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1.  Inducible Defenses Stay Up Late: Temporal Patterns of Immune Gene Expression in Tenebrio molitor 
G3: Genes|Genomes|Genetics  2014;4(6):947-955.
The course of microbial infection in insects is shaped by a two-stage process of immune defense. Constitutive defenses, such as engulfment and melanization, act immediately and are followed by inducible defenses, archetypically the production of antimicrobial peptides, which eliminate or suppress the remaining microbes. By applying RNAseq across a 7-day time course, we sought to characterize the long-lasting immune response to bacterial challenge in the mealworm beetle Tenebrio molitor, a model for the biochemistry of insect immunity and persistent bacterial infection. By annotating a hybrid de novo assembly of RNAseq data, we were able to identify putative orthologs for the majority of components of the conserved insect immune system. Compared with Tribolium castaneum, the most closely related species with a reference genome sequence and a manually curated immune system annotation, the T. molitor immune gene count was lower, with lineage-specific expansions of genes encoding serine proteases and their countervailing inhibitors accounting for the majority of the deficit. Quantitative mapping of RNAseq reads to the reference assembly showed that expression of genes with predicted functions in cellular immunity, wound healing, melanization, and the production of reactive oxygen species was transiently induced immediately after immune challenge. In contrast, expression of genes encoding antimicrobial peptides or components of the Toll signaling pathway and iron sequestration response remained elevated for at least 7 days. Numerous genes involved in metabolism and nutrient storage were repressed, indicating a possible cost of immune induction. Strikingly, the expression of almost all antibacterial peptides followed the same pattern of long-lasting induction, regardless of their spectra of activity, signaling possible interactive roles in vivo.
doi:10.1534/g3.113.008516
PMCID: PMC4065263  PMID: 24318927
RNAseq time course; costs of immunity; insect immunity; persistent infection; innate immunity; complex genetics; tolerance; complex immunity; infection; resistance
2.  Follow Up for Emergency Department Patients After Intravenous Contrast and Risk of Nephropathy 
Introduction: Contrast-induced nephropathy (CIN), defined as an increase in serum creatinine (SCr) greater than 25% or ≥0.5 mg/dL within 3 days of intravenous (IV) contrast administration in the absence of an alternative cause, is the third most common cause of new acute renal failure in hospitalized patients. It is known to increase in-hospital mortality up to 27%. The purpose of this study was to investigate the rate of outpatient follow up and the occurrence of CIN in patients who presented to the emergency department (ED) and were discharged home after computed tomography (CT) of the abdomen and pelvis (AP) with IV contrast.
Methods: We conducted a single center retrospective review of charts for patients who required CT of AP with IV contrast and who were discharged home. Patients' clinical data included the presence of diabetes mellitus, hypertension, chronic kidney disease (CKD) and congestive heart failure (CHF).
Results: Five hundred and thirty six patients underwent CT of AP with IV contrast in 2011 and were discharged home. Diabetes mellitus was documented in 96 patients (18%). Hypertension was present in 141 patients (26.3%), and 82 patients (15.3%) were on angiotensin-converting-enzyme inhibitors (ACEI). Five patients (0.9%) had documented CHF and all of them were taking furosemide. Seventy patients (13%) had a baseline SCr >1.2 mg/dL. One hundred fifty patients (28%) followed up in one of the clinics or the ED within one week after discharge, but only 40 patients (7.5%) had laboratory workup. Out of 40 patients who followed up within 1 week after discharge, 9 patients (22.5%) developed CIN. One hundred ninety patients (35.4%) followed up in one of the clinics or the ED after 7 days and within 1 month after discharge, but only 71 patients (13.2%) had laboratory workup completed. Out of 71 patients who followed up within 1 month, 11 patients (15%) developed CIN. The overall incidence of CIN was 15.3% (17 out of 111 patients).
Conclusion: There was a poor outpatient follow up after CT of AP with IV contrast and biochemically CIN appears to be present in some patients. Unlike previous reports that CKD is the major risk factor for CIN, our results demonstrated that risk factors such as advanced age, DM and hypertension seem to predispose patients to CIN rather than abnormal baseline SCr. [West J Emerg Med. 2014;15(3):276–281.]
doi:10.5811/westjem.2013.8.17915
PMCID: PMC4025523  PMID: 24868304
3.  Effector Kinase Coupling Enables High-Throughput Screens for Direct HIV-1 Nef Antagonists with Anti-retroviral Activity 
Chemistry & biology  2013;20(1):82-91.
HIV-1 Nef, a critical AIDS progression factor, represents an important target protein for antiretroviral drug discovery. Because Nef lacks intrinsic enzymatic activity, we developed an assay that couples Nef to the activation of Hck, a Src-family member and Nef effector protein. Using this assay, we screened a large, diverse chemical library and identified small molecules that block Nef-dependent Hck activity with low micromolar potency. Of these, a diphenylpyrazolo compound demonstrated sub-micromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound binds directly to Nef via a pocket formed by the Nef dimerization interface and disrupts Nef dimerization in cells. Coupling of non-enzymatic viral accessory factors to host cell effector proteins amenable to high-throughput screening may represent a general strategy for the discovery of new antimicrobial agents.
doi:10.1016/j.chembiol.2012.11.005
PMCID: PMC3559019  PMID: 23352142
4.  Development and Validation of a High-Content Screening Assay to Identify Inhibitors of Cytoplasmic Dynein-Mediated Transport of Glucocorticoid Receptor to the Nucleus 
Abstract
Rapid ligand-induced trafficking of glucocorticoid nuclear hormone receptor (GR) from the cytoplasm to the nucleus is an extensively studied model for intracellular retrograde cargo transport employed in constructive morphogenesis and many other cellular functions. Unfortunately, potent and selective small-molecule disruptors of this process are lacking, which has restricted pharmacological investigations. We describe here the development and validation of a 384-well high-content screening (HCS) assay to identify inhibitors of the rapid ligand-induced retrograde translocation of cytoplasmic glucocorticoid nuclear hormone receptor green fluorescent fusion protein (GR-GFP) into the nuclei of 3617.4 mouse mammary adenocarcinoma cells. We selected 3617.4 cells, because they express GR-GFP under the control of a tetracycline (Tet)-repressible promoter and are exceptionally amenable to image acquisition and analysis procedures. Initially, we investigated the time-dependent expression of GR-GFP in 3617.4 cells under Tet-on and Tet-off control to determine the optimal conditions to measure dexamethasone (Dex)-induced GR-GFP nuclear translocation on the ArrayScan-VTI automated imaging platform. We then miniaturized the assay into a 384-well format and validated the performance of the GR-GFP nuclear translocation HCS assay in our 3-day assay signal window and dimethylsulfoxide validation tests. The molecular chaperone heat shock protein 90 (Hsp90) plays an essential role in the regulation of GR steroid binding affinity and ligand-induced retrograde trafficking to the nucleus. We verified that the GR-GFP HCS assay captured the concentration-dependent inhibition of GR-GFP nuclear translocation by 17-AAG, a benzoquinone ansamycin that selectively blocks the binding and hydrolysis of ATP by Hsp90. We screened the 1280 compound library of pharmacologically active compounds set in the Dex-induced GR-GFP nuclear translocation assay and used the multi-parameter HCS data to eliminate cytotoxic compounds and fluorescent outliers. We identified five qualified hits that inhibited the rapid retrograde trafficking of GR-GFP in a concentration-dependent manner: Bay 11-7085, 4-phenyl-3-furoxancarbonitrile, parthenolide, apomorphine, and 6-nitroso-1,2-benzopyrone. The data presented here demonstrate that the GR-GFP HCS assay provides an effective phenotypic screen and support the proposition that screening a larger library of diversity compounds will yield novel small-molecule probes that will enable the further exploration of intracellular retrograde transport of cargo along microtubules, a process which is essential to the morphogenesis and function of all cells.
doi:10.1089/adt.2012.456
PMCID: PMC3464420  PMID: 22830992
5.  Targeting sub-cellular localization through the Polo-Box Domain: non-ATP competitive Inhibitors recapitulate a PLK1 phenotype 
Molecular cancer therapeutics  2012;11(8):1683-1692.
The polo-box domain (PBD) has critical roles in the mitotic functions of PLK1. The REPLACE strategy to develop inhibitors of protein-protein interactions has identified alternatives for the N-terminal tripeptide of a Cdc25C substrate. In addition, a peptide structure activity relationship described key determinants and novel information useful for drug design. Fragment ligated inhibitory peptides (FLIPs) were generated with comparable affinity to peptide PBD inhibitors and possessed anti-proliferative phenotypes in cells consistent with the observed decrease in PLK1 centrosomal localization. These FLIPs demonstrated evidence of enhanced PLK1 inhibition in cells relative to peptides and induced monopolar and multipolar spindles, which stands in contrast to previously reported small molecule PBD inhibitors that display phenotypes only partially representative of PLK1 knockdown. Progress obtained applying REPLACE validates this approach for identifying fragment alternatives for determinants of the Cdc25C binding motif and extends its applicability of the strategy for discovering protein-protein interaction inhibitors. In addition, the described PBD inhibitors retain high specificity for PLK1 over PLK3 and therefore show promise as isotype selective, non-ATP competitive kinase inhibitors that provide new impetus for the development of PLK1 selective anti-tumor therapeutics.
doi:10.1158/1535-7163.MCT-12-0006-T
PMCID: PMC3711794  PMID: 22848093
Phosphorylation and proteolysis in cell cycle control; Protein serine-threonine kinases; Molecular modelling; In silico evaluation of targets and design of libraries; Protein/protein interactions; Cell cycle mechanisms of anticancer drug action; Kinase and phosphatase inhibitors; Novel antitumor agents
6.  Characterization of Inhibitors of Glucocorticoid Receptor Nuclear Translocation: A Model of Cytoplasmic Dynein-Mediated Cargo Transport 
Abstract
Agonist-induced glucocorticoid receptor [GR] transport from the cytoplasm to the nucleus was used as a model to identify dynein-mediated cargo transport inhibitors. Cell-based screening of the library of pharmacologically active compound (LOPAC)-1280 collection identified several small molecules that stalled the agonist-induced transport of GR-green fluorescent protein (GFP) in a concentration-dependent manner. Fluorescent images of microtubule organization, nuclear DNA staining, expression of GR-GFP, and its subcellular distribution were inspected and quantified by image analysis to evaluate the impact of compounds on cell morphology, toxicity, and GR transport. Given the complexity of the multi-protein complex involved in dynein-mediated cargo transport and the variety of potential mechanisms for interruption of that process, we therefore developed and validated a panel of biochemical assays to investigate some of the more likely intracellular target(s) of the GR transport inhibitors. Although the apomorphine enantiomers exhibited the most potency toward the ATPase activities of cytoplasmic dynein, myosin, and the heat-shock proteins (HSPs), their apparent lack of specificity made them unattractive for further study in our quest. Other molecules appeared to be nonspecific inhibitors that targeted reactive cysteines of proteins. Ideally, specific retrograde transport inhibitors would either target dynein itself or one of the other important proteins associated with the transport process. Although the hits from the cell-based screen of the LOPAC-1280 collection did not exhibit this desired profile, this screening platform provided a promising phenotypic system for the discovery of dynein/HSP modulators.
doi:10.1089/adt.2010.0367
PMCID: PMC3277738  PMID: 21919741
7.  Redox cycling compounds generate H2O2 in HTS buffers containing strong reducing reagents – real hits or promiscuous artifacts? 
Redox cycling compounds (RCCs) generate µM concentrations of hydrogen peroxide (H2O2) in the presence of strong reducing agents, common buffer components used to maintain the catalytic activity and/or folding of target proteins for high throughput screening (HTS) assays. H2O2 generated by RCCs can indirectly inhibit the catalytic activity of proteins by oxidizing accessible cysteine, tryptophan, methionine, histidine or selenocysteine residues, and indeed several important classes of protein targets are susceptible to H2O2-mediated inactivation; protein tyrosine phosphatases, cysteine proteases, and metalloenzymes. The main sources of H2O2 in cells are the Nox enzyme/SOD systems, peroxisome metabolism, and the autoxidation of reactive chemicals by enzyme mediated redox cycling at both the microsomal and mitochondrial sites of electron transport. Given the role of H2O2 as a second messenger involved in the regulation of many signaling pathways it is hardly surprising that compounds which can generate intracellular H2O2 by enzyme mediated redox cycling would have pleiotropic effects. RCCs can therefore have serious negative consequences for the probe and/or lead generation process: primary HTS assay hit rates may be inflated by RCC false positives; critical resources will be diverted to develop and implement follow up assays to distinguish RCCs from real hits; and screening databases will become annotated with the promiscuous activity of RCCs. In an attempt to mitigate the serious impact of RCCs on probe and lead generation, two groups have independently developed assays to indentify RCCs.
doi:10.1016/j.cbpa.2010.10.022
PMCID: PMC3040250  PMID: 21075044
8.  Characterization and Optimization of a Novel Protein–Protein Interaction Biosensor High-Content Screening Assay to Identify Disruptors of the Interactions Between p53 and hDM2 
Abstract
We present here the characterization and optimization of a novel imaging-based positional biosensor high-content screening (HCS) assay to identify disruptors of p53-hDM2 protein–protein interactions (PPIs). The chimeric proteins of the biosensor incorporated the N-terminal PPI domains of p53 and hDM2, protein targeting sequences (nuclear localization and nuclear export sequence), and fluorescent reporters, which when expressed in cells could be used to monitor p53-hDM2 PPIs through changes in the subcellular localization of the hDM2 component of the biosensor. Coinfection with the recombinant adenovirus biosensors was used to express the NH-terminal domains of p53 and hDM2, fused to green fluorescent protein and red fluorescent protein, respectively, in U-2 OS cells. We validated the p53-hDM2 PPI biosensor (PPIB) HCS assay with Nutlin-3, a compound that occupies the hydrophobic pocket on the surface of the N-terminus of hDM2 and blocks the binding interactions with the N-terminus of p53. Nutlin-3 disrupted the p53-hDM2 PPIB in a concentration-dependent manner and provided a robust, reproducible, and stable assay signal window that was compatible with HCS. The p53-hDM2 PPIB assay was readily implemented in HCS and we identified four (4) compounds in the 1,280-compound Library of Pharmacologically Active Compounds that activated the p53 signaling pathway and elicited biosensor signals that were clearly distinct from the responses of inactive compounds. Anthracycline (topoisomerase II inhibitors such as mitoxantrone and ellipticine) and camptothecin (topoisomerase I inhibitor) derivatives including topotecan induce DNA double strand breaks, which activate the p53 pathway through the ataxia telangiectasia mutated-checkpoint kinase 2 (ATM-CHK2) DNA damage response pathway. Although mitoxantrone, ellipticine, camptothecin, and topotecan all exhibited concentration-dependent disruption of the p53-hDM2 PPIB, they were much less potent than Nutlin-3. Further, their corresponding cellular images and quantitative HCS data did not completely match the Nutlin-3 phenotypic profile.
doi:10.1089/adt.2010.0281
PMCID: PMC2929144  PMID: 20662736
9.  Design and Synthesis of a Library of Tetracyclic Hydroazulenoisoindoles 
Forty-four tetracyclic hydroazulenoisoindoles were synthesized via a tandem cyclopropanation/Cope rearrangement followed by a Diels-Alder sequence from easily available five-membered cyclic cross-conjugated trienones. These trienones were obtained from two different routes depending upon whether R1 and R2 are alkyl or amino acid derived functional groups, via a rhodium(I)-catalyzed cycloisomerization reaction. In order to increase diversity, four maleimides and two 1,2,4-triazoline-3,5-diones were used as dienophiles in the Diels-Alder step. Several Diels-Alder adducts were further reacted under palladium-catalyzed hydrogenation conditions, leading to a diastereoselective reduction of the trisubstituted double bond. This library has demonstrated rapid access to a variety of structurally complex natural product-like compounds via stereochemical diversity and building block diversity approaches.
doi:10.1021/cc900024p
PMCID: PMC3104412  PMID: 19366169
10.  Profiling the NIH Small Molecule Repository for Compounds That Generate H2O2 by Redox Cycling in Reducing Environments 
We have screened the Library of Pharmacologically Active Compounds (LOPAC) and the National Institutes of Health (NIH) Small Molecule Repository (SMR) libraries in a horseradish peroxidase–phenol red (HRP-PR) H2O2 detection assay to identify redox cycling compounds (RCCs) capable of generating H2O2 in buffers containing dithiothreitol (DTT). Two RCCs were identified in the LOPAC set, the ortho-naphthoquinone β-lapachone and the para-naphthoquinone NSC 95397. Thirty-seven (0.02%) concentration-dependent RCCs were identified from 195,826 compounds in the NIH SMR library; 3 singleton structures, 9 ortho-quinones, 2 para-quinones, 4 pyrimidotriazinediones, 15 arylsulfonamides, 2 nitrothiophene-2-carboxylates, and 2 tolyl hydrazides. Sixty percent of the ortho-quinones and 80% of the pyrimidotriazinediones in the library were confirmed as RCCs. In contrast, only 3.9% of the para-quinones were confirmed as RCCs. Fifteen of the 251 arylsulfonamides in the library were confirmed as RCCs, and since we screened 17,868 compounds with a sulfonamide functional group we conclude that the redox cycling activity of the arylsulfonamide RCCs is due to peripheral reactive enone, aromatic, or heterocyclic functions. Cross-target queries of the University of Pittsburgh Drug Discovery Institute (UPDDI) and PubChem databases revealed that the RCCs exhibited promiscuous bioactivity profiles and have populated both screening databases with significantly higher numbers of active flags than non-RCCs. RCCs were promiscuously active against protein targets known to be susceptible to oxidation, but were also active in cell growth inhibition assays, and against other targets thought to be insensitive to oxidation. Profiling compound libraries or the hits from screening campaigns in the HRP-PR H2O2 detection assay significantly reduce the timelines and resources required to identify and eliminate promiscuous nuisance RCCs from the candidates for lead optimization.
doi:10.1089/adt.2009.0247
PMCID: PMC3098569  PMID: 20070233
11.  Percutaneous management of a saphenous vein graft perforation using a covered stent and final coil embolization technique 
Coronary artery perforation as a result of percutaneous coronary intervention is a rare complication which may result in cardiac tamponade, myocardial infarction and death. Perforation of a saphenous vein graft is unusual and generally requires surgical intervention. We describe a novel percutaneous approach that facilitated the successful management of a potentially catastrophic saphenous vein graft (SVG) perforation.
doi:10.5114/aoms.2010.19317
PMCID: PMC3302718  PMID: 22427780
vein graft perforation
12.  Automated High-Content Live Animal Drug Screening Using C. elegans Expressing the Aggregation Prone Serpin α1-antitrypsin Z 
PLoS ONE  2010;5(11):e15460.
The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.
doi:10.1371/journal.pone.0015460
PMCID: PMC2980495  PMID: 21103396
13.  Cdc25B Dual-Specificity Phosphatase Inhibitors Identified in a High-Throughput Screen of the NIH Compound Library 
Abstract
The University of Pittsburgh Molecular Library Screening Center (Pittsburgh, PA) conducted a screen with the National Institutes of Health compound library for inhibitors of in vitro cell division cycle 25 protein (Cdc25) B activity during the pilot phase of the Molecular Library Screening Center Network. Seventy-nine (0.12%) of the 65,239 compounds screened at 10 μM met the active criterion of ≥50% inhibition of Cdc25B activity, and 25 (31.6%) of these were confirmed as Cdc25B inhibitors with 50% inhibitory concentration (IC50) values <50 μM. Thirteen of the Cdc25B inhibitors were represented by singleton chemical structures, and 12 were divided among four clusters of related structures. Thirteen (52%) of the Cdc25B inhibitor hits were quinone-based structures. The Cdc25B inhibitors were further characterized in a series of in vitro secondary assays to confirm their activity, to determine their phosphatase selectivity against two other dual-specificity phosphatases, mitogen-activated protein kinase phosphatase (MKP)-1 and MKP-3, and to examine if the mechanism of Cdc25B inhibition involved oxidation and inactivation. Nine Cdc25B inhibitors did not appear to affect Cdc25B through a mechanism involving oxidation because they did not generate detectable amounts of H2O2 in the presence of dithiothreitol, and their Cdc25B IC50 values were not significantly affected by exchanging the dithiothreitol for β-mercaptoethanol or reduced glutathione or by adding catalase to the assay. Six of the nonoxidative hits were selective for Cdc25B inhibition versus MKP-1 and MKP-3, but only the two bisfuran-containing hits, PubChem substance identifiers 4258795 and 4260465, significantly inhibited the growth of human MBA-MD-435 breast and PC-3 prostate cancer cell lines. To confirm the structure and biological activity of 4260465, the compound was resynthesized along with two analogs. Neither of the substitutions to the two analogs was tolerated, and only the resynthesized hit 26683752 inhibited Cdc25B activity in vitro (IC50 = 13.83 ± 1.0 μM) and significantly inhibited the growth of the MBA-MD-435 breast and PC-3 prostate cancer cell lines (IC50 = 20.16 ± 2.0 μM and 24.87 ± 2.25 μM, respectively). The two bis-furan-containing hits identified in the screen represent novel nonoxidative Cdc25B inhibitor chemotypes that block tumor cell proliferation. The availability of non-redox active Cdc25B inhibitors should provide valuable tools to explore the inhibition of the Cdc25 phosphatases as potential mono- or combination therapies for cancer.
doi:10.1089/adt.2008.186
PMCID: PMC2956648  PMID: 19530895
14.  Gut Bacteria Are Not Required for the Insecticidal Activity of Bacillus thuringiensis toward the Tobacco Hornworm, Manduca sexta▿ †  
Applied and Environmental Microbiology  2009;75(15):5094-5099.
It was recently proposed that gut bacteria are required for the insecticidal activity of the Bacillus thuringiensis-based insecticide, DiPel, toward the lepidopterans Manduca sexta, Pieris rapae, Vanessa cardui, and Lymantria dispar. Using a similar methodology, it was found that gut bacteria were not required for the toxicity of DiPel or Cry1Ac or for the synergism of an otherwise sublethal concentration of Cry1Ac toward M. sexta. The toxicities of DiPel and of B. thuringiensis HD73 Cry− spore/Cry1Ac synergism were attenuated by continuously exposing larvae to antibiotics before bioassays. Attenuation could be eliminated by exposing larvae to antibiotics only during the first instar without altering larval sterility. Prior antibiotic exposure did not attenuate Cry1Ac toxicity. The presence of enterococci in larval guts slowed mortality resulting from DiPel exposure and halved Cry1Ac toxicity but had little effect on B. thuringiensis HD73 Cry− spore/Cry1Ac synergism. B. thuringiensis Cry− cells killed larvae after intrahemocoelic inoculation of M. sexta, Galleria mellonella, and Spodoptera litura and grew rapidly in plasma from M. sexta, S. litura, and Tenebrio molitor. These findings suggest that gut bacteria are not required for B. thuringiensis insecticidal activity toward M. sexta but that B. thuringiensis lethality is reduced in larvae that are continuously exposed to antibiotics before bioassay.
doi:10.1128/AEM.00966-09
PMCID: PMC2725506  PMID: 19525273
15.  Computational design, synthesis and biological evaluation of para-quinone-based inhibitors for redox regulation of the dual-specificity phosphatase Cdc25B 
Organic & biomolecular chemistry  2008;6(18):3256-3263.
Quinoid inhibitors of Cdc25B were designed based on the Linear Combination of Atomic Potentials (LCAP) methodology. In contrast to a published hypothesis, the biological activities and hydrogen peroxide generation in reducing media of three synthetic models did not correlate with the quinone half-wave potential, E1/2.
doi:10.1039/b806712k
PMCID: PMC2752828  PMID: 18802630
16.  Development of a 384-Well Colorimetric Assay to Quantify Hydrogen Peroxide Generated by the Redox Cycling of Compounds in the Presence of Reducing Agents 
We report here the development and optimization of a simple 384-well colorimetric assay to measure H2O2 generated by the redox cycling of compounds incubated with reducing agents in high-throughput screening (HTS) assay buffers. The phenol red-horseradish peroxidase (HRP) assay readily detected H2O2 either added exogenously or generated by the redox cycling of compounds in dithiothreitol (DTT). The generation of H2O2 was dependent on the concentration of both the compound and DTT and was abolished by catalase. Although both DTT and tris(2-carboxyethyl)-phosphine sustain the redox cycling generation of H2O2 by a model quinolinedione, 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione (NSC 663284; DA3003-1), other reducing agents such as β-mercaptoethanol, glutathione, and cysteine do not. The assay is compatible with HTS. Once terminated, the assay signal was stable for at least 5 h, allowing for a reasonable throughput. The assay tolerated up to 20% dimethyl sulfoxide, allowing a wide range of compound concentrations to be tested. The assay signal window was robust and reproducible with average Z-factors of ≥0.8, and the redox cycling generation of H2O2 by DA3003-1 in DTT exhibited an average 50% effective concentration of 0.830 ± 0.068 μM. Five of the mitogen-activated protein kinase phosphatase (MKP) 1 inhibitors identified in an HTS were shown to generate H2O2 in the presence of DTT, and their inhibition of MKP-1 activity was shown to be time dependent and was abolished or significantly reduced by either 100 U of catalase or by higher DTT levels. A cross-target query of the PubChem database with three structurally related pyrimidotriazinediones revealed active flags in 36–39% of the primary screening assays. Activity was confirmed against a number of targets containing active site cysteines, including protein tyrosine phosphatases, cathepsins, and caspases, as well as a number of cellular cytotoxicity assays. Rather than utilize resources to conduct a hit characterization effort involving several secondary assays, the phenol red-HRP assay provides a simple, rapid, sensitive, and inexpensive method to identify compounds that redox cycle in DTT or tris(2-carboxyethyl)phosphine to produce H2O2 that may indirectly modulate target activity and represent promiscuous false-positives from a primary screen.
doi:10.1089/adt.2008.151
PMCID: PMC2752819  PMID: 18699726
17.  Development of a 384-Well Colorimetric Assay to Quantify Hydrogen Peroxide Generated by the Redox Cycling of Compounds in the Presence of Reducing Agents 
Abstract
We report here the development and optimization of a simple 384-well colorimetric assay to measure H2O2 generated by the redox cycling of compounds incubated with reducing agents in high-throughput screening (HTS) assay buffers. The phenol red-horseradish peroxidase (HRP) assay readily detected H2O2 either added exogenously or generated by the redox cycling of compounds in dithiothreitol (DTT). The generation of H2O2 was dependent on the concentration of both the compound and DTT and was abolished by catalase. Although both DTT and tris(2-carboxyethyl)-phosphine sustain the redox cycling generation of H2O2 by a model quinolinedione, 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione (NSC 663284; DA3003-1), other reducing agents such as β-mercaptoethanol, glutathione, and cysteine do not. The assay is compatible with HTS. Once terminated, the assay signal was stable for at least 5 h, allowing for a reasonable throughput. The assay tolerated up to 20% dimethyl sulfoxide, allowing a wide range of compound concentrations to be tested. The assay signal window was robust and reproducible with average Z-factors of ≥0.8, and the redox cycling generation of H2O2 by DA3003-1 in DTT exhibited an average 50% effective concentration of 0.830 μ 0.068 μM. Five of the mitogen-activated protein kinase phosphatase (MKP) 1 inhibitors identified in an HTS were shown to generate H2O2 in the presence of DTT, and their inhibition of MKP-1 activity was shown to be time dependent and was abolished or significantly reduced by either 100 U of catalase or by higher DTT levels. A cross-target query of the PubChem database with three structurally related pyrimidotriazinediones revealed active flags in 36–39% of the primary screening assays. Activity was confirmed against a number of targets containing active site cysteines, including protein tyrosine phosphatases, cathepsins, and caspases, as well as a number of cellular cytotoxicity assays. Rather than utilize resources to conduct a hit characterization effort involving several secondary assays, the phenol red-HRP assay provides a simple, rapid, sensitive, and inexpensive method to identify compounds that redox cycle in DTT or tris(2-carboxyethyl)phosphine to produce H2O2 that may indirectly modulate target activity and represent promiscuous false-positives from a primary screen.
doi:10.1089/adt.2008.151
PMCID: PMC2752819  PMID: 18699726
18.  Perforated Appendicitis After Colonoscopy 
Background:
Acute appendicitis is a rare complication of colonoscopy that has been reported only 12 times in the English-language literature and is usually associated with obstruction of the appendiceal lumen with fecal matter during colonoscopy. None of the previous reports have described findings of perforation of the appendix within 24 hours of colonoscopy.
Methods:
We present the case report of a patient who underwent urgent laparotomy within 16 hours of colonos-copy for findings of free intraabdominal air and peritonitis from acute perforated appendicitis.
Results:
Laparoscopy confirmed 2 perforations of the appendix and diffuse peritonitis. Laparotomy was necessary to perform appendectomy, exclude a right colonic injury, and control intraabdominal sepsis.
Conclusion:
In patients with abdominal pain who have had a recent colonoscopy, a high index of suspicion is necessary for accurate diagnosis of perforated appendicitis. Perforation can occur hours after colonoscopy even when a biopsy is not performed.
PMCID: PMC3015887  PMID: 18765066
Perforated appendicitis; Colonoscopy
19.  Percutaneous Coronary Intervention in the Elderly 
The Ulster Medical Journal  2007;76(1):18-21.
Objective
To determine the clinical risks and procedural outcomes for elderly patients undergoing percutaneous coronary intervention (PCI).
Method
A retrospective case-load analysis was performed of all patients over the age of 80 years, undergoing PCI, over a two-year period, in a tertiary referral hospital. Patient demographics, procedural details and in-hospital complications were obtained by reviewing patient notes. Twelve-month outcomes were obtained from telephone follow-up to the general practitioners and all surviving patients.
Results
There were 55 procedures. Procedural risk was high, with a median TIMI risk score of four (IQR 3-6) and a median additive EuroSCORE of nine (IQR 8-10). There was a 95% angiographic success rate. There were no in-hospital complications. Median Canadian Cardiovascular Society angina class fell from four (IQR 3-4) to one (IQR 0-1). At one year there were twelve deaths (10 cardiovascular), eight of these occurred in patients who had incomplete revascularisation.
Conclusion
PCI can be performed in an elderly, high-risk population with a low in-hospital mortality and marked symptomatic benefit. However, there is a significant 1-year mortality, particularly in patients who are only suitable for partial revascularisation.
PMCID: PMC1940296  PMID: 17288300
20.  Secretion of Proinflammatory Cytokines and Chemokines during Amphotericin B Exposure Is Mediated by Coactivation of Toll-Like Receptors 1 and 2 
Amphotericin B (AmB) is a ligand of toll-like receptor 2 (TLR2). Here, we demonstrate the participation of TLR1 in AmB-induced cell activation that led to the secretion of tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-8. Hence, TLR2-TLR1 coactivation serves as the underlying mechanism for the proinflammatory toxicities associated with AmB.
doi:10.1128/AAC.49.4.1617-1621.2005
PMCID: PMC1068636  PMID: 15793154
21.  Effect of Volume Expansion on Hemodynamics of the Hypoperfused Rat Kidney 
Journal of Clinical Investigation  1979;64(2):550-558.
The hemodynamics of the rat kidney were studied during reduction of renal arterial pressure to 35-40 mm Hg (H), and after volume expansion at that pressure with 0.9% NaCl (IS), 1.7% NaCl (HS), 5% mannitol in 0.9% NaCl (MS), 5% mannitol in water (MW), or 50 mM mannitol + 125 mM NaCl. During H, left renal blood flow (RBF) was 0.8±0.1 ml/min. Expansion with IS did not alter RBF, but expansion with HS, MS, MW, and 50 + 125 mM NaCl elevated RBF to 200-250% of hypoperfusion values. Glomerular capillary pressure rose significantly from 15.7±0.7 mm Hg during H to 22.3±1.1, 24.4±0.7, and 26.6±0.7 mm Hg following expansion with HS, MS, or MW, respectively. Efferent arteriolar pressure also rose significantly to 6.9±0.5, 9.7±0.8, and 9.5±0.9 mm Hg, respectively. Preglomerular resistance fell to 18-24% of H values, and postglomerular resistance fell to 58-74% of H values after expansion with HS, MS, or MW. Glomerular filtration (GFR) could not be detected during H or after IS expansion. HS and mannitol-containing solutions restored GFR to 0.10±0.02-0.15±0.02 ml/min, and single nephron glomerular filtration to 6-12 nl/min. Papaverine, acetylcholine, and kinins had no effect on RBF or GFR at a perfusion pressure of 35-40 mm Hg. We conclude that mannitol and HS have the capacity to augment RBF during hypoperfusion by reducing arteriolar resistance. The mechanism of the rise in RBF is uncertain; it may be due to changes in effective osmolality of the extracellular fluid or to a direct action of mannitol on vascular smooth muscle. Other potent vasodilators were ineffective during hypoperfusion. Restoration of GFR occurs as a result of the combined effects of augmented RBF and elevated net filtration pressure.
PMCID: PMC372150  PMID: 457868
22.  Taxonomic Features of Seven Serotypes of Simian and Ape Foamy Viruses 
Infection and Immunity  1971;3(6):793-799.
Two new simian viruses were recovered from squirrel monkeys and galagos. They possess foamy virus group properties which appear distinct from other virus groups. Serologically they were designated types 4 and 5 and are completely specific from each other and from types 1, 2, 3, 6, and 7 by neutralization tests. The study includes a comparison of the properties of all seven types in one laboratory.
Images
PMCID: PMC416239  PMID: 16558056
23.  BILLROTH I RESECTION FOR PEPTIC ULCER 
California Medicine  1956;84(3):157-161.
The Billroth I gastric resection, with and without vagotomy, was used in 20 selected cases of peptic ulcer.
Vagotomy and pyloroplasty is considered the operation of first choice for duodenal ulcer. The cases for Billroth I resections were selected from cases not suitable for pyloroplasty.
Operations for peptic ulcer which preserve the gastrointestinal continuity are considered to be physiologically superior. Vagotomy and pyloroplasty, and Billroth I gastric resection both qualify in this regard. The postoperative digestive symptoms after Billroth I gastric resection in the present series were minimal, which tends to confirm this theoretical superiority.
PMCID: PMC1532899  PMID: 13304669

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