antagonists with the most potent FPR1 agonist and FPR2 antagonist identified to date with an EC₅₀ of 131 nM (4 nM K(i)) and an IC₅₀ of 81 nM (1 nM K(i)), respectively, in intracellular Ca²⁺ response determinations. Comparative analyses of other previous screening approaches clearly illustrate the efficiency of identifying receptor selective, individual compounds from mixture-based combinatorial libraries.

High-throughput flow cytometry exploits a novel many-samples/one-file approach to dramatically speed data acquisition, limit aspirated sample volume to as little as 2 μl/well and produce multisample data sets that facilitate automated analysis of samples in groups as well as individually. It has been successfully applied to both cell- and microsphere-based bioassays in 96- and 384-well formats, to screen tens-of-thousands of compounds and identify novel bioactive structures. High-content multiparametric analysis capabilities have been exploited for assay multiplexing, allowing the assessment of biologic selectivity and specificity to be an integral component of primary screens. These and other advances in the last decade have contributed to the application of flow cytometry as a uniquely powerful tool for probing biologic and chemical diversity and complex systems biology.

This paper provides a comprehensive exploration of the types of accounting fraud committed by firms over the period 1995-2009. Using detailed data from US SEC Accounting and Auditing Enforcement Releases (AAER), we examine the likelihood and timing of analyst coverage decisions and recommendation revisions related to fraud firms versus firms without accounting fraud. We find that analysts have a higher probability of taking the more severe action of dropping coverage rather than only revising down recommendations for firms with any type of accounting fraud and also for specific egregious types of accounting fraud. Through the use of competing hazards models, we also find that accounting frauds and their egregiousness are positively (negatively) associated with the timeliness of the analysts’ action to drop coverage (revise only). Overall, we find that analysts’ actions may be useful in determining the occurrence of accounting fraud prior to the public announcement of the fraud.

Increasingly, shareholders and regulators have been calling for a reigning in of executive salaries. Most of this discussion has focused on bonuses and stock options, the more observable portions of an executive compensation package. However long term incentive pay, such as supplemental executive retirement plans (SERPs), has become a significant portion of executive compensation and is more difficult to monitor. This paper examines whether managers use real earnings management in the form of Accelerated Share repurchases (ASR) to increase total compensation through this more obscure or ‘stealth’, area of pay. This is because ASRs tend to have a more immediate and significant impact on EPS versus open market repurchases (OMRs). While SERPs provide an opportunity for CEOs to hide compensation, stronger managerial power may enhance this opportunity. We find evidence that managers who have SERPs in place are significantly more likely to choose ASRs versus OMRs. We also find that as executives’ horizon shorten, they are also more likely to use this stronger form of earnings management (ASR). Additionally, we find that, on average, ASR firms have higher managerial power than OMR firms. Finally, we are able to provide direct evidence of the economic significance of SERP programs. Additional analyses provide further support for the link between SERPs and real earnings management in the form of share repurchase choice.

Reaction of the molecular cleft-containing triphosphazane C6H4N2[P(S)(NEt2)2]2PCl (2) with LiBH4 yields the borane-coordinated triphosphazane hydride C6H4N2[P(S)(NEt2)2]2P(BH3)H (3). The complex is...

Academia and small business research units are poised to play an increasing role in drug discovery, with drug repurposing as one of the major areas of activity. Here we summarize project status for a number of drugs or classes of drugs: raltegravir, cyclobenzaprine, benzbromarone, mometasone furoate, astemizole, R-naproxen, ketorolac, tolfenamic acid, phenothiazines, methylergonovine maleate and beta-adrenergic receptor drugs, respectively. Based on this multi-year, multi-project experience we discuss strengths and weaknesses of academic-based drug repurposing research. Translational, target and disease foci are strategic advantages fostered by close proximity and frequent interactions between basic and clinical scientists, which often result in discovering new modes of action for approved drugs. On the other hand, lack of integration with pharmaceutical sciences and toxicology, lack of appropriate intellectual coverage and issues related to dosing and safety may lead to significant drawbacks. The development of a more streamlined regulatory process world-wide, and the development of pre-competitive knowledge transfer systems such as a global healthcare database focused on regulatory and scientific information for drugs world-wide, are among the ideas proposed to improve the process of academic drug discovery and repurposing, and to overcome the “valley of death” by bridging basic to clinical sciences.

TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high-throughput flow cytometry multiplexed screen using five GFP-tagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast. Each GFP-tagged clone was differentially color-coded, and the GFP signal of each clone was measured simultaneously by flow cytometry, which allows rapid prioritization of compounds that likely act through direct modulation of TORC1 or proximal signaling components. A total of 255 compounds were confirmed in dose–response analysis to alter GFP expression in one or more clones. To validate the concept of the high- throughput screen, we have characterized CID 3528206, a small molecule most likely to act on TORC1 as it alters GFP expression in all five GFP clones in a manner analogous to that of rapamycin. We have shown that CID 3528206 inhibited yeast cell growth and that CID 3528206 inhibited TORC1 activity both in vitro and in vivo with EC50's of 150 nM and 3.9 μM, respectively. The results of microarray analysis and yeast GFP collection screen further support the notion that CID 3528206 and rapamycin modulate similar cellular pathways. Together, these results indicate that the HTS has identified a potentially useful small molecule for further development of TOR inhibitors.

Regioselective reactivity, molecular cleft selectivity, and conformational properties have been examined in the skeletally stabilized triphosphazanes C 6 H 4 N 2 [P(NEt 2 ) 2 ] 2 PNEt 2 (4), C 6 H 4 N 2 -[P(S)(NEt 2 ) 2 ] 2 PCl (5), and C 6 H 4 N 2 [P(S)(NEt 2 ) 2 ] 2 P(S)NEt 2 (9) and in a series of new derivatives. 3 is oxidized/coordinated regioselectively by O 3 , Se, PhN 3 , and BH 3 to C 6 H 4 N 2 [P(O)(NEt 2 ) 2 ] 2 PNEt 2 and C 6 H 4 N 4 [P(O)(NEt 2 ) 2 ] 2 P(O)NEtz

Skeletal P-N-P unit conformational preferences of the unsymmetrically substituted bis(phosphino)amines erythro-i-PrNi-PrN[PhP(i-PrNH)] [PhP(EtNH)] (4A), meso- and d,l-i-PrN[PhP(i-PrNH)] 2 (5A and 5B), erythro-i-PrN-[PhP(i-PrNH)] [PhP(t-BuNH)] (6), erythro-i-PrN [PhP(i-PrNH)] [PhP(PhNH)] (7), and erythro-i-PrN [PhP(PhNH)] 2 (8) have been examined

Rho family GTPases and their effector proteins regulate a wide range of cell signaling pathways. In normal physiological conditions, their activity is tightly controlled and it is not surprising that their aberrant activation contributes to tumorigenesis or other diseases. For this reason, the identification of small, cell permeable molecules capable of inhibition of Rho GTPases can be extraordinarily useful, particularly if they are specific and act reversibly.Herein, we describe a flow cytometric assay, which allows us to measure the activity of six small GTPases simultaneously. GST-tagged small GTPases are bound to six glutathione bead sets each set having a different intensity of red fluorescence at a fixed wavelength. The coated bead sets were washed, combined, and dispensed into 384-well plates with test compounds, and fluorescent-GTP binding was used as the read-out.This multiplex bead-based assay was successfully used for to identify both general and selective inhibitors of Rho family GTPases.

This article describes the creation of a discovery team in an academic environment. The team is specifically structured to perform four integrated activities: characterizing membrane protein, formatting the materials for analysis of cell physiology and molecular interactions, performing screens of small-molecule activity on a novel high-throughput flow cytometric instrument platform, and integrating virtual screening with activity screening. These activities improve the overall efficiency of the discovery process. Keywords: discovery team; virtual screening; cell physiology; molecular interactions

The HyperCyt high-throughput (HT) flow cytometry sampling platform uses a peristaltic pump, in combination with an autosampler, and a novel approach to data collection, to circumvent time-delay bottlenecks of conventional flow cytometry. This approach also dramatically reduces the amount of sample aspirated for each analysis, typically requiring ~2 microL per sample while making quantitative fluorescence measurements of 40 or more samples per minute with thousands to tens of thousands of cells in each sample. Here, we describe a simple robust screening assay that exploits the high-content measurement capabilities of the flow cytometer to simicroltaneously probe the binding of test compounds to two different receptors in a common assay volume, a duplex assay format. The ability of the flow cytometer to distinguish cell-bound from free fluorophore is also exploited to eliminate wash steps during assay setup. HT flow cytometry with this assay has allowed efficient screening of tens of thousands of small molecules from the NIH Small-Molecule Repository to identify selective ligands for two related G-protein-coupled receptors, the formylpeptide receptor and formylpeptide receptor-like 1.

The orthocyclophane cyclotetraphosphazanes [C 6 H 4 N 2 (PR) 2 ] 2 (R=Ph (4), Me) have been obtained from RPCl 2 /1,2-(NH 2 ) 2 C 6 H 4 /Et 3 N reactions. S 8 oxidation yields [C 6 H 4 N 2 (PhP)(PhPS)] 2 and [C 6 H 4 N 2 ] 2 (PhPS) 3 (PhP) (7); are characterized by spectral (MS, IR, 1 H and 31 P NMR) data. 4 and 6 are further characterized by X-ray single-crystal analyses. Monoclinic, C2/c

This unit describes a system for the automated high-throughput analysis of cell cytotoxicity in 96-well and 384-well microplates. Discrete cell cultures are analyzed at rates of 40/min (∼2.5 min/96 wells, ∼10 min/384 wells) and cytotoxicity is quantified on the basis of a combination of propidium iodide (PI) fluorescence analysis and cell counting performed by the flow cytometer. Only 2 µl is aspirated from a culture for analysis so that assays can be performed in small volumes to minimize reagent cost and usage. Keywords: high-throughput screening; cytotoxicity; propidium iodide; cell viability assay