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Sample records for screen identifies not3

  1. A screening cascade to identify ERβ ligands

    PubMed Central

    Filgueira, Carly S.; Benod, Cindy; Lou, Xiaohua; Gunamalai, Prem S.; Villagomez, Rosa A.; Strom, Anders; Gustafsson, Jan-Åke; Berkenstam, Anders L.; Webb, Paul

    2014-01-01

    The establishment of effective high throughput screening cascades to identify nuclear receptor (NR) ligands that will trigger defined, therapeutically useful sets of NR activities is of considerable importance. Repositioning of existing approved drugs with known side effect profiles can provide advantages because de novo drug design suffers from high developmental failure rates and undesirable side effects which have dramatically increased costs. Ligands that target estrogen receptor β (ERβ) could be useful in a variety of diseases ranging from cancer to neurological to cardiovascular disorders. In this context, it is important to minimize cross-reactivity with ERα, which has been shown to trigger increased rates of several types of cancer. Because of high sequence similarities between the ligand binding domains of ERα and ERβ, preferentially targeting one subtype can prove challenging. Here, we describe a sequential ligand screening approach comprised of complementary in-house assays to identify small molecules that are selective for ERβ. Methods include differential scanning fluorimetry, fluorescence polarization and a GAL4 transactivation assay. We used this strategy to screen several commercially-available chemical libraries, identifying thirty ERβ binders that were examined for their selectivity for ERβ versus ERα, and tested the effects of selected ligands in a prostate cancer cell proliferation assay. We suggest that this approach could be used to rapidly identify candidates for drug repurposing. PMID:25422593

  2. Zebrafish phenotypic screen identifies novel Notch antagonists.

    PubMed

    Velaithan, Vithya; Okuda, Kazuhide Shaun; Ng, Mei Fong; Samat, Norazwana; Leong, Sze Wei; Faudzi, Siti Munirah Mohd; Abas, Faridah; Shaari, Khozirah; Cheong, Sok Ching; Tan, Pei Jean; Patel, Vyomesh

    2017-04-01

    Zebrafish represents a powerful in vivo model for phenotype-based drug discovery to identify clinically relevant small molecules. By utilizing this model, we evaluated natural product derived compounds that could potentially modulate Notch signaling that is important in both zebrafish embryogenesis and pathogenic in human cancers. A total of 234 compounds were screened using zebrafish embryos and 3 were identified to be conferring phenotypic alterations similar to embryos treated with known Notch inhibitors. Subsequent secondary screens using HEK293T cells overexpressing truncated Notch1 (HEK293TΔE) identified 2 compounds, EDD3 and 3H4MB, to be potential Notch antagonists. Both compounds reduced protein expression of NOTCH1, Notch intracellular domain (NICD) and hairy and enhancer of split-1 (HES1) in HEK293TΔE and downregulated Notch target genes. Importantly, EDD3 treatment of human oral cancer cell lines demonstrated reduction of Notch target proteins and genes. EDD3 also inhibited proliferation and induced G0/G1 cell cycle arrest of ORL-150 cells through inducing p27(KIP1). Our data demonstrates the utility of the zebrafish phenotypic screen and identifying EDD3 as a promising Notch antagonist for further development as a novel therapeutic agent.

  3. Identifying reference chemicals for thyroid bioactivity screening.

    PubMed

    Wegner, Susanna; Browne, Patience; Dix, David

    2016-10-01

    Reference chemicals were selected based on thyroid bioactivity in 'Tier 1' screening assays used by the U.S. EPA's Endocrine Disruptor Screening Program. Active reference chemicals had significant effects on thyroid-responsive endpoints in the amphibian metamorphosis assay, and the male and female pubertal rat assays. In the absence of thyroid weight or histopathological effects, additional published studies providing mechanistic data on thyroid activity were required for active chemicals. Inactive reference chemicals had no significant effects on thyroid-responsive endpoints in Tier 1 assays, or in amphibian or rodent studies from several online databases. The 34 reference chemicals (29 active and five inactive) will be useful for performance-based validation of alternative, high throughput screening assays for thyroid bioactivity.

  4. Congenital adrenal hyperplasia cases identified by newborn screening in one- and two-screen states

    PubMed Central

    Held, Patrice K.; Shapira, Stuart K.; Hinton, Cynthia F.; Jones, Elizabeth; Hannon, W. Harry; Ojodu, Jelili

    2015-01-01

    There is no clear consensus among state newborn screening programs on whether routine second screening of newborns identifies clinically relevant cases of congenital adrenal hyperplasia. This retrospective study evaluated laboratory practices, along with biochemical and medical characteristics of congenital adrenal hyperplasia (CAH) cases (1) detected on the first newborn screen in one-screen compared to two-screen states, and (2) detected on the first versus the second screen in the two-screen states, to determine the effectiveness of a second screen. A total of 374 confirmed cases of CAH from 2 one-screen states and 5 two-screen states were included in this study. Demographic data and diagnostic information on each reported case were collected and analyzed. Additionally, laboratory data, including screening methodologies and algorithms, were evaluated. The one-screen states reported 99 cases of CAH out of 1,740,586 (1 in 17,500) newborns screened: 88 (89%) identified on first screen and 5 (5%) identified on targeted second screen. The two-screen states reported 275 cases of CAH out of 2,629,627 (1 in 9,500) newborns screened: 165 (60%) identified on first screen and 99 (36%) identified on second screen. Using a multivariate model, the only significant predictor of whether a case was identified on the first or second screen in the two-screen states was the type of CAH. Compared with classical salt-wasting CAH, classical simple virilizing and non-classical CAH cases were less likely to be detected on the first versus the second screen. The routine second newborn screen is important for identifying children with CAH, particularly simple virilizing and non-classical forms, which might otherwise not be captured through a single screen. PMID:26296712

  5. Congenital adrenal hyperplasia cases identified by newborn screening in one- and two-screen states.

    PubMed

    Held, Patrice K; Shapira, Stuart K; Hinton, Cynthia F; Jones, Elizabeth; Hannon, W Harry; Ojodu, Jelili

    2015-11-01

    There is no clear consensus among state newborn screening programs on whether routine second screening of newborns identifies clinically relevant cases of congenital adrenal hyperplasia. This retrospective study evaluated laboratory practices, along with biochemical and medical characteristics of congenital adrenal hyperplasia (CAH) cases (1) detected on the first newborn screen in one-screen compared to two-screen states, and (2) detected on the first versus the second screen in the two-screen states, to determine the effectiveness of a second screen. A total of 374 confirmed cases of CAH from 2 one-screen states and 5 two-screen states were included in this study. Demographic data and diagnostic information on each reported case were collected and analyzed. Additionally, laboratory data, including screening methodologies and algorithms, were evaluated. The one-screen states reported 99 cases of CAH out of 1,740,586 (1 in 17,500) newborns screened: 88 (89%) identified on the first screen and 5 (5%) identified on the targeted second screen. The two-screen states reported 275 cases of CAH out of 2,629,627 (1 in 9500) newborns screened: 165 (60%) identified on the first screen and 99 (36%) identified on the second screen. Using a multivariate model, the only significant predictor of whether a case was identified on the first or the second screen in the two-screen states was the type of CAH. Compared with classical salt-wasting CAH, classical simple virilizing and non-classical CAH cases were less likely to be detected on the first versus the second screen. The routine second newborn screen is important for identifying children with CAH, particularly simple virilizing and non-classical forms, which might otherwise not be captured through a single screen. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Selecting Universal Screening Measures to Identify Students at Risk Academically

    ERIC Educational Resources Information Center

    Salinger, Rachel L.

    2016-01-01

    Universal screening measures can be used to identify students at risk academically due to learning disabilities or other difficulties. Research and legislation support the use of screening measures early in students' education to ensure they receive any supports necessary to bolster their academic achievement. When selecting a screening measure,…

  7. Selecting Universal Screening Measures to Identify Students at Risk Academically

    ERIC Educational Resources Information Center

    Salinger, Rachel L.

    2016-01-01

    Universal screening measures can be used to identify students at risk academically due to learning disabilities or other difficulties. Research and legislation support the use of screening measures early in students' education to ensure they receive any supports necessary to bolster their academic achievement. When selecting a screening measure,…

  8. Tiered High-Throughput Screening Approach to Identify ...

    EPA Pesticide Factsheets

    High-throughput screening (HTS) for potential thyroid–disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limited in the US EPA ToxCast screening assay portfolio. To fill one critical screening gap, the Amplex UltraRed-thyroperoxidase (AUR-TPO) assay was developed to identify chemicals that inhibit TPO, as decreased TPO activity reduces TH synthesis. The ToxCast Phase I and II chemical libraries, comprised of 1,074 unique chemicals, were initially screened using a single, high concentration to identify potential TPO inhibitors. Chemicals positive in the single concentration screen were retested in concentration-response. Due to high false positive rates typically observed with loss-of-signal assays such as AUR-TPO, we also employed two additional assays in parallel to identify possible sources of nonspecific assay signal loss, enabling stratification of roughly 300 putative TPO inhibitors based upon selective AUR-TPO activity. A cell-free luciferase inhibition assay was used to identify nonspecific enzyme inhibition among the putative TPO inhibitors, and a cytotoxicity assay using a human cell line was used to estimate the cellular tolerance limit. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO and an orthogonal peroxidase oxidation assay using

  9. Identifying Unbiased Items for Screening Preschoolers for Disruptive Behavior Problems.

    PubMed

    Studts, Christina R; Polaha, Jodi; van Zyl, Michiel A

    2016-10-25

    OBJECTIVE : Efficient identification and referral to behavioral services are crucial in addressing early-onset disruptive behavior problems. Existing screening instruments for preschoolers are not ideal for pediatric primary care settings serving diverse populations. Eighteen candidate items for a new brief screening instrument were examined to identify those exhibiting measurement bias (i.e., differential item functioning, DIF) by child characteristics. METHOD : Parents/guardians of preschool-aged children (N = 900) from four primary care settings completed two full-length behavioral rating scales. Items measuring disruptive behavior problems were tested for DIF by child race, sex, and socioeconomic status using two approaches: item response theory-based likelihood ratio tests and ordinal logistic regression. RESULTS : Of 18 items, eight were identified with statistically significant DIF by at least one method. CONCLUSIONS : The bias observed in 8 of 18 items made them undesirable for screening diverse populations of children. These items were excluded from the new brief screening tool.

  10. A Screening Tool to Identify Spasticity in Need of Treatment

    PubMed Central

    Zorowitz, Richard D.; Wein, Theodore H.; Dunning, Kari; Deltombe, Thierry; Olver, John H.; Davé, Shashank J.; Dimyan, Michael A.; Kelemen, John; Pagan, Fernando L.; Evans, Christopher J.; Gillard, Patrick J.; Kissela, Brett M.

    2017-01-01

    Objective To develop a clinically useful patient-reported screening tool for health care providers to identify patients with spasticity in need of treatment regardless of etiology. Design Eleven spasticity experts participated in a modified Delphi panel and reviewed and revised 2 iterations of a screening tool designed to identify spasticity symptoms and impact on daily function and sleep. Spasticity expert panelists evaluated items pooled from existing questionnaires to gain consensus on the screening tool content. The study also included cognitive interviews of 20 patients with varying spasticity etiologies to determine if the draft screening tool was understandable and relevant to patients with spasticity. Results The Delphi panel reached an initial consensus on 21 of 47 items for the screening tool and determined that the tool should have no more than 11 to 15 items and a 1-month recall period for symptom and impact items. After 2 rounds of review, 13 items were selected and modified by the expert panelists. Most patients (n = 16 [80%]) completed the cognitive interview and interpreted the items as intended. Conclusions Through the use of a Delphi panel and patient interviews, a 13-item spasticity screening tool was developed that will be practical and easy to use in routine clinical practice. PMID:27552355

  11. A Triangulation Method for Identifying Hydrostratigraphic Locations of Well Screens

    SciTech Connect

    Whiteside, T. S.

    2015-01-31

    A method to identify the hydrostratigraphic location of well screens was developed using triangulation with known locations. This method was applied to all of the monitor wells being used to develop the new GSA groundwater model. Results from this method are closely aligned with those from an alternate method which uses a mesh surface.

  12. Phenotypic lentivirus screens to identify functional single domain antibodies

    PubMed Central

    Schmidt, Florian I.; Hanke, Leo; Morin, Benjamin; Brewer, Rebeccah; Brusic, Vesna; Whelan, Sean P.J.; Ploegh, Hidde L.

    2016-01-01

    Manipulation of proteins is key in assessing their in vivo function. While genetic ablation is straightforward, reversible and specific perturbation of protein function remains a challenge. Single domain antibody fragments, such as camelid-derived VHHs, can serve as inhibitors or activators of intracellular protein function, but functional testing of identified VHHs is laborious. To address this challenge, we developed a lentiviral screening approach to identify VHHs that elicit a phenotype when expressed intracellularly. We identified 19 antiviral VHHs that protect human A549 cells from lethal infection with influenza A virus (IAV) or vesicular stomatitis virus (VSV), respectively. Both negative-sense RNA viruses are vulnerable to VHHs uniquely specific for their respective nucleoproteins. Antiviral VHHs prevented nuclear import of viral ribonucleoproteins or mRNA transcription, respectively, and may provide clues for novel antiviral reagents. In principle, the screening approach described here should be applicable to identify inhibitors of any pathogen or biological pathway. PMID:27573105

  13. Newborn screening for SCID identifies patients with ataxia telangiectasia.

    PubMed

    Mallott, Jacob; Kwan, Antonia; Church, Joseph; Gonzalez-Espinosa, Diana; Lorey, Fred; Tang, Ling Fung; Sunderam, Uma; Rana, Sadhna; Srinivasan, Rajgopal; Brenner, Steven E; Puck, Jennifer

    2013-04-01

    Severe combined immunodeficiency (SCID) is characterized by failure of T lymphocyte development and absent or very low T cell receptor excision circles (TRECs), DNA byproducts of T cell maturation. Newborn screening for TRECs to identify SCID is now performed in several states using PCR of DNA from universally collected dried blood spots (DBS). In addition to infants with typical SCID, TREC screening identifies infants with T lymphocytopenia who appear healthy and in whom a SCID diagnosis cannot be confirmed. Deep sequencing was employed to find causes of T lymphocytopenia in such infants. Whole exome sequencing and analysis were performed in infants and their parents. Upon finding deleterious mutations in the ataxia telangiectasia mutated (ATM) gene, we confirmed the diagnosis of ataxia telangiectasia (AT) in two infants and then tested archival newborn DBS of additional AT patients for TREC copy number. Exome sequencing and analysis led to 2 unsuspected gene diagnoses of AT. Of 13 older AT patients for whom newborn DBS had been stored, 7 samples tested positive for SCID under the criteria of California's newborn screening program. AT children with low neonatal TRECs had low CD4 T cell counts subsequently detected (R = 0.64). T lymphocytopenia in newborns can be a feature of AT, as revealed by TREC screening and exome sequencing. Although there is no current cure for the progressive neurological impairment of AT, early detection permits avoidance of infectious complications, while providing information for families regarding reproductive recurrence risks and increased cancer risks in patients and carriers.

  14. Dietary screening tool identifies nutritional risk in older adults123

    PubMed Central

    Miller, Paige E; Mitchell, Diane C; Hartman, Terryl J; Lawrence, Frank R; Sempos, Christopher T; Smiciklas-Wright, Helen

    2009-01-01

    Background: No rapid methods exist for screening overall dietary intakes in older adults. Objective: The purpose of this study was to develop and evaluate a scoring system for a diet screening tool to identify nutritional risk in community-dwelling older adults. Design: This cross-sectional study in older adults (n = 204) who reside in rural areas examined nutrition status by using an in-person interview, biochemical measures, and four 24-h recalls that included the use of dietary supplements. Results: The dietary screening tool was able to characterize 3 levels of nutritional risk: at risk, possible risk, and not at risk. Individuals classified as at nutritional risk had significantly lower indicators of diet quality (Healthy Eating Index and Mean Adequacy Ratio) and intakes of protein, most micronutrients, dietary fiber, fruit, and vegetables. The at-risk group had higher intakes of fats and oils and refined grains. The at-risk group also had the lowest serum vitamin B-12, folate, β-cryptoxanthin, lutein, and zeaxanthin concentrations. The not-at-nutritional-risk group had significantly higher lycopene and β-carotene and lower homocysteine and methylmalonic acid concentrations. Conclusion: The dietary screening tool is a simple and practical tool that can help to detect nutritional risk in older adults. PMID:19458013

  15. Axon Regeneration Genes Identified by RNAi Screening in C. elegans

    PubMed Central

    Nix, Paola; Hammarlund, Marc; Hauth, Linda; Lachnit, Martina; Jorgensen, Erik M.

    2014-01-01

    Axons of the mammalian CNS lose the ability to regenerate soon after development due to both an inhibitory CNS environment and the loss of cell-intrinsic factors necessary for regeneration. The complex molecular events required for robust regeneration of mature neurons are not fully understood, particularly in vivo. To identify genes affecting axon regeneration in Caenorhabditis elegans, we performed both an RNAi-based screen for defective motor axon regeneration in unc-70/β-spectrin mutants and a candidate gene screen. From these screens, we identified at least 50 conserved genes with growth-promoting or growth-inhibiting functions. Through our analysis of mutants, we shed new light on certain aspects of regeneration, including the role of β-spectrin and membrane dynamics, the antagonistic activity of MAP kinase signaling pathways, and the role of stress in promoting axon regeneration. Many gene candidates had not previously been associated with axon regeneration and implicate new pathways of interest for therapeutic intervention. PMID:24403161

  16. Membrane Lipid Screen to Identify Molecular Targets of Biomolecules.

    PubMed

    Jimah, John R; Schlesinger, Paul H; Tolia, Niraj H

    2017-08-05

    Proteins that bind to and disrupt cell membranes may target specific phospholipids. Here we describe a protocol to identify the lipid targets of proteins and biomolecules. First, we describe a screen to identify lipids in membranes that are specifically bound by the biomolecule of interest. Second, we describe a method for determining if the presence of these lipids within membranes is necessary for membrane disruption. The methods described here were used to determine that the malaria vaccine candidate CelTOS disrupts cell membranes by specifically targeting phosphatidic acid (Jimah et al., 2016). This protocol has a companion protocol: 'Liposome disruption assay to examine lytic properties of biomolecules' which can be applied to examine the ability of the biomolecule to disrupt membranes composed of the lipid target identified by following this protocol (Jimah et al., 2017).

  17. First Steps Screening Module: Procedures for Screening and Identifying At-Risk Kindergartners.

    ERIC Educational Resources Information Center

    Walker, Hill; Stiller, Bruce

    This report describes the first module of the "First Steps" program, an early intervention program for at-risk kindergartners who show the early signs of antisocial behavior patterns. This module presents four options for screening and identifying kindergarten students at risk for antisocial behavior patterns. In option one, the…

  18. Chemical screening identifies ATM as a target for alleviating senescence.

    PubMed

    Kang, Hyun Tae; Park, Joon Tae; Choi, Kobong; Kim, Yongsub; Choi, Hyo Jei Claudia; Jung, Chul Won; Lee, Young-Sam; Park, Sang Chul

    2017-06-01

    Senescence, defined as irreversible cell-cycle arrest, is the main driving force of aging and age-related diseases. Here, we performed high-throughput screening to identify compounds that alleviate senescence and identified the ataxia telangiectasia mutated (ATM) inhibitor KU-60019 as an effective agent. To elucidate the mechanism underlying ATM's role in senescence, we performed a yeast two-hybrid screen and found that ATM interacted with the vacuolar ATPase V1 subunits ATP6V1E1 and ATP6V1G1. Specifically, ATM decreased E-G dimerization through direct phosphorylation of ATP6V1G1. Attenuation of ATM activity restored the dimerization, thus consequently facilitating assembly of the V1 and V0 domains with concomitant reacidification of the lysosome. In turn, this reacidification induced the functional recovery of the lysosome/autophagy system and was coupled with mitochondrial functional recovery and metabolic reprogramming. Together, our data reveal a new mechanism through which senescence is controlled by the lysosomal-mitochondrial axis, whose function is modulated by the fine-tuning of ATM activity.

  19. Small-Molecule Screening Identifies Modulators of Aquaporin-2 Trafficking

    PubMed Central

    Bogum, Jana; Faust, Dörte; Zühlke, Kerstin; Eichhorst, Jenny; Moutty, Marie C.; Furkert, Jens; Eldahshan, Adeeb; Neuenschwander, Martin; von Kries, Jens Peter; Wiesner, Burkhard; Trimpert, Christiane; Deen, Peter M.T.; Valenti, Giovanna; Rosenthal, Walter

    2013-01-01

    In the principal cells of the renal collecting duct, arginine vasopressin (AVP) stimulates the synthesis of cAMP, leading to signaling events that culminate in the phosphorylation of aquaporin-2 water channels and their redistribution from intracellular domains to the plasma membrane via vesicular trafficking. The molecular mechanisms that control aquaporin-2 trafficking and the consequent water reabsorption, however, are not completely understood. Here, we used a cell-based assay and automated immunofluorescence microscopy to screen 17,700 small molecules for inhibitors of the cAMP-dependent redistribution of aquaporin-2. This approach identified 17 inhibitors, including 4-acetyldiphyllin, a selective blocker of vacuolar H+-ATPase that increases the pH of intracellular vesicles and causes accumulation of aquaporin-2 in the Golgi compartment. Although 4-acetyldiphyllin did not inhibit forskolin-induced increases in cAMP formation and downstream activation of protein kinase A (PKA), it did prevent cAMP/PKA-dependent phosphorylation at serine 256 of aquaporin-2, which triggers the redistribution to the plasma membrane. It did not, however, prevent cAMP-induced changes to the phosphorylation status at serines 261 or 269. Last, we identified the fungicide fluconazole as an inhibitor of cAMP-mediated redistribution of aquaporin-2, but its target in this pathway remains unknown. In conclusion, our screening approach provides a method to begin dissecting molecular mechanisms underlying AVP-mediated water reabsorption, evidenced by our identification of 4-acetyldiphyllin as a modulator of aquaporin-2 trafficking. PMID:23559583

  20. Quantitative High-throughput Luciferase Screening in Identifying CAR Modulators

    PubMed Central

    Lynch, Caitlin; Zhao, Jinghua; Wang, Hongbing; Xia, Menghang

    2017-01-01

    Summary The constitutive androstane receptor (CAR, NR1I3) is responsible for the transcription of multiple drug metabolizing enzymes and transporters. There are two possible methods of activation for CAR, direct ligand binding and a ligand-independent method, which makes this a unique nuclear receptor. Both of these mechanisms require translocation of CAR from the cytoplasm into the nucleus. Interestingly, CAR is constitutively active in immortalized cell lines due to the basal nuclear location of this receptor. This creates an important challenge in most in vitro assay models because immortalized cells cannot be used without inhibiting the basal activity. In this book chapter, we go into detail of how to perform quantitative high-throughput screens to identify hCAR1 modulators through the employment of a double stable cell line. Using this line, we are able to identify activators, as well as deactivators, of the challenging nuclear receptor, CAR. PMID:27518621

  1. A Chemical Screen Identifies Small Molecules that Regulate Hepcidin Expression

    PubMed Central

    Gaun, Vera; Patchen, Bonnie; Volovetz, Josephine; Zhen, Aileen W.; Andreev, Aleksandr; Pollastri, Michael P.; Fraenkel, Paula G.

    2014-01-01

    Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Bone morphogenic protein and Stat3 signaling regulate Hepcidin's transcription. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing 2.7 kilobases of the human Hepcidin promoter upstream of a firefly reporter gene. We used high throughput methods to screen 10,169 chemicals in duplicate for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change >3 standard deviations above or >1.5 standard deviations below the mean of the other chemicals (z-score >3 or <-1.5), while not adversely affecting cell viability, quantified by fluorescence assay. Following validation assays, we identified 16 chemicals in a broad range of functional classes that promote Hepcidin expression. All of the chemicals identified increased expression of bone morphogenic protein-dependent and/or Stat3-dependent genes, however none of them strongly increased phosphorylation of Smad1,5,8 or Stat3. PMID:24998898

  2. A chemical screen identifies small molecules that regulate hepcidin expression.

    PubMed

    Gaun, Vera; Patchen, Bonnie; Volovetz, Josephine; Zhen, Aileen W; Andreev, Aleksandr; Pollastri, Michael P; Fraenkel, Paula G

    2014-12-01

    Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Bone morphogenic protein and Stat3 signaling regulate Hepcidin's transcription. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing 2.7kb of the human Hepcidin promoter upstream of a firefly reporter gene. We used high throughput methods to screen 10,169 chemicals in duplicate for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change >3 standard deviations above or >1 standard deviation below the mean of the other chemicals (z-score >3 or <1), while not adversely affecting cell viability, quantified by fluorescence assay. Following validation assays, we identified 16 chemicals in a broad range of functional classes that promote Hepcidin expression. All of the chemicals identified increased expression of bone morphogenic protein-dependent and/or Stat3-dependent genes, however none of them strongly increased phosphorylation of Smad1,5,8 or Stat3.

  3. Large screen approaches to identify novel malaria vaccine candidates

    PubMed Central

    Davies, D. Huw; Duffy, Patrick; Bodmer, Jean-Luc; Felgner, Philip L.; Doolan, Denise L.

    2016-01-01

    Until recently, malaria vaccine development efforts have focused almost exclusively on a handful of well characterized Plasmodium falciparum antigens. Despite dedicated work by many researchers on different continents spanning more than half a century, a successful malaria vaccine remains elusive. Sequencing of the P. falciparum genome has revealed more than five thousand genes, providing the foundation for systematic approaches to discover candidate vaccine antigens. We are taking advantage of this wealth of information to discover new antigens that may be more effective vaccine targets. Herein, we describe different approaches to large-scale screening of the P. falciparum genome to identify targets of either antibody responses or T cell responses using human specimens collected in Controlled Human Malaria Infections (CHMI) or under conditions of natural exposure in the field. These genome, proteome and transcriptome based approaches offer enormous potential for the development of an efficacious malaria vaccine. PMID:26428458

  4. Identifying a borderline personality disorder prodrome: Implications for community screening.

    PubMed

    Stepp, Stephanie D; Lazarus, Sophie A

    2017-08-01

    Elucidating early signs and symptoms of borderline personality disorder (BPD) has important implications for screening and identifying youth appropriate for early intervention. The purpose of this study was to identify dimensions of child temperament and psychopathology symptom severity that predict conversion to a positive screen for BPD over a 14-year follow-up period in a large, urban community sample of girls (n = 2 450). Parent and teacher reports of child temperament and psychopathology symptom severity assessed when girls were ages 5-8 years were examined as predictors of new-onset BPD cases when girls were ages 14-22 years. In the final model, parent and teacher ratings of emotionality remained significant predictors of new-onset BPD. Additionally, parent ratings of hyperactivity/impulsivity and depression severity, as well as teacher ratings of inattention severity, were also predictive. Results also revealed that elevations in these dimensions pose a notable increase in risk for conversion to BPD over the follow-up period. Supplementary analyses revealed that with the exception of parent-reported depression severity, these same predictors were associated with increases in BPD symptom severity over the follow-up period. These findings suggest BPD onset in adolescence and early adulthood can be detected from parent and teacher reports of temperament and symptom severity dimensions assessed in childhood. The identification of this prodrome holds promise for advancing early detection of children at risk prior to the development of the full-blown disorder. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  5. High-Throughput Screening of Small Molecules Identifies Hepcidin Antagonists

    PubMed Central

    Fung, Eileen; Sugianto, Priscilla; Hsu, Jason; Damoiseaux, Robert; Ganz, Tomas

    2013-01-01

    Anemia of inflammation (AI) is common in patients with infection, autoimmune diseases, cancer, and chronic kidney disease. Unless the underlying condition can be reversed, treatment options are limited to erythropoiesis-stimulating agents with or without intravenous iron therapy, modalities that are not always effective and can cause serious adverse effects. Hepcidin, the iron regulatory hormone, has been identified as a pathogenic factor in the development of AI. To explore new therapeutic options for AI and other iron-related disorders caused by hepcidin excess, we developed a cell-based screen to identify hepcidin antagonists. Of the 70,000 small molecules in the library, we identified 14 compounds that antagonized the hepcidin effect on ferroportin. One of these was fursultiamine, a Food and Drug Administration (FDA)–approved thiamine derivative. Fursultiamine directly interfered with hepcidin binding to its receptor, ferroportin, by blocking ferroportin C326 thiol residue essential for hepcidin binding. Consequently, fursultiamine prevented hepcidin-induced ferroportin ubiquitination, endocytosis, and degradation in vitro and allowed continuous cellular iron export despite the presence of hepcidin, with IC50 in the submicromolar range. Thiamine, the fursultiamine metabolite, and benfotiamine, another thiamine derivative, did not interfere with the effect of hepcidin on ferroportin. Other FDA-approved thiol-reactive compounds were at least 1000-fold less potent than fursultiamine in antagonizing hepcidin. In vivo, fursultiamine did not reproducibly antagonize the effect of hepcidin on serum iron, likely because of its rapid conversion to inactive metabolites. Fursultiamine is a unique antagonist of hepcidin in vitro that could serve as a template for the development of drug candidates that inhibit the hepcidin-ferroportin interaction. PMID:23292796

  6. Human Picobirnaviruses Identified by Molecular Screening of Diarrhea Samples▿ †

    PubMed Central

    van Leeuwen, Marije; Williams, Marisol M. W.; Koraka, Penelope; Simon, James H.; Smits, Saskia L.; Osterhaus, Albert D. M. E.

    2010-01-01

    The global threat of (re)emerging infectious viruses requires a more effective approach regarding virus surveillance and diagnostic assays, as current diagnostics are often virus species specific and not able to detect highly divergent or unknown viruses. A systematic exploration of viruses that infect humans is the key to effectively counter the potential public health threat caused by new and emerging infectious diseases. The human gut is a known reservoir of a wide variety of microorganisms, including viruses. In this study, Dutch clinical diarrhea samples for which no etiological agent could be identified by available cell culture, serological, or nucleic acid-based tests were gathered. Large-scale molecular RNA virus screening based on host nucleic acid depletion, sequence-independent amplification, and sequencing of partially purified viral RNA from a limited number of clinical diarrhea samples revealed four eukaryotic virus species. Among the detected viruses were a rhinovirus and a new picobirnavirus variant. In total, ∼20% of clinical diarrhea samples contained human picobirnavirus sequences. The Dutch picobirnaviruses belonged to different phylogenetic clades and did not group with other picobirnaviruses according to year of isolation or host species. Interestingly, the average age of patients infected with picobirnavirus was significantly higher than that of uninfected patients. Our data show that sequence-independent amplification of partially purified viral RNA is an efficient procedure for identification of known and highly divergent new RNA viruses in clinical diarrhea samples. PMID:20335418

  7. MLH1 methylation screening is effective in identifying epimutation carriers.

    PubMed

    Pineda, Marta; Mur, Pilar; Iniesta, María Dolores; Borràs, Ester; Campos, Olga; Vargas, Gardenia; Iglesias, Sílvia; Fernández, Anna; Gruber, Stephen B; Lázaro, Conxi; Brunet, Joan; Navarro, Matilde; Blanco, Ignacio; Capellá, Gabriel

    2012-12-01

    Recently, constitutional MLH1 epimutations have been identified in a subset of Lynch syndrome (LS) cases. The aim of this study was the identification of patients harboring constitutional MLH1 epimutations in a set of 34 patients with a clinical suspicion of LS, MLH1-methylated tumors and non-detected germline mutations in mismatch repair (MMR) genes. MLH1 promoter methylation was analyzed in lymphocyte DNA samples by MS-MLPA (Methylation-specific multiplex ligation-dependent probe amplification). Confirmation of MLH1 constitutional methylation was performed by MS-MCA (Methylation-specific melting curve analysis), bisulfite sequencing and pyrosequencing in different biological samples. Allelic expression was determined using heterozygous polymorphisms. Vertical transmission was evaluated by MS-MLPA and haplotype analyses. MS-MLPA analysis detected constitutional MLH1 methylation in 2 of the 34 individuals whose colorectal cancers showed MLH1 methylation (5.9%). These results were confirmed by bisulfite-based methods. Both epimutation carriers had developed metachronous early-onset LS tumors, with no family history of LS-associated cancers in their first-degree relatives. In one of the cases, the identified MLH1 constitutional methylation was monoallelic and results in MLH1 and EPM2AIP1 allele-specific transcriptional silencing. It was present in normal somatic tissues and absent in spermatozoa. The methylated MLH1 allele was maternally transmitted and methylation was reversed in a daughter who inherited the same allele. MLH1 methylation screening in lymphocyte DNA from patients with early-onset MLH1-methylated LS-associated tumors allows the identification of epimutation carriers. The present study adds further evidence to the emerging entity of soma-wide MLH1 epimutation and its heritability.

  8. School-Based Screening to Identify At-Risk Students Not Already Known to School Professionals: The Columbia Suicide Screen

    PubMed Central

    Wilcox, Holly C.; Schonfeld, Irvin Sam; Davies, Mark; Hicks, Roger C.; Turner, J. Blake; Shaffer, David

    2009-01-01

    Objectives. We sought to determine the degree of overlap between students identified through school-based suicide screening and those thought to be at risk by school administrative and clinical professionals. Methods. Students from 7 high schools in the New York metropolitan area completed the Columbia Suicide Screen; 489 of the 1729 students screened had positive results. The clinical status of 641 students (73% of those who had screened positive and 23% of those who had screened negative) was assessed with modules from the Diagnostic Interview Schedule for Children. School professionals nominated by their principal and unaware of students' screening and diagnostic status were asked to indicate whether they were concerned about the emotional well-being of each participating student. Results. Approximately 34% of students with significant mental health problems were identified only through screening, 13.0% were identified only by school professionals, 34.9% were identified both through screening and by school professionals, and 18.3% were identified neither through screening nor by school professionals. The corresponding percentages among students without mental health problems were 9.1%, 24.0%, 5.5%, and 61.3%. Conclusions. School-based screening can identify suicidal and emotionally troubled students not recognized by school professionals. PMID:19059865

  9. Screening Procedures to Identify Visual Arts Giftedness. Session V.

    ERIC Educational Resources Information Center

    Amram, Raphi; And Others

    1991-01-01

    This discussion from the Indiana University Summer Arts Institute finds panelists considering screening methods for arts students, including portfolio preparation, site-specific creation of art work, and batteries of tests and tasks. Procedures used at the Israel academy, developmental processes in creative students, and differences in student…

  10. Screening Procedures to Identify Visual Arts Giftedness. Session V.

    ERIC Educational Resources Information Center

    Amram, Raphi; And Others

    1991-01-01

    This discussion from the Indiana University Summer Arts Institute finds panelists considering screening methods for arts students, including portfolio preparation, site-specific creation of art work, and batteries of tests and tasks. Procedures used at the Israel academy, developmental processes in creative students, and differences in student…

  11. Identifying producers of antibacterial compounds by screening for antibiotic resistance.

    PubMed

    Thaker, Maulik N; Wang, Wenliang; Spanogiannopoulos, Peter; Waglechner, Nicholas; King, Andrew M; Medina, Ricardo; Wright, Gerard D

    2013-10-01

    Microbially derived natural products are major sources of antibiotics and other medicines, but discovering new antibiotic scaffolds and increasing the chemical diversity of existing ones are formidable challenges. We have designed a screen to exploit the self-protection mechanism of antibiotic producers to enrich microbial libraries for producers of selected antibiotic scaffolds. Using resistance as a discriminating criterion we increased the discovery rate of producers of both glycopeptide and ansamycin antibacterial compounds by several orders of magnitude in comparison with historical hit rates. Applying a phylogeny-based screening filter for biosynthetic genes enabled the binning of producers of distinct scaffolds and resulted in the discovery of a glycopeptide antibacterial compound, pekiskomycin, with an unusual peptide scaffold. This strategy provides a means to readily sample the chemical diversity available in microbes and offers an efficient strategy for rapid discovery of microbial natural products and their associated biosynthetic enzymes.

  12. Screening practices for identifying type 2 diabetes in adolescents.

    PubMed

    Lee, Joyce M; Eason, Ashley; Nelson, Courtney; Kazzi, Nayla G; Cowan, Anne E; Tarini, Beth A

    2014-02-01

    To characterize pediatrician and family physician (FP) screening practices for type 2 diabetes among adolescents and to examine the impact of the 2010 American Diabetes Association (ADA) guidelines, recommending use of Hemoglobin A1c (HbA1c). We conducted a cross-sectional mail survey of a random sample of 1,400 U.S. pediatricians and FPs and we received 604 eligible responses. Our main outcome measure was the types of tests ordered by physicians, particularly HbA1c, when presented with a hypothetical scenario. The overall response rate was 52% (57% for pediatricians and 48% for FPs). Fasting glucose and HbA1c were the most commonly ordered tests. Overall, at least 58% of physicians ordered HbA1c; 35% ordered HbA1c in conjunction with fasting tests; and 22% ordered HbA1c alone or with nonfasting tests. Only 38% of providers were aware of the new ADA recommended HbA1c screening guidelines. However, a majority (67%) said they would change their screening practices. In the context of the guidelines, 84% of physicians would now order HbA1c. Furthermore, there was a large increase in the proportion of physicians who would shift to using HbA1c only or with other nonfasting tests. When screening adolescents for type 2 diabetes, providers are more likely to order HbA1c and order fewer fasting tests in response to the new ADA guidelines. HbA1c has lower sensitivity and higher costs than other testing modalities in children, therefore increasing uptake of this test (HbA1c) in children may have implications for both detection rates and healthcare costs. Copyright © 2014 Society for Adolescent Health and Medicine. Published by Elsevier Inc. All rights reserved.

  13. Genetic Screens in Yeast to Identify BRCA1 Modifiers

    DTIC Science & Technology

    2005-12-01

    EUG1 YDR518W protein folding FLO9 YAL063C inv in flocculation FOB1 YDR110W,HRM1 DNA replication fork blocking GCR1 YPL075W transcriptional activator... initiated . The PI of this proposal is participating in a multi-institutional study through the Cancer Genetics Network to collect DNA samples and...insertional mutagenesis screen using Synder Library DNA into strains developed in Tasks 1 and 2. a. Assay for changes in chromosome loss rate. Progress

  14. RNAi screens in mice identify physiological regulators of oncogenic growth

    PubMed Central

    Beronja, Slobodan; Janki, Peter; Heller, Evan; Lien, Wen-Hui; Keyes, Brice; Oshimori, Naoki; Fuchs, Elaine

    2013-01-01

    Summary Tissue growth is the multifaceted outcome of a cell’s intrinsic capabilities and its interactions with the surrounding environment. Decoding these complexities is essential for understanding human development and tumorigenesis. Here, we tackle this problem by carrying out the first genome-wide RNAi-mediated screens in mice. Focusing on skin development and oncogenic (HrasG12V-induced) hyperplasia, our screens uncover novel as well as anticipated regulators of embryonic epidermal growth. Among top oncogenic screen hits are Mllt6 and the Wnt effector β-catenin; they maintain HrasG12V-dependent hyperproliferation. We also expose β-catenin as an unanticipated antagonist of normal epidermal growth, functioning through Wnt-independent intercellular adhesion. Finally, we document physiological relevance to mouse and human cancers, thereby establishing the feasibility of in vivo mammalian genome-wide investigations to dissect tissue development and tumorigenesis. By documenting some oncogenic growth regulators, we pave the way for future investigations of other hits and raise promise for unearthing new targets for cancer therapies. PMID:23945586

  15. Supporting patient screening to identify suitable clinical trials.

    PubMed

    Bucur, Anca; Van Leeuwen, Jasper; Chen, Njin-Zu; Claerhout, Brecht; De Schepper, Kristof; Perez-Rey, David; Alonso-Calvo, Raul; Pugliano, Lina; Saini, Kamal

    2014-01-01

    To support the efficient execution of post-genomic multi-centric clinical trials in breast cancer we propose a solution that streamlines the assessment of the eligibility of patients for available trials. The assessment of the eligibility of a patient for a trial requires evaluating whether each eligibility criterion is satisfied and is often a time consuming and manual task. The main focus in the literature has been on proposing different methods for modelling and formalizing the eligibility criteria. However the current adoption of these approaches in clinical care is limited. Less effort has been dedicated to the automatic matching of criteria to the patient data managed in clinical care. We address both aspects and propose a scalable, efficient and pragmatic patient screening solution enabling automatic evaluation of eligibility of patients for a relevant set of trials. This covers the flexible formalization of criteria and of other relevant trial metadata and the efficient management of these representations.

  16. An in vivo screening system to identify tumorigenic genes.

    PubMed

    Ihara, T; Hosokawa, Y; Kumazawa, K; Ishikawa, K; Fujimoto, J; Yamamoto, M; Muramkami, T; Goshima, N; Ito, E; Watanabe, S; Semba, K

    2017-04-06

    Screening for oncogenes has mostly been performed by in vitro transformation assays. However, some oncogenes might not exhibit their transforming activities in vitro unless putative essential factors from in vivo microenvironments are adequately supplied. Here, we have developed an in vivo screening system that evaluates the tumorigenicity of target genes. This system uses a retroviral high-efficiency gene transfer technique, a large collection of human cDNA clones corresponding to ~70% of human genes and a luciferase-expressing immortalized mouse mammary epithelial cell line (NMuMG-luc). From 845 genes that were highly expressed in human breast cancer cell lines, we focused on 205 genes encoding membrane proteins and/or kinases as that had the greater possibility of being oncogenes or drug targets. The 205 genes were divided into five subgroups, each containing 34-43 genes, and then introduced them into NMuMG-luc cells. These cells were subcutaneously injected into nude mice and monitored for tumor development by in vivo imaging. Tumors were observed in three subgroups. Using DNA microarray analyses and individual tumorigenic assays, we found that three genes, ADORA2B, PRKACB and LPAR3, were tumorigenic. ADORA2B and LPAR3 encode G-protein-coupled receptors and PRKACB encodes a protein kinase A catalytic subunit. Cells overexpressing ADORA2B, LPAR3 or PRKACB did not show transforming phenotypes in vitro, suggesting that transformation by these genes requires in vivo microenvironments. In addition, several clinical data sets, including one for breast cancer, showed that the expression of these genes correlated with lower overall survival rate.

  17. Identifying Communication Barriers to Colorectal Cancer Screening Adherence among Appalachian Kentuckians.

    PubMed

    Bachman, Audrey Smith; Cohen, Elisia L; Collins, Tom; Hatcher, Jennifer; Crosby, Richard; Vanderpool, Robin C

    2017-08-18

    Utilizing data from 40 in-depth interviews, this article identifies both barriers and facilitators to colorectal screening guideline adherence among Appalachian Kentucky adults recruited through a community-based research network. Key findings identify (a) varying levels of knowledge about screening guidelines, (b) reticence to engage in screening processes, and (c) nuanced communication with healthcare providers and family members regarding screening adherence. What participants knew about the screening process was often derived from personal stories or recalled stories from family members about their screening experiences. Reticence to engage in screening processes reflected reports of cumbersome preparation, privacy issues, embarrassment, medical mistrust, fear of receiving a cancer diagnosis, and lack of symptoms. Participants cited many ways to enhance patient-centered communication, and the findings from this study have implications for health communication message design and communication strategies for healthcare practices in Appalachian Kentucky clinics.

  18. Screening methods to identify TALEN-mediated knockout mice.

    PubMed

    Nakagawa, Yoshiko; Yamamoto, Takashi; Suzuki, Ken-Ichi; Araki, Kimi; Takeda, Naoki; Ohmuraya, Masaki; Sakuma, Tetsushi

    2014-01-01

    Genome editing with site-specific nucleases, such as zinc-finger nucleases or transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases, such as the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, is becoming the new standard for targeted genome modification in various organisms. Application of these techniques to the manufacture of knockout mice would be greatly aided by simple and easy methods for genotyping of mutant and wild-type pups among litters. However, there are no detailed or comparative reports concerning the identification of mutant mice generated using genome editing technologies. Here, we genotyped TALEN-derived enhanced green fluorescent protein (eGFP) knockout mice using a combination of approaches, including fluorescence observation, heteroduplex mobility assay, restriction fragment length polymorphism analysis and DNA sequencing. The detection sensitivities for TALEN-induced mutations differed among these methods, and we therefore concluded that combinatorial testing is necessary for the screening and determination of mutant genotypes. Since the analytical methods tested can be carried out without specialized equipment, costly reagents and/or sophisticated protocols, our report should be of interest to a broad range of researchers who are considering the application of genome editing technologies in various organisms.

  19. A Focused Screen Identifies Antifolates with Activity on Mycobacterium tuberculosis

    PubMed Central

    Kumar, Anuradha; Colmenarejo, Gonzalo; Pérez, Esther; Gonzalez, Ruben R.; Torres, Pedro; Calvo, David; Gómez, Ruben M.; Ortega, Fátima; Jiménez, Elena; Gabarro, Raquel C.; Rullás, Joaquín; Ballell, Lluis

    2015-01-01

    Antifolates are widely used to treat several diseases but are not currently used in the first-line treatment of tuberculosis, despite evidence that some of these molecules can target Mycobacterium tuberculosis (Mtb) bacilli in vitro. To identify new antifolate candidates for animal-model efficacy studies of tuberculosis, we paired knowledge and tools developed in academia with the infrastructure and chemistry resources of a large pharmaceutical company. Together we curated a focused library of 2508 potential antifolates, which were then tested for activity against live Mtb. We identified 210 primary hits, confirmed the on-target activity of potent compounds, and now report the identification and characterization of 5 hit compounds, representative of 5 different chemical scaffolds. These antifolates have potent activity against Mtb and represent good starting points for improvement that could lead to in vivo efficacy studies. PMID:26771003

  20. Identifying Nursing Interventions in a Cancer Screening Program Using Nursing Interventions Classification Taxonomy.

    PubMed

    Benito, Llucia; Lluch, María Teresa; Falcó, Anna Marta; García, Montse; Puig, Montse

    2017-04-01

    This study aimed to investigate which Nursing Interventions Classification (NIC) labels correspond to specific nursing interventions provided during cancer screening to establish a nursing documentation system. This descriptive study was conducted to identify and classify the interventions that cancer screening nurses perform based on an initial list. The initial list was grouped into 15 interventions that corresponded to four domains and eight classes. The study found expert consensus regarding the duties of cancer screening nurses and identified 15 interventions that should be implemented in clinical practice for cancer screening care, according to the NIC taxonomy. This study is the first step in developing indicators to assess nursing performance in cancer screening, and it helps to establish the core competency requirements for cancer screening nurses. © 2015 NANDA International, Inc.

  1. The Slingerland Screening Tests for Identifying Children with Specific Language Disability: Screening for Learning Disabilities in First Grade.

    ERIC Educational Resources Information Center

    Dinero, Thomas E.; And Others

    1979-01-01

    The responses on the Slingerland Screening Tests for identifying children with specific learning disabilities of 29 learning disabled and 11 nondisabled children in Grade 1 distinguished the two groups, except for copying (near vision). Copying (far vision) and auditory, visual, and kinesthetic perception and discrimination together were the…

  2. Novel screening cascade identifies MKK4 as key kinase regulating Tau phosphorylation at Ser422.

    PubMed

    Grueninger, Fiona; Bohrmann, Bernd; Christensen, Klaus; Graf, Martin; Roth, Doris; Czech, Christian

    2011-11-01

    Phosphorylation of Tau at serine 422 promotes Tau aggregation. The kinase that is responsible for this key phosphorylation event has so far not been identified but could be a potential drug target for Alzheimer's disease. We describe here an assay strategy to identify this kinase. Using a combination of screening a library of 65'000 kinase inhibitors and in vitro inhibitor target profiling of the screening hits using the Ambit kinase platform, MKK4 was identified as playing a key role in Tau-S422 phosphorylation in human neuroblastoma cells.

  3. High Throughput Screening Identifies Novel Lead Compounds with Activity against Larval, Juvenile and Adult Schistosoma mansoni

    PubMed Central

    Gardner, J. Mark F.; Bell, Andrew S.; Parkinson, Tanya; Bickle, Quentin

    2016-01-01

    An estimated 600 million people are affected by the helminth disease schistosomiasis caused by parasites of the genus Schistosoma. There is currently only one drug recommended for treating schistosomiasis, praziquantel (PZQ), which is effective against adult worms but not against the juvenile stage. In an attempt to identify improved drugs for treating the disease, we have carried out high throughput screening of a number of small molecule libraries with the aim of identifying lead compounds with balanced activity against all life stages of Schistosoma. A total of almost 300,000 compounds were screened using a high throughput assay based on motility of worm larvae and image analysis of assay plates. Hits were screened against juvenile and adult worms to identify broadly active compounds and against a mammalian cell line to assess cytotoxicity. A number of compounds were identified as promising leads for further chemical optimization. PMID:27128493

  4. Identifying Breast Tumor Suppressors Using in Vitro and in Vivo RNAi Screens

    DTIC Science & Technology

    2011-10-01

    identification of novel genes involved in the initiation and development of tumors are critical. During this research period we conducted the first whole human ... genome in vivo RNA interference screen to identify functionally important tumor suppressor genes. Using our novel approach, we identified previously

  5. A modified reverse one-hybrid screen identifies transcriptional activation in Phyochrome-Interacting Factor 3

    USDA-ARS?s Scientific Manuscript database

    Transcriptional activation domains (TAD) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput...

  6. Utility of a focused vancomycin-resistant enterococci screening protocol to identify colonization in hospitalized children.

    PubMed

    Weddle, Gina; Jackson, Mary Anne; Selvarangan, Rangaraj

    2012-11-01

    Screening for vancomycin-resistant enterococci (VRE) is controversial, and disagreement exists on policy implementation. This study investigated the likelihood of a positive test using 1, 2, or 3 rectal screenings for VRE colonization. In this descriptive study of positive VRE screening cultures, a total of 1211 VRE screens identified 41 positive results. The mean age of these positive patients was 5.7 years. Thirty-nine of the 41 had a chronic illness, and only 2 were healthy. Diagnoses included pulmonary disease in 11 patients and chronic gastrointestinal abnormality in 7. Six patients had been born preterm, and 12 had been treated in a neonatal intensive care unit within the previous 6 months. Thirty-six of the 41 positive results were identified on the first screen. The likelihood of subsequently having a positive screen after a negative screen was 0.43% (95% confidence interval, 0.15%-1.02%). The cost of cultures plus isolation was $50,000 for the study period. Our data show that the likelihood of detecting a positive VRE culture after an initial negative was low, particularly in otherwise healthy children.

  7. Successful outcomes of older adolescents and adults with profound biotinidase deficiency identified by newborn screening.

    PubMed

    Wolf, Barry

    2017-04-01

    We began screening newborns for biotinidase deficiency disorder in 1984, and now all states in the United States and many countries perform this screening. The purpose of this study was to determine the outcomes of older adolescent and adult individuals with the disorder identified by newborn screening. We located and surveyed, by questionnaire and telephone interviews, 44 individuals with profound biotinidase deficiency identified by newborn screening with a mean age of 23.1 years. All individuals had successfully completed high school, and many were attending or had completed college or graduate school. Compliance in using biotin has been excellent. Several individuals developed a variety of symptoms when they discontinued biotin for days or weeks. These features readily resolved when biotin was resumed. In addition, five treated women had nine uneventful pregnancies and deliveries. Newborn screening for profound biotinidase deficiency and early treatment with biotin result in excellent outcomes for older adolescents and adults with the disorder. In addition, mothers with profound biotinidase deficiency who were treated with biotin had pregnancies with good outcomes. These outcome results indicate that newborn screening for biotinidase deficiency is one of the most successful newborn screening programs.Genet Med 19 4, 396-402.

  8. Modifying culture conditions in chemical library screening identifies alternative inhibitors of mycobacteria.

    PubMed

    Miller, Christopher H; Nisa, Shahista; Dempsey, Sandi; Jack, Cameron; O'Toole, Ronan

    2009-12-01

    In this study, application of a dual absorbance/fluorescence assay to a chemical library screen identified several previously unknown inhibitors of mycobacteria. In addition, growth conditions had a significant effect on the activity profile of the library. Some inhibitors such as Se-methylselenocysteine were detected only when screening was performed under nutrient-limited culture conditions as opposed to nutrient-rich culture conditions. We propose that multiple culture condition library screening is required for complete inhibitory profiling and for maximal antimycobacterial compound detection.

  9. Screening strategies to identify new chemical diversity for drug development to treat kinetoplastid infections.

    PubMed

    Don, Rob; Ioset, Jean-Robert

    2014-01-01

    The Drugs for Neglected Diseases initiative (DNDi) has defined and implemented an early discovery strategy over the last few years, in fitting with its virtual R&D business model. This strategy relies on a medium- to high-throughput phenotypic assay platform to expedite the screening of compound libraries accessed through its collaborations with partners from the pharmaceutical industry. We review the pragmatic approaches used to select compound libraries for screening against kinetoplastids, taking into account screening capacity. The advantages, limitations and current achievements in identifying new quality series for further development into preclinical candidates are critically discussed, together with attractive new approaches currently under investigation.

  10. Iterative Focused Screening with Biological Fingerprints Identifies Selective Asc-1 Inhibitors Distinct from Traditional High Throughput Screening.

    PubMed

    Kutchukian, Peter S; Warren, Lee; Magliaro, Brian C; Amoss, Adam; Cassaday, Jason A; O'Donnell, Gregory; Squadroni, Brian; Zuck, Paul; Pascarella, Danette; Culberson, J Chris; Cooke, Andrew J; Hurzy, Danielle; Schlegel, Kelly-Ann Sondra; Thomson, Fiona; Johnson, Eric N; Uebele, Victor N; Hermes, Jeffrey D; Parmentier-Batteur, Sophie; Finley, Michael

    2017-02-17

    N-methyl-d-aspartate receptors (NMDARs) mediate glutamatergic signaling that is critical to cognitive processes in the central nervous system, and NMDAR hypofunction is thought to contribute to cognitive impairment observed in both schizophrenia and Alzheimer's disease. One approach to enhance the function of NMDAR is to increase the concentration of an NMDAR coagonist, such as glycine or d-serine, in the synaptic cleft. Inhibition of alanine-serine-cysteine transporter-1 (Asc-1), the primary transporter of d-serine, is attractive because the transporter is localized to neurons in brain regions critical to cognitive function, including the hippocampus and cortical layers III and IV, and is colocalized with d-serine and NMDARs. To identify novel Asc-1 inhibitors, two different screening approaches were performed with whole-cell amino acid uptake in heterologous cells stably expressing human Asc-1: (1) a high-throughput screen (HTS) of 3 M compounds measuring (35)S l-cysteine uptake into cells attached to scintillation proximity assay beads in a 1536 well format and (2) an iterative focused screen (IFS) of a 45 000 compound diversity set using a (3)H d-serine uptake assay with a liquid scintillation plate reader in a 384 well format. Critically important for both screening approaches was the implementation of counter screens to remove nonspecific inhibitors of radioactive amino acid uptake. Furthermore, a 15 000 compound expansion step incorporating both on- and off-target data into chemical and biological fingerprint-based models for selection of additional hits enabled the identification of novel Asc-1-selective chemical matter from the IFS that was not identified in the full-collection HTS.

  11. Enhancing depression screening to identify college students at risk for persistent depressive symptoms.

    PubMed

    Hill, Ryan M; Yaroslavsky, Ilya; Pettit, Jeremy W

    2015-03-15

    Depressive symptoms in college students are prevalent and are associated with considerable academic impairment. Many universities have implemented depressive symptom screening programs and the number of students identified as in need of services following screening greatly exceeds available mental health resources. The present study sought to refine depressive symptom screening programs by identifying predictors of a persistent course of depressive symptoms and developing cut-scores for accurately identifying students who will experience a persistent symptom course. Students (n=262) who reported elevated depressive symptoms both an initial screening and baseline assessment (n=150) were invited to participate in telephone-based follow-up assessments 4, 8, and 12 months post-baseline. Two depressive symptom courses were identified: a persistently elevated depressive symptoms course and a decreasing depressive symptoms course. Baseline social disconnection and negative feedback-seeking both significantly predicted membership in the persistently elevated depressive symptoms course. Cut-scores that robustly discriminated between the two symptom courses were identified. The present sample was predominantly female and Hispanic; the four-month spacing of assessments may have resulted in a failure to identify individuals who experience brief, yet impairing, recurrent depressive episodes. These findings can inform approaches to identifying college students most in need of mental health services for depressive symptoms based on the presence of social disconnection and/or negative feedback-seeking. Screening cut-points on social disconnection and negative feedback-seeking measures can reduce the number of cases identified as needing mental health services while retaining the majority of cases who will experience a persistent depressive symptom course. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. High-throughput drug library screening identifies colchicine as a thyroid cancer inhibitor

    PubMed Central

    Zhang, Le; Yang, Zhaoying; Granieri, Letizia; Pasculescu, Adrian; Datti, Alessandro; Asa, Sylvia L.; Xu, Zheli; Ezzat, Shereen

    2016-01-01

    We employed a high-throughput drug library screening platform to identify novel agents affecting thyroid cancer cells. We used human thyroid cancer cell lines to screen a collection of approximately 5200 small molecules with biological and/or pharmacologial properties. Parallel primary screens yielded a number of hits differentially active between thyroid and melanoma cells. Amongst compounds specifically targeting thyroid cancer cells, colchicine emerged as an effective candidate. Colchicine inhibited cell growth which correlated with G2 cell cycle arrest and apoptosis. These effects were hampered through inhibition of MEK1/2 and JNK. In contrast, inhibition of p38-MAPK had little effect, and AKT had no impact on colchicine action. Systemic colchicine inhibited thyroid cancer progression in xenografted mice. These findings demonstrate that our screening platform is an effective vehicle for drug reposition and show that colchicine warrants further attention in well-defined clinical niches such as thyroid cancer. PMID:26942566

  13. High-throughput drug library screening identifies colchicine as a thyroid cancer inhibitor.

    PubMed

    Zhang, Le; Yang, Zhaoying; Granieri, Letizia; Pasculescu, Adrian; Datti, Alessandro; Asa, Sylvia L; Xu, Zheli; Ezzat, Shereen

    2016-04-12

    We employed a high-throughput drug library screening platform to identify novel agents affecting thyroid cancer cells. We used human thyroid cancer cell lines to screen a collection of approximately 5200 small molecules with biological and/or pharmacologial properties. Parallel primary screens yielded a number of hits differentially active between thyroid and melanoma cells. Amongst compounds specifically targeting thyroid cancer cells, colchicine emerged as an effective candidate. Colchicine inhibited cell growth which correlated with G2 cell cycle arrest and apoptosis. These effects were hampered through inhibition of MEK1/2 and JNK. In contrast, inhibition of p38-MAPK had little effect, and AKT had no impact on colchicine action. Systemic colchicine inhibited thyroid cancer progression in xenografted mice. These findings demonstrate that our screening platform is an effective vehicle for drug reposition and show that colchicine warrants further attention in well-defined clinical niches such as thyroid cancer.

  14. Small-molecule screen in adult Drosophila identifies VMAT as a regulator of sleep.

    PubMed

    Nall, Aleksandra H; Sehgal, Amita

    2013-05-08

    Sleep is an important physiological state, but its function and regulation remain elusive. In Drosophila melanogaster, a useful model organism for studying sleep, forward genetic screens have identified important sleep-modulating genes and pathways; however, the results of such screens may be limited by developmental abnormalities or lethality associated with mutation of certain genes. To circumvent these limitations, we used a small-molecule screen to identify sleep-modulating genes and pathways. We administered 1280 pharmacologically active small molecules to adult flies and monitored their sleep. We found that administration of reserpine, a small-molecule inhibitor of the vesicular monoamine transporter (VMAT) that repackages monoamines into presynaptic vesicles, resulted in an increase in sleep. Supporting the idea that VMAT is the sleep-relevant target of reserpine, we found that VMAT-null mutants have an increased sleep phenotype, as well as an increased arousal threshold and resistance to the effects of reserpine. However, although the VMAT mutants are consistently resistant to reserpine, other aspects of their sleep phenotype are dependent on genetic background. These findings indicate that small-molecule screens can be used effectively to identify sleep-modulating genes whose phenotypes may be suppressed in traditional genetic screens. Mutations affecting single monoamine pathways did not affect reserpine sensitivity, suggesting that effects of VMAT/reserpine on sleep are mediated by multiple monoamines. Overall, we identify VMAT as an important regulator of sleep in Drosophila and demonstrate that small-molecule screens provide an effective approach to identify genes and pathways that impact adult Drosophila behavior.

  15. Cost-effectiveness of screening strategies for identifying pediatric diabetes mellitus and dysglycemia.

    PubMed

    Wu, En-Ling; Kazzi, Nayla G; Lee, Joyce M

    2013-01-01

    To conduct a cost-effectiveness analysis of screening strategies for identifying children with type 2 diabetes mellitus and dysglycemia (prediabetes/diabetes). Cost simulation study. A one-time US screening program. A total of 2.5 million children aged 10 to 17 years. Screening strategies for identifying diabetes and dysglycemia. Effectiveness (proportion of cases identified), total costs (direct and indirect), and efficiency (cost per case identified) of each screening strategy based on test performance data from a pediatric cohort and cost data from Medicare and the US Bureau of Labor Statistics. In the base-case model, 500 and 400 000 US adolescents had diabetes and dysglycemia, respectively. For diabetes, the cost per case was extremely high ($312 000-$831 000 per case identified) because of the low prevalence of disease. For dysglycemia, the cost per case was in a more reasonable range. For dysglycemia, preferred strategies were the 2-hour oral glucose tolerance test (100% effectiveness; $390 per case), 1-hour glucose challenge test (63% effectiveness; $571), random glucose test (55% effectiveness; $498), or a hemoglobin A1c threshold of 5.5% (45% effectiveness; $763). Hemoglobin A1c thresholds of 5.7% and 6.5% were the least effective and least efficient (ranges, 7%-32% and $938-$3370) of all strategies evaluated. Sensitivity analyses for diabetes revealed that disease prevalence was a major driver of cost-effectiveness. Sensitivity analyses for dysglycemia did not lead to appreciable changes in overall rankings among tests. For diabetes, the cost per case is extremely high because of the low prevalence of the disease in the pediatric population. Screening for diabetes could become more cost-effective if dysglycemia is explicitly considered as a screening outcome.

  16. Cost-effectiveness of Screening Strategies for Identifying Pediatric Diabetes Mellitus and Dysglycemia

    PubMed Central

    Wu, En-Ling; Kazzi, Nayla G.; Lee, Joyce M.

    2013-01-01

    Objective To conduct a cost-effectiveness analysis of screening strategies for identifying children with type 2 diabetes mellitus and dysglycemia (prediabetes/diabetes). Design Cost simulation study. Setting A one-time US screening program. Study Participants A total of 2.5 million children aged 10 to 17 years. Intervention Screening strategies for identifying diabetes and dysglycemia. Main Outcome Measures Effectiveness (proportion of cases identified), total costs (direct and indirect), and efficiency (cost per case identified) of each screening strategy based on test performance data from a pediatric cohort and cost data from Medicare and the US Bureau of Labor Statistics. Results In the base-case model, 500 and 400 000 US adolescents had diabetes and dysglycemia, respectively. For diabetes, the cost per case was extremely high ($312 000–$831 000 per case identified) because of the low prevalence of disease. For dysglycemia, the cost per case was in a more reasonable range. For dysglycemia, preferred strategies were the 2-hour oral glucose tolerance test (100% effectiveness; $390 per case), 1-hour glucose challenge test (63% effectiveness; $571), random glucose test (55% effectiveness; $498), or a hemoglobin A1c threshold of 5.5% (45% effectiveness; $763). Hemoglobin A1c thresholds of 5.7% and 6.5% were the least effective and least efficient (ranges, 7%–32% and $938– $3370) of all strategies evaluated. Sensitivity analyses for diabetes revealed that disease prevalence was a major driver of cost-effectiveness. Sensitivity analyses for dysglycemia did not lead to appreciable changes in overall rankings among tests. Conclusions For diabetes, the cost per case is extremely high because of the low prevalence of the disease in the pediatric population. Screening for diabetes could become more cost-effective if dysglycemia is explicitly considered as a screening outcome. PMID:23403824

  17. Comparison of traditional trigger tool to data warehouse based screening for identifying hospital adverse events.

    PubMed

    O'Leary, Kevin J; Devisetty, Vikram K; Patel, Amitkumar R; Malkenson, David; Sama, Pradeep; Thompson, William K; Landler, Matthew P; Barnard, Cynthia; Williams, Mark V

    2013-02-01

    Research supports medical record review using screening triggers as the optimal method to detect hospital adverse events (AE), yet the method is labour-intensive. This study compared a traditional trigger tool with an enterprise data warehouse (EDW) based screening method to detect AEs. We created 51 automated queries based on 33 traditional triggers from prior research, and then applied them to 250 randomly selected medical patients hospitalised between 1 September 2009 and 31 August 2010. Two physicians each abstracted records from half the patients using a traditional trigger tool and then performed targeted abstractions for patients with positive EDW queries in the complementary half of the sample. A third physician confirmed presence of AEs and assessed preventability and severity. Traditional trigger tool and EDW based screening identified 54 (22%) and 53 (21%) patients with one or more AE. Overall, 140 (56%) patients had one or more positive EDW screens (total 366 positive screens). Of the 137 AEs detected by at least one method, 86 (63%) were detected by a traditional trigger tool, 97 (71%) by EDW based screening and 46 (34%) by both methods. Of the 11 total preventable AEs, 6 (55%) were detected by traditional trigger tool, 7 (64%) by EDW based screening and 2 (18%) by both methods. Of the 43 total serious AEs, 28 (65%) were detected by traditional trigger tool, 29 (67%) by EDW based screening and 14 (33%) by both. We found relatively poor agreement between traditional trigger tool and EDW based screening with only approximately a third of all AEs detected by both methods. A combination of complementary methods is the optimal approach to detecting AEs among hospitalised patients.

  18. Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method

    PubMed Central

    Fischbach, Jens; Loh, Qiuting; Bier, Frank F.; Lim, Theam Soon; Frohme, Marcus; Glökler, Jörn

    2017-01-01

    We identified Alizarin Red S and other well known fluorescent dyes useful for the online detection of pyrophosphate in enzymatic assays, including the loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays. An iterative screening was used for a selected set of compounds to first secure enzyme compatibility, evaluate inorganic pyrophosphate sensitivity in the presence of manganese as quencher and optimize conditions for an online detection. Of the selected dyes, the inexpensive alizarin red S was found to selectively detect pyrophosphate under LAMP and PCR conditions and is superior with respect to its defined red-shifted spectrum, long shelf life and low toxicity. In addition, the newly identified properties may also be useful in other enzymatic assays which do not generate nucleic acids but are based on inorganic pyrophosphate. Finally, we propose that our screening method may provide a blueprint for rapid screening of compounds for detecting inorganic pyrophosphate. PMID:28338022

  19. A multistep screening method to identify genes using evolutionary transcriptome of plants.

    PubMed

    Kim, Chang-Kug; Lim, Hye-Min; Na, Jong-Kuk; Choi, Ji-Weon; Sohn, Seong-Han; Park, Soo-Chul; Kim, Young-Hwan; Kim, Yong-Kab; Kim, Dool-Yi

    2014-01-01

    We introduced a multistep screening method to identify the genes in plants using microarrays and ribonucleic acid (RNA)-seq transcriptome data. Our method describes the process for identifying genes using the salt-tolerance response pathways of the potato (Solanum tuberosum) plant. Gene expression was analyzed using microarrays and RNA-seq experiments that examined three potato lines (high, intermediate, and low salt tolerance) under conditions of salt stress. We screened the orthologous genes and pathway genes involved in salinity-related biosynthetic pathways, and identified nine potato genes that were candidates for salinity-tolerance pathways. The nine genes were selected to characterize their phylogenetic reconstruction with homologous genes of Arabidopsis thaliana, and a Circos diagram was generated to understand the relationships among the selected genes. The involvement of the selected genes in salt-tolerance pathways was verified by reverse transcription polymerase chain reaction analysis. One candidate potato gene was selected for physiological validation by generating dehydration-responsive element-binding 1 (DREB1)-overexpressing transgenic potato plants. The DREB1 overexpression lines exhibited increased salt tolerance and plant growth when compared to that of the control. Although the nine genes identified by our multistep screening method require further characterization and validation, this study demonstrates the power of our screening strategy after the initial identification of genes using microarrays and RNA-seq experiments.

  20. Quick Screen for Voice and Supplementary Documents for Identifying Pediatric Voice Disorders

    ERIC Educational Resources Information Center

    Lee, Linda; Stemple, Joseph C.; Glaze, Leslie; Kelchner, Lisa N.

    2004-01-01

    Three documents are provided to help the speech-language pathologist (SLP) identify children with voice disorders and educate family members. The first is a quickly administered screening test that covers multiple aspects of voice, respiration, and resonance. It was tested on 3,000 children in kindergarten and first and fifth grades, and on 47…

  1. A library screening approach identifies naturally occurring RNA sequences for a G-quadruplex binding ligand.

    PubMed

    Mirihana Arachchilage, Gayan; Morris, Mark J; Basu, Soumitra

    2014-02-07

    An RNA G-quadruplex library was synthesised and screened against kanamycin A as the ligand. Naturally occurring G-quadruplex forming sequences that differentially bind to kanamycin A were identified and characterized. This provides a simple and effective strategy for identification of potential intracellular G-quadruplex targets for a ligand.

  2. Rigorous Screening Technology for Identifying Suitable CO2 Storage Sites II

    SciTech Connect

    George J. Koperna Jr.; Vello A. Kuuskraa; David E. Riestenberg; Aiysha Sultana; Tyler Van Leeuwen

    2009-06-01

    This report serves as the final technical report and users manual for the 'Rigorous Screening Technology for Identifying Suitable CO2 Storage Sites II SBIR project. Advanced Resources International has developed a screening tool by which users can technically screen, assess the storage capacity and quantify the costs of CO2 storage in four types of CO2 storage reservoirs. These include CO2-enhanced oil recovery reservoirs, depleted oil and gas fields (non-enhanced oil recovery candidates), deep coal seems that are amenable to CO2-enhanced methane recovery, and saline reservoirs. The screening function assessed whether the reservoir could likely serve as a safe, long-term CO2 storage reservoir. The storage capacity assessment uses rigorous reservoir simulation models to determine the timing, ultimate storage capacity, and potential for enhanced hydrocarbon recovery. Finally, the economic assessment function determines both the field-level and pipeline (transportation) costs for CO2 sequestration in a given reservoir. The screening tool has been peer reviewed at an Electrical Power Research Institute (EPRI) technical meeting in March 2009. A number of useful observations and recommendations emerged from the Workshop on the costs of CO2 transport and storage that could be readily incorporated into a commercial version of the Screening Tool in a Phase III SBIR.

  3. Neurotrapping: Cellular Screens to Identify the Neural Substrates of Behavior in Drosophila

    PubMed Central

    White, Benjamin H.; Peabody, Nathan C.

    2009-01-01

    The availability of new tools for manipulating neuronal activity, coupled with the development of increasingly sophisticated techniques for targeting these tools to subsets of cells in living, behaving animals, is permitting neuroscientists to tease apart brain circuits by a method akin to classical mutagenesis. Just as mutagenesis can be used to introduce changes into an organism's DNA to identify the genes required for a given biological process, changes in activity can be introduced into the nervous system to identify the cells required for a given behavior. If the changes are introduced randomly, the cells can be identified without any prior knowledge of their properties. This strategy, which we refer to here as “neurotrapping,” has been implemented most effectively in Drosophila, where transgenes capable of either suppressing or stimulating neuronal activity can be reproducibly targeted to arbitrary subsets of neurons using so-called “enhancer-trap” techniques. By screening large numbers of enhancer-trap lines, experimenters have been able to identify groups of neurons which, when suppressed (or, in some cases, activated), alter a specific behavior. Parsing these groups of neurons to identify the minimal subset required for generating a behavior has proved difficult, but emerging tools that permit refined transgene targeting are increasing the resolution of the screening techniques. Some of the most recent neurotrapping screens have identified physiological substrates of behavior at the single neuron level. PMID:19949456

  4. Validation of a Medicare Claims-based Algorithm for Identifying Breast Cancers Detected at Screening Mammography

    PubMed Central

    Fenton, Joshua J.; Onega, Tracy; Zhu, Weiwei; Balch, Steven; Smith-Bindman, Rebecca; Henderson, Louise; Sprague, Brian L.; Kerlikowske, Karla; Hubbard, Rebecca A.

    2013-01-01

    Background The breast cancer detection rate is a benchmark measure of screening mammography quality, but its computation requires linkage of mammography interpretive performance information with cancer incidence data. A Medicare claims-based measure of detected breast cancers could simplify measurement of this benchmark and facilitate mammography quality assessment and research. Objectives To validate a claims-based algorithm that can identify with high positive predictive value (PPV) incident breast cancers that were detected at screening mammography. Research Design Development of a claims-derived algorithm using classification and regression tree analyses within a random half-sample of Medicare screening mammography claims followed by validation of the algoritm in the remaining half-sample using clinical data on mammography results and cancer incidence from the Breast Cancer Surveillance Consortium (BCSC). Subjects Female fee-for-service Medicare enrollees age 68 years and older who underwent screening mammography from 2001 to 2005 within BCSC registries in four states (CA, NC, NH, and VT), enabling linkage of claims and BCSC mammography data (N=233,044 mammograms obtained by 104,997 women). Measures Sensitivity, specificity, and PPV of algorithmic identification of incident breast cancers that were detected by radiologists relative to a reference standard based on BCSC mammography and cancer incidence data. Results An algorithm based on subsequent codes for breast cancer diagnoses and treatments and follow-up mammography identified incident screen-detected breast cancers with 92.9% sensitivity (95% CI: 91.0%-94.8%), 99.9% specificity (95% CI: 99.9%-99.9%), and a PPV of 88.0% (95% CI: 85.7%-90.4%). Conclusions A simple claims-based algorithm can accurately identify incident breast cancers detected at screening mammography among Medicare enrollees. The algorithm may enable mammography quality assessment using Medicare claims alone. PMID:23929404

  5. Yeast functional screen to identify genetic determinants capable of conferring abiotic stress tolerance in Jatropha curcas

    PubMed Central

    2010-01-01

    Background Environmentally inflicted stresses such as salinity and drought limit the plant productivity both in natural and agricultural system. Increasing emphasis has been directed to molecular breeding strategies to enhance the intrinsic ability of plant to survive stress conditions. Functional screens in microorganisms with heterologous genes are a rapid, effective and powerful tool to identify stress tolerant genes in plants. Jatropha curcas (Physic nut) has been identified as a potential source of biodiesel plant. In order to improve its productivity under stress conditions to benefit commercial plantations, we initiated prospecting of novel genes expressed during stress in J. curcas that can be utilized to enhance stress tolerance ability of plant. Results To identify genes expressed during salt tolerance, cDNA expression libraries were constructed from salt-stressed roots of J. curcas, regulated under the control of the yeast GAL1 system. Using a replica based screening, twenty thousand yeast transformants were screened to identify transformants expressing heterologous gene sequences from J. curcas with enhanced ability to tolerate stress. From the screen we obtained 32 full length genes from J. curcas [GenBank accession numbers FJ489601-FJ489611, FJ619041-FJ619057 and FJ623457-FJ623460] that can confer abiotic stress tolerance. As a part of this screen, we optimized conditions for salt stress in J. curcas, defined parameters for salt stress in yeast, as well as isolated three salt hypersensitive yeast strains shs-2, shs-6 and shs-8 generated through a process of random mutagenesis, and exhibited growth retardation beyond 750 mM NaCl. Further, we demonstrated complementation of the salt sensitive phenotypes in the shs mutants, and analyzed the expression patterns for selected J. curcas genes obtained from the screen in both leaf and root tissues after salt stress treatments. Conclusions The approach described in this report provides a rapid and universal

  6. Human Adenine Nucleotide Translocase (ANT) Modulators Identified by High-Throughput Screening of Transgenic Yeast.

    PubMed

    Zhang, Yujian; Tian, Defeng; Matsuyama, Hironori; Hamazaki, Takashi; Shiratsuchi, Takayuki; Terada, Naohiro; Hook, Derek J; Walters, Michael A; Georg, Gunda I; Hawkinson, Jon E

    2016-04-01

    Transport of ADP and ATP across mitochondria is one of the primary points of regulation to maintain cellular energy homeostasis. This process is mainly mediated by adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane. There are four human ANT isoforms, each having a unique tissue-specific expression pattern and biological function, highlighting their potential as drug targets for diverse clinical indications, including male contraception and cancer. In this study, we present a novel yeast-based high-throughput screening (HTS) strategy to identify compounds inhibiting the function of ANT. Yeast strains generated by deletion of endogenous proteins with ANT activity followed by insertion of individual human ANT isoforms are sensitive to cell-permeable ANT inhibitors, which reduce proliferation. Screening hits identified in the yeast proliferation assay were characterized in ADP/ATP exchange assays employing recombinant ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis as well as by oxygen consumption rate in mammalian cells. Using this approach, closantel and CD437 were identified as broad-spectrum ANT inhibitors, whereas leelamine was found to be a modulator of ANT function. This yeast "knock-out/knock-in" screening strategy is applicable to a broad range of essential molecular targets that are required for yeast survival. © 2016 Society for Laboratory Automation and Screening.

  7. Whole-organism screening for gluconeogenesis identifies activators of fasting metabolism

    PubMed Central

    Gut, Philipp; Baeza-Raja, Bernat; Andersson, Olov; Hasenkamp, Laura; Hsiao, Joseph; Hesselson, Daniel; Akassoglou, Katerina; Verdin, Eric; Hirschey, Matthew D.; Stainier, Didier Y.R.

    2012-01-01

    Improving the control of energy homeostasis can lower cardiovascular risk in metabolically compromised individuals. To identify new regulators of whole-body energy control, we conducted a high-throughput screen in transgenic reporter zebrafish for small molecules that modulate the expression of the fasting-inducible gluconeogenic gene pck1. We show that this in vivo strategy identified several drugs that impact gluconeogenesis in humans, as well as metabolically uncharacterized compounds. Most notably, we find that the Translocator Protein (TSPO) ligands PK 11195 and Ro5-4864 are glucose lowering agents despite a strong inductive effect on pck1 expression. We show that these drugs are activators of a fasting-like energy state, and importantly that they protect high-fat diet induced obese mice from hepatosteatosis and glucose intolerance, two pathological manifestations of metabolic dysregulation. Thus, using a whole-organism screening strategy, this study has identified new small molecule activators of fasting metabolism. PMID:23201900

  8. Chemical screen identifies FDA-approved drugs and target pathways that induce precocious pancreatic endocrine differentiation.

    PubMed

    Rovira, Meritxell; Huang, Wei; Yusuff, Shamila; Shim, Joong Sup; Ferrante, Anthony A; Liu, Jun O; Parsons, Michael J

    2011-11-29

    Pancreatic β-cells are an essential source of insulin and their destruction because of autoimmunity causes type I diabetes. We conducted a chemical screen to identify compounds that would induce the differentiation of insulin-producing β-cells in vivo. To do this screen, we brought together the use of transgenic zebrafish as a model of β-cell differentiation, a unique multiwell plate that allows easy visualization of lateral views of swimming larval fish and a library of clinical drugs. We identified six hits that can induce precocious differentiation of secondary islets in larval zebrafish. Three of these six hits were known drugs with a considerable background of published data on mechanism of action. Using pharmacological approaches, we have identified and characterized two unique pathways in β-cell differentiation in the zebrafish, including down-regulation of GTP production and retinoic acid biosynthesis.

  9. Chemical screen identifies FDA-approved drugs and target pathways that induce precocious pancreatic endocrine differentiation

    PubMed Central

    Rovira, Meritxell; Huang, Wei; Yusuff, Shamila; Shim, Joong Sup; Ferrante, Anthony A.; Liu, Jun O.; Parsons, Michael J.

    2011-01-01

    Pancreatic β-cells are an essential source of insulin and their destruction because of autoimmunity causes type I diabetes. We conducted a chemical screen to identify compounds that would induce the differentiation of insulin-producing β-cells in vivo. To do this screen, we brought together the use of transgenic zebrafish as a model of β-cell differentiation, a unique multiwell plate that allows easy visualization of lateral views of swimming larval fish and a library of clinical drugs. We identified six hits that can induce precocious differentiation of secondary islets in larval zebrafish. Three of these six hits were known drugs with a considerable background of published data on mechanism of action. Using pharmacological approaches, we have identified and characterized two unique pathways in β-cell differentiation in the zebrafish, including down-regulation of GTP production and retinoic acid biosynthesis. PMID:22084084

  10. High-throughput matrix screening identifies synergistic and antagonistic antimalarial drug combinations

    PubMed Central

    Mott, Bryan T.; Eastman, Richard T.; Guha, Rajarshi; Sherlach, Katy S.; Siriwardana, Amila; Shinn, Paul; McKnight, Crystal; Michael, Sam; Lacerda-Queiroz, Norinne; Patel, Paresma R.; Khine, Pwint; Sun, Hongmao; Kasbekar, Monica; Aghdam, Nima; Fontaine, Shaun D.; Liu, Dongbo; Mierzwa, Tim; Mathews-Griner, Lesley A.; Ferrer, Marc; Renslo, Adam R.; Inglese, James; Yuan, Jing; Roepe, Paul D.; Su, Xin-zhuan; Thomas, Craig J.

    2015-01-01

    Drug resistance in Plasmodium parasites is a constant threat. Novel therapeutics, especially new drug combinations, must be identified at a faster rate. In response to the urgent need for new antimalarial drug combinations we screened a large collection of approved and investigational drugs, tested 13,910 drug pairs, and identified many promising antimalarial drug combinations. The activity of known antimalarial drug regimens was confirmed and a myriad of new classes of positively interacting drug pairings were discovered. Network and clustering analyses reinforced established mechanistic relationships for known drug combinations and identified several novel mechanistic hypotheses. From eleven screens comprising >4,600 combinations per parasite strain (including duplicates) we further investigated interactions between approved antimalarials, calcium homeostasis modulators, and inhibitors of phosphatidylinositide 3-kinases (PI3K) and the mammalian target of rapamycin (mTOR). These studies highlight important targets and pathways and provide promising leads for clinically actionable antimalarial therapy. PMID:26403635

  11. Whole-organism screening for gluconeogenesis identifies activators of fasting metabolism.

    PubMed

    Gut, Philipp; Baeza-Raja, Bernat; Andersson, Olov; Hasenkamp, Laura; Hsiao, Joseph; Hesselson, Daniel; Akassoglou, Katerina; Verdin, Eric; Hirschey, Matthew D; Stainier, Didier Y R

    2013-02-01

    Improving the control of energy homeostasis can lower cardiovascular risk in metabolically compromised individuals. To identify new regulators of whole-body energy control, we conducted a high-throughput screen in transgenic reporter zebrafish for small molecules that modulate the expression of the fasting-inducible gluconeogenic gene pck1. We show that this in vivo strategy identified several drugs that affect gluconeogenesis in humans as well as metabolically uncharacterized compounds. Most notably, we find that the translocator protein ligands PK 11195 and Ro5-4864 are glucose-lowering agents despite a strong inductive effect on pck1 expression. We show that these drugs are activators of a fasting-like energy state and, notably, that they protect high-fat diet-induced obese mice from hepatosteatosis and glucose intolerance, two pathological manifestations of metabolic dysregulation. Thus, using a whole-organism screening strategy, this study has identified new small-molecule activators of fasting metabolism.

  12. A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs

    PubMed Central

    Karlas, Alexander; Berre, Stefano; Couderc, Thérèse; Varjak, Margus; Braun, Peter; Meyer, Michael; Gangneux, Nicolas; Karo-Astover, Liis; Weege, Friderike; Raftery, Martin; Schönrich, Günther; Klemm, Uwe; Wurzlbauer, Anne; Bracher, Franz; Merits, Andres; Meyer, Thomas F.; Lecuit, Marc

    2016-01-01

    Chikungunya virus (CHIKV) is a globally spreading alphavirus against which there is no commercially available vaccine or therapy. Here we use a genome-wide siRNA screen to identify 156 proviral and 41 antiviral host factors affecting CHIKV replication. We analyse the cellular pathways in which human proviral genes are involved and identify druggable targets. Twenty-one small-molecule inhibitors, some of which are FDA approved, targeting six proviral factors or pathways, have high antiviral activity in vitro, with low toxicity. Three identified inhibitors have prophylactic antiviral effects in mouse models of chikungunya infection. Two of them, the calmodulin inhibitor pimozide and the fatty acid synthesis inhibitor TOFA, have a therapeutic effect in vivo when combined. These results demonstrate the value of loss-of-function screening and pathway analysis for the rational identification of small molecules with therapeutic potential and pave the way for the development of new, host-directed, antiviral agents. PMID:27177310

  13. Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets.

    PubMed

    Eggert, Ulrike S; Kiger, Amy A; Richter, Constance; Perlman, Zachary E; Perrimon, Norbert; Mitchison, Timothy J; Field, Christine M

    2004-12-01

    Cytokinesis involves temporally and spatially coordinated action of the cell cycle and cytoskeletal and membrane systems to achieve separation of daughter cells. To dissect cytokinesis mechanisms it would be useful to have a complete catalog of the proteins involved, and small molecule tools for specifically inhibiting them with tight temporal control. Finding active small molecules by cell-based screening entails the difficult step of identifying their targets. We performed parallel chemical genetic and genome-wide RNA interference screens in Drosophila cells, identifying 50 small molecule inhibitors of cytokinesis and 214 genes important for cytokinesis, including a new protein in the Aurora B pathway (Borr). By comparing small molecule and RNAi phenotypes, we identified a small molecule that inhibits the Aurora B kinase pathway. Our protein list provides a starting point for systematic dissection of cytokinesis, a direction that will be greatly facilitated by also having diverse small molecule inhibitors, which we have identified. Dissection of the Aurora B pathway, where we found a new gene and a specific small molecule inhibitor, should benefit particularly. Our study shows that parallel RNA interference and small molecule screening is a generally useful approach to identifying active small molecules and their target pathways.

  14. Parallel Chemical Genetic and Genome-Wide RNAi Screens Identify Cytokinesis Inhibitors and Targets

    PubMed Central

    Kiger, Amy A; Richter, Constance; Perlman, Zachary E; Perrimon, Norbert; Mitchison, Timothy J; Field, Christine M

    2004-01-01

    Cytokinesis involves temporally and spatially coordinated action of the cell cycle and cytoskeletal and membrane systems to achieve separation of daughter cells. To dissect cytokinesis mechanisms it would be useful to have a complete catalog of the proteins involved, and small molecule tools for specifically inhibiting them with tight temporal control. Finding active small molecules by cell-based screening entails the difficult step of identifying their targets. We performed parallel chemical genetic and genome-wide RNA interference screens in Drosophila cells, identifying 50 small molecule inhibitors of cytokinesis and 214 genes important for cytokinesis, including a new protein in the Aurora B pathway (Borr). By comparing small molecule and RNAi phenotypes, we identified a small molecule that inhibits the Aurora B kinase pathway. Our protein list provides a starting point for systematic dissection of cytokinesis, a direction that will be greatly facilitated by also having diverse small molecule inhibitors, which we have identified. Dissection of the Aurora B pathway, where we found a new gene and a specific small molecule inhibitor, should benefit particularly. Our study shows that parallel RNA interference and small molecule screening is a generally useful approach to identifying active small molecules and their target pathways. PMID:15547975

  15. Diagnostic Value of Screening Instruments for Identifying Obstructive Sleep Apnea in Kidney Failure

    PubMed Central

    Nicholl, David D. M.; Ahmed, Sofia B.; Loewen, Andrea H. S.; Hemmelgarn, Brenda R.; Sola, Darlene Y.; Beecroft, Jaime M.; Turin, Tanvir C.; Hanly, Patrick J.

    2013-01-01

    Background: Patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD) have a high prevalence of obstructive sleep apnea (OSA) that can have significant clinical implications. An accurate clinical screening tool for OSA that identifies patients for further diagnostic testing would assist in the identification of this comorbidity. The Berlin Questionnaire (BQ), Adjusted Neck Circumference (ANC), and STOP-BANG questionnaire are 3 such instruments that have been validated in patients with normal kidney function. Objective: The objective of this study was to determine the validity of these screening instruments in patients with CKD and ESRD, using overnight cardiopulmonary monitoring to diagnose OSA. Methods: One hundred seventy-two patients were recruited from nephrology clinics and hemodialysis units (CKD: n = 109; ESRD: n = 63). All patients completed the BQ, ANC, STOP-BANG, and overnight cardiopulmonary monitoring to diagnose OSA (respiratory disturbance index [RDI] ≥ 15). Sensitivity, specificity, positive and negative predictive values, and accuracy were calculated for the BQ, ANC, and STOP-BANG. Results: Obstructive sleep apnea was present in 41 CKD patients (38%) and 32 ESRD patients (51%). All screening instruments had satisfactory sensitivity (56% to 94%) but poor specificity (29% to 77%) and low accuracy (51% to 69%) in both CKD and ESRD patients with RDI ≥ 15. Using an RDI ≥ 30 yielded similar results. Conclusions: Current screening questionnaires do not accurately identify patients at high risk for OSA or rule out the presence of OSA in patients with CKD and ESRD. Consequently, objective monitoring during sleep is required to reliably identify sleep apnea in these patient populations. Citation: Nicholl DDM; Ahmed SB; Loewen AHS; Hemmelgarn BR; Sola DY; Beecroft JM; Turin TC; Hanly PJ. Diagnostic value of screening instruments for identifying obstructive sleep apnea in kidney failure. J Clin Sleep Med 2013;9(1):31-38. PMID:23319902

  16. A small-molecule screening strategy to identify suppressors of statin myopathy.

    PubMed

    Wagner, Bridget K; Gilbert, Tamara J; Hanai, Jun-ichi; Imamura, Shintaro; Bodycombe, Nicole E; Bon, Robin S; Waldmann, Herbert; Clemons, Paul A; Sukhatme, Vikas P; Mootha, Vamsi K

    2011-09-16

    The reduction of plasma low-density lipoprotein levels by HMG-CoA reductase inhibitors, or statins, has had a revolutionary impact in medicine, but muscle-related side effects remain a dose-limiting toxicity in many patients. We describe a chemical epistasis approach that can be useful in refining the mechanism of statin muscle toxicity, as well as in screening for agents that suppress muscle toxicity while preserving the ability of statins to increase the expression of the low-density lipoprotein receptor. Using this approach, we identified one compound that attenuates the muscle side effects in both cellular and animal models of statin toxicity, likely by influencing Rab prenylation. Our proof-of-concept screen lays the foundation for truly high-throughput screens that could help lead to the development of clinically useful adjuvants that can one day be co-administered with statins.

  17. High prevalence of modifiable stroke risk factors identified in a pharmacy-based screening programme

    PubMed Central

    Sandhu, Roopinder K; Dolovich, Lisa; Deif, Bishoy; Barake, Walid; Agarwal, Gina; Grinvalds, Alex; Lim, Ting; Quinn, F Russell; Gladstone, David; Conen, David; Connolly, Stuart J; Healey, Jeff S

    2016-01-01

    Background Population-based screening for atrial fibrillation (AF) is a promising public health strategy to prevent stroke. However, none of the published reports have evaluated comprehensive screening for additional stroke risk factors such as hypertension and diabetes in a pharmacy setting. Methods The Program for the Identification of ‘Actionable’ Atrial Fibrillation in the Pharmacy Setting (PIAAF-Pharmacy) screened individuals aged ≥65 years, attending community pharmacies in Canada, who were not receiving oral anticoagulation (OAC). Participants were screened for AF using a hand-held ECG device, had blood pressure (BP) measured, and diabetes risk estimated using the Canadian Diabetes Risk Assessment Questionnaire (CANRISK) questionnaire. ‘Actionable’ AF was defined as unrecognised or undertreated AF. A 6-week follow-up visit with the family physician was suggested for participants with ‘actionable’ AF and a scheduled 3-month visit occurred at an AF clinic. Results During 6 months, 1145 participants were screened at 30 pharmacies. ‘Actionable’ AF was identified in 2.5% (95% CI 1.7 to 3.6; n=29); of these, 96% were newly diagnosed. Participants with ‘actionable AF’ had a mean age of 77.2±6.8 years, 58.6% were male and 93.1% had a CHA2DS2-VASc score ≥2. A BP>140/90 was found in 54.9% (616/1122) of participants and 44.4% (214/492) were found to be at high risk of diabetes. At 3 months, only 17% of participants were started on OAC, 50% had improved BP and 71% had confirmatory diabetes testing. Conclusions Integrated stroke screening identifies a high prevalence of individuals who could benefit from stroke prevention therapies but must be coupled with a defined care pathway. PMID:28123758

  18. High-Content Screening in Zebrafish Embryos Identifies Butafenacil as a Potent Inducer of Anemia

    PubMed Central

    Leet, Jessica K.; Lindberg, Casey D.; Bassett, Luke A.; Isales, Gregory M.; Yozzo, Krystle L.; Raftery, Tara D.; Volz, David C.

    2014-01-01

    Using transgenic zebrafish (fli1:egfp) that stably express enhanced green fluorescent protein (eGFP) within vascular endothelial cells, we recently developed and optimized a 384-well high-content screening (HCS) assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization. After ranking developmental toxicity data generated from the U.S. Environmental Protection Agency's (EPA's) zebrafish teratogenesis assay, we screened 26 of the most acutely toxic chemicals within EPA's ToxCast Phase-I library in concentration-response format (0.05–50 µM) using this HCS assay. Based on this screen, we identified butafenacil as a potent inducer of anemia, as exposure from 0.39 to 3.125 µM butafenacil completely abolished arterial circulation in the absence of effects on all other endpoints evaluated. Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPO) – an enzyme necessary for heme production in vertebrates. Using o-dianisidine staining, we then revealed that severe butafenacil-induced anemia in zebrafish was due to a complete loss of hemoglobin following exposure during early development. Therefore, six additional PPO inhibitors within the ToxCast Phase-I library were screened to determine whether anemia represents a common adverse outcome for these herbicides. Embryonic exposure to only one of these PPO inhibitors – flumioxazin – resulted in a similar phenotype as butafenacil, albeit not as severe as butafenacil. Overall, this study highlights the potential utility of this assay for (1) screening chemicals for cardiovascular toxicity and (2) prioritizing chemicals for future hypothesis

  19. High-content screening in zebrafish embryos identifies butafenacil as a potent inducer of anemia.

    PubMed

    Leet, Jessica K; Lindberg, Casey D; Bassett, Luke A; Isales, Gregory M; Yozzo, Krystle L; Raftery, Tara D; Volz, David C

    2014-01-01

    Using transgenic zebrafish (fli1:egfp) that stably express enhanced green fluorescent protein (eGFP) within vascular endothelial cells, we recently developed and optimized a 384-well high-content screening (HCS) assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization. After ranking developmental toxicity data generated from the U.S. Environmental Protection Agency's (EPA's) zebrafish teratogenesis assay, we screened 26 of the most acutely toxic chemicals within EPA's ToxCast Phase-I library in concentration-response format (0.05-50 µM) using this HCS assay. Based on this screen, we identified butafenacil as a potent inducer of anemia, as exposure from 0.39 to 3.125 µM butafenacil completely abolished arterial circulation in the absence of effects on all other endpoints evaluated. Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPO)--an enzyme necessary for heme production in vertebrates. Using o-dianisidine staining, we then revealed that severe butafenacil-induced anemia in zebrafish was due to a complete loss of hemoglobin following exposure during early development. Therefore, six additional PPO inhibitors within the ToxCast Phase-I library were screened to determine whether anemia represents a common adverse outcome for these herbicides. Embryonic exposure to only one of these PPO inhibitors--flumioxazin--resulted in a similar phenotype as butafenacil, albeit not as severe as butafenacil. Overall, this study highlights the potential utility of this assay for (1) screening chemicals for cardiovascular toxicity and (2) prioritizing chemicals for future hypothesis-driven and mechanism

  20. Identifying Regulators for EAG1 Channels with a Novel Electrophysiology and Tryptophan Fluorescence Based Screen

    PubMed Central

    Davies, Douglas R.; Stewart, Lance J.; Zagotta, William N.

    2010-01-01

    Background Ether-à-go-go (EAG) channels are expressed throughout the central nervous system and are also crucial regulators of cell cycle and tumor progression. The large intracellular amino- and carboxy- terminal domains of EAG1 each share similarity with known ligand binding motifs in other proteins, yet EAG1 channels have no known regulatory ligands. Methodology/Principal Findings Here we screened a library of small biologically relevant molecules against EAG1 channels with a novel two-pronged screen to identify channel regulators. In one arm of the screen we used electrophysiology to assess the functional effects of the library compounds on full-length EAG1 channels. In an orthogonal arm, we used tryptophan fluorescence to screen for binding of the library compounds to the isolated C-terminal region. Conclusions/Significance Several compounds from the flavonoid, indole and benzofuran chemical families emerged as binding partners and/or regulators of EAG1 channels. The two-prong screen can aid ligand and drug discovery for ligand-binding domains of other ion channels. PMID:20824064

  1. Characteristics of hypertensive children identified by primary care referral compared with school-based screening.

    PubMed

    Sorof, Jonathan M; Turner, Jennifer; Franco, Kathy; Portman, Ronald J

    2004-04-01

    To determine whether there are clinical differences between children referred for hypertension evaluation from a primary care practice and children with hypertension detected through school-based screening. Study design Referral patients (n=58) were compared with 44 screening patients with hypertension from school-based screening of 5102 students. All subjects underwent 24-hour ambulatory blood pressure (BP) monitoring. White coat hypertension was defined as 24-hour mean BP <95th percentile and BP load <25%. Referral subjects were more likely to be male and had higher body mass index than screening subjects but did not differ by age or ethnic distribution. Average clinic BP values tended to be higher among referral patients (140/79 vs 135/76 mm Hg, P=.07); however, the hypertension severity was closely matched when clinic BP was indexed to the subject-specific 95th percentile. Ambulatory mean BP, BP indices, and BP loads showed no differences by subject source for 24-hour, wake, or sleep periods. White coat hypertension prevalence did not differ between referral and screening subjects (28% vs 30%, P=.83). These findings suggest that hypertensive children identified by subspecialty referral are representative of the overall population of hypertensive children in the community, thereby supporting the generalizability of clinic-based research in pediatric hypertension.

  2. Newborn Screening Quality Assurance Program for CFTR Mutation Detection and Gene Sequencing to Identify Cystic Fibrosis

    PubMed Central

    Hendrix, Miyono M.; Foster, Stephanie L.; Cordovado, Suzanne K.

    2016-01-01

    All newborn screening laboratories in the United States and many worldwide screen for cystic fibrosis. Most laboratories use a second-tier genotyping assay to identify a panel of mutations in the CF transmembrane regulator (CFTR) gene. Centers for Disease Control and Prevention’s Newborn Screening Quality Assurance Program houses a dried blood spot repository of samples containing CFTR mutations to assist newborn screening laboratories and ensure high-quality mutation detection in a high-throughput environment. Recently, CFTR mutation detection has increased in complexity with expanded genotyping panels and gene sequencing. To accommodate the growing quality assurance needs, the repository samples were characterized with several multiplex genotyping methods, Sanger sequencing, and 3 next-generation sequencing assays using a high-throughput, low-concentration DNA extraction method. The samples performed well in all of the assays, providing newborn screening laboratories with a resource for complex CFTR mutation detection and next-generation sequencing as they transition to new methods. PMID:28261631

  3. Combinatorial drug screening identifies compensatory pathway interactions and adaptive resistance mechanisms

    PubMed Central

    Axelrod, Mark; Gordon, Vicki L.; Conaway, Mark; Tarcsafalvi, Adel; Neitzke, Daniel J.; Gioeli, Daniel; Weber, Michael J.

    2013-01-01

    Constitutively activated signaling molecules are often the primary drivers of malignancy, and are favored targets for therapeutic intervention. However, the effectiveness of targeted inhibition of cell signaling can be blunted by compensatory signaling which generates adaptive resistance mechanisms and reduces therapeutic responses. Therefore, it is important to identify and target these compensatory pathways with combinations of targeted agents to achieve durable clinical benefit. In this report, we demonstrate the use of high-throughput combinatorial drug screening as a discovery tool to identify compensatory pathways that generate resistance to the cytotoxic effects of targeted therapy. We screened 420 drug combinations in 14 different cell lines representing three cancer lineages, and assessed the ability of each combination to cause synergistic cytotoxicity. Drug substitution studies were used to validate the functionally important drug targets. Of the 84 combinations that caused robust synergy in multiple cell lines, none were synergistic in more than half of the lines tested, and we observed no pattern of lineage specificity in the observed synergies. This reflects the plasticity of cell signaling networks, even among cell lines of the same tissue of origin. Mechanistic analysis of one novel synergistic combination identified in the screen, the multi-kinase inhibitor Ro31-8220 and lapatinib, demonstrated compensatory crosstalk between the p70S6 kinase and EGF receptor pathways. In addition, we identified BAD as a node of convergence between these two pathways that may be playing a role in the enhanced apoptosis observed upon combination treatment. PMID:23599172

  4. Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast.

    PubMed

    Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L; Hallström, Björn M; Liu, Zihe; Petranovic, Dina; Uhlén, Mathias; Joensson, Haakan N; Andersson-Svahn, Helene; Nielsen, Jens

    2015-08-25

    There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.

  5. An in vitro screening cascade to identify neuroprotective antioxidants in ALS

    PubMed Central

    Barber, Siân C.; Higginbottom, Adrian; Mead, Richard J.; Barber, Stuart; Shaw, Pamela J.

    2009-01-01

    Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease, characterized by progressive dysfunction and death of motor neurons. Although evidence for oxidative stress in ALS pathogenesis is well described, antioxidants have generally shown poor efficacy in animal models and human clinical trials. We have developed an in vitro screening cascade to identify antioxidant molecules capable of rescuing NSC34 motor neuron cells expressing an ALS-associated mutation of superoxide dismutase 1. We have tested known antioxidants and screened a library of 2000 small molecules. The library screen identified 164 antioxidant molecules, which were refined to the 9 most promising molecules in subsequent experiments. Analysis of the in silico properties of hit compounds and a review of published literature on their in vivo effectiveness have enabled us to systematically identify molecules with antioxidant activity combined with chemical properties necessary to penetrate the central nervous system. The top-performing molecules identified include caffeic acid phenethyl ester, esculetin, and resveratrol. These compounds were tested for their ability to rescue primary motor neuron cultures after trophic factor withdrawal, and the mechanisms of action of their antioxidant effects were investigated. Subsequent in vivo studies can be targeted using molecules with the greatest probability of success. PMID:19439221

  6. Screening of Pharmacologically Active Small Molecule Compounds Identifies Antifungal Agents Against Candida Biofilms

    PubMed Central

    Watamoto, Takao; Egusa, Hiroshi; Sawase, Takashi; Yatani, Hirofumi

    2015-01-01

    Candida species have emerged as important and common opportunistic human pathogens, particularly in immunocompromised individuals. The current antifungal therapies either have toxic side effects or are insufficiently effect. The aim of this study is develop new small-molecule antifungal compounds by library screening methods using Candida albicans, and to evaluate their antifungal effects on Candida biofilms and cytotoxic effects on human cells. Wild-type C. albicans strain SC5314 was used in library screening. To identify antifungal compounds, we screened a small-molecule library of 1,280 pharmacologically active compounds (LOPAC1280TM) using an antifungal susceptibility test (AST). To investigate the antifungal effects of the hit compounds, ASTs were conducted using Candida strains in various growth modes, including biofilms. We tested the cytotoxicity of the hit compounds using human gingival fibroblast (hGF) cells to evaluate their clinical safety. Only 35 compounds were identified by screening, which inhibited the metabolic activity of C. albicans by >50%. Of these, 26 compounds had fungistatic effects and nine compounds had fungicidal effects on C. albicans. Five compounds, BAY11-7082, BAY11-7085, sanguinarine chloride hydrate, ellipticine and CV-3988, had strong fungicidal effects and could inhibit the metabolic activity of Candida biofilms. However, BAY11-7082, BAY11-7085, sanguinarine chloride hydrate and ellipticine were cytotoxic to hGF cells at low concentrations. CV-3988 showed no cytotoxicity at a fungicidal concentration. Four of the compounds identified, BAY11-7082, BAY11-7085, sanguinarine chloride hydrate and ellipticine, had toxic effects on Candida strains and hGF cells. In contrast, CV-3988 had fungicidal effects on Candida strains, but low cytotoxic effects on hGF cells. Therefore, this screening reveals agent, CV-3988 that was previously unknown to be antifungal agent, which could be a novel therapies for superficial mucosal candidiasis. PMID

  7. Viral load in children with congenital cytomegalovirus infection identified on newborn hearing screening.

    PubMed

    Kawada, Jun-ichi; Torii, Yuka; Kawano, Yoshihiko; Suzuki, Michio; Kamiya, Yasuko; Kotani, Tomomi; Kikkawa, Fumitaka; Kimura, Hiroshi; Ito, Yoshinori

    2015-04-01

    Congenital cytomegalovirus (CMV) infection is the most common non-genetic cause of sensorineural hearing loss (SNHL) in children. However, congenital SNHL without other clinical abnormalities is rarely diagnosed as CMV-related in early infancy. The aim of this study was to identify and treat patients with congenital CMV-related SNHL or CMV-related clinical abnormalities other than SNHL. The association between CMV load and SNHL was also evaluated. Newborns who had abnormal hearing screening results or other clinical abnormalities were screened for congenital CMV infection by PCR of saliva or urine specimens, and identified infected patients were treated with valganciclovir (VGCV) for 6 weeks. The CMV load of patients with or without SNHL was compared at regular intervals during as well as after VGCV treatment. Of 127 infants with abnormal hearing screening results, and 31 infants with other clinical abnormalities, CMV infection was identified in 6 and 3 infants, respectively. After VGCV treatment, 1 case had improved hearing but the other 5 SNHL cases had little or no improvement. Among these 9 patients with or without SNHL at 1 year of age, there was no significant difference in CMV blood or urine load at diagnosis, but both were significantly higher in patients with SNHL during VGCV treatment. Selective CMV screening of newborns having an abnormal hearing screening result would be a reasonable strategy for identification of symptomatic congenital CMV infection. Prolonged detection of CMV in blood could be a risk factor for SNHL. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Identifying Barriers to Delirium Screening and Prevention in the Pediatric ICU: Evaluation of PICU Staff Knowledge.

    PubMed

    Flaigle, Melanie Cooper; Ascenzi, Judy; Kudchadkar, Sapna R

    2016-01-01

    Delirium in the pediatric intensive care unit (PICU) setting is often unrecognized and undertreated. The importance of screening and identification of ICU delirium has been identified in both adult and pediatric literature. Delirium increases ICU morbidity, length of mechanical ventilation and length of stay. The objective of this study was to determine the current knowledge level about delirium and its risk factors among pediatric critical care nurses through a short questionnaire. We hypothesized that before a targeted educational intervention, PICU care providers do not have an adequate knowledge base for accurate screening and diagnosis of delirium in critically ill children. A 17 question online survey was given to all nurses in a tertiary 36-bed PICU to assess current knowledge about delirium in children. The response rate was 73% (105/143). When asked to identify the correct way to diagnose pediatric delirium, 11.4% of nurses surveyed (12/105) incorrectly believed that Glasgow Coma Score is the appropriate screening tool. A large proportion of respondents (40/105) believed that benzodiazepines are helpful in treatment of delirium. The results of the survey identified specific knowledge gaps about risk factors and treatment of pediatric delirium in the critically ill child. There is a critical need for education about pediatric delirium and its risk factors among PICU staff prior to unit-wide implementation of a delirium screening and prevention program, specifically with regards to screening methods and pharmacologic risk factors. These results are likely generalizable to all physicians, nurses and staff who care for critically ill children.

  9. Large-Scale RNA Interference Screening in Mammalian Cells Identifies Novel Regulators of Mutant Huntingtin Aggregation

    PubMed Central

    Tosaki, Asako; Bauer, Peter O.; Wada, Koji; Kurosawa, Masaru; Shimogori, Tomomi; Hattori, Nobutaka; Nukina, Nobuyuki

    2014-01-01

    In polyglutamine (polyQ) diseases including Huntington's disease (HD), mutant proteins containing expanded polyQ stretch form aggregates in neurons. Genetic or RNAi screenings in yeast, C. elegans or Drosophila have identified multiple genes modifying polyQ aggregation, a few of which are confirmed effective in mammals. However, the overall molecular mechanism underlying polyQ protein aggregation in mammalian cells still remains obscure. We here perform RNAi screening in mouse neuro2a cells to identify mammalian modifiers for aggregation of mutant huntingtin, a causative protein of HD. By systematic cell transfection and automated cell image analysis, we screen ∼12000 shRNA clones and identify 111 shRNAs that either suppress or enhance mutant huntingtin aggregation, without altering its gene expression. Classification of the shRNA-targets suggests that genes with various cellular functions such as gene transcription and protein phosphorylation are involved in modifying the aggregation. Subsequent analysis suggests that, in addition to the aggregation-modifiers sensitive to proteasome inhibition, some of them, such as a transcription factor Tcf20, and kinases Csnk1d and Pik3c2a, are insensitive to it. As for Tcf20, which contains polyQ stretches at N-terminus, its binding to mutant huntingtin aggregates is observed in neuro2a cells and in HD model mouse neurons. Notably, except Pik3c2a, the rest of the modifiers identified here are novel. Thus, our first large-scale RNAi screening in mammalian system identifies previously undescribed genetic players that regulate mutant huntingtin aggregation by several, possibly mammalian-specific mechanisms. PMID:24705917

  10. A High-Throughput Screen Identifies a New Natural Product with Broad-Spectrum Antibacterial Activity

    PubMed Central

    Ymele-Leki, Patrick; Cao, Shugeng; Sharp, Jared; Lambert, Kathleen G.; McAdam, Alexander J.; Husson, Robert N.; Tamayo, Giselle; Clardy, Jon; Watnick, Paula I.

    2012-01-01

    Due to the inexorable invasion of our hospitals and communities by drug-resistant bacteria, there is a pressing need for novel antibacterial agents. Here we report the development of a sensitive and robust but low-tech and inexpensive high-throughput metabolic screen for novel antibiotics. This screen is based on a colorimetric assay of pH that identifies inhibitors of bacterial sugar fermentation. After validation of the method, we screened over 39,000 crude extracts derived from organisms that grow in the diverse ecosystems of Costa Rica and identified 49 with reproducible antibacterial effects. An extract from an endophytic fungus was further characterized, and this led to the discovery of three novel natural products. One of these, which we named mirandamycin, has broad-spectrum antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae, methicillin-resistant Staphylococcus aureus, and Mycobacterium tuberculosis. This demonstrates the power of simple high throughput screens for rapid identification of new antibacterial agents from environmental samples. PMID:22359585

  11. Development and validation of a screening tool to identify eating disorders in female athletes.

    PubMed

    McNulty, K Y; Adams, C H; Anderson, J M; Affenito, S G

    2001-08-01

    To develop and validate a screening tool to identify eating pathology in women athletes. Three cross-sectional subject groups were established: college women athletes diagnosed with eating disorders; college women athletes without eating pathology; college women who did not participate in athletics but were diagnosed with an eating disorder. The Female Athlete Screening Tool (FAST), and 3 valid psychometric measures were administered to subjects in all groups. Internal reliability, discriminant and concurrent validity were established. Women college students between the ages of 18 and 23 years (n=41) from the University of Connecticut and St Joseph College were recruited. The athletes were screened for eating disorders by a sports medicine team. Cronbach's alpha, one-way analysis of variance, and correlation analyses. Reliability analysis indicated a high internal consistency of the FAST (Cronbach's alpha = 0.87). Athletes with eating disorders scored significantly higher on the FAST as compared with athletes without eating pathology and nonathletes with eating disorders (P<.001), which demonstrated discriminant validity. Correlation analyses showed that the FAST was strongly correlated to the Eating Disorder Examination-Questionnaire (0.60, P<.05) and Eating Disorder Inventory (0.89, P<.001). Early detection of eating disorders can help prevent the onset or severity of a clinical eating disorder. By using the screening tool that has been validated for use in women athletes, dietetic professionals can quickly identify those athletes who need assistance with their aberrant eating habits.

  12. A loss of function screen identifies nine new radiation susceptibility genes

    SciTech Connect

    Sudo, Hitomi; Tsuji, Atsushi B. Sugyo, Aya; Imai, Takashi; Saga, Tsuneo; Harada, Yoshi-nobu

    2007-12-21

    Genomic instability is considered a hallmark of carcinogenesis, and dysfunction of DNA repair and cell cycle regulation in response to DNA damage caused by ionizing radiation are thought to be important factors in the early stages of genomic instability. We performed cell-based functional screening using an RNA interference library targeting 200 genes in human cells. We identified three known and nine new radiation susceptibility genes, eight of which are linked directly or potentially with cell cycle progression. Cell cycle analysis on four of the genes not previously linked to cell cycle progression demonstrated that one, ZDHHC8, was associated with the G{sub 2}/M checkpoint in response to DNA damage. Further study of the 12 radiation susceptibility genes identified in this screen may help to elucidate the molecular mechanisms of cell cycle progression, DNA repair, cell death, cell growth and genomic instability, and to develop new radiation sensitizing agents for radiotherapy.

  13. Augmented meaningful use criteria to identify patients eligible for lung cancer screening.

    PubMed

    Raz, Dan J; Dunham, Rachel; Tiep, Brian; Sandoval, Argelia; Grannis, Frederic; Rotter, Arnold; Kim, Jae Y

    2014-09-01

    Lung cancer screening (LCS) with low-dose-radiation (low-dose computed tomography [LDCT]) saves lives. Despite recent US Preventive Services Task Force (USPTF) draft endorsement of LCS, a minority of eligible patients get screened. Meaningful use is a set of standards for electronic health records (EHR) established by the Centers for Medicare and Medicaid Services and includes reporting of smoking status. We sought to improve rates of LCS among patients treated at our institution by identifying eligible patients using augmented smoking-related meaningful use criteria. We launched an LCS program at our institution, a National Comprehensive Cancer Network (NCCN) cancer center, in January 2013. We developed a "tobacco screen," administered by clinic staff to all adult outpatients every 6 months and entered into the EHR. This contained smoking-related meaningful use criteria as well as a pack-year calculation and quit date if applicable. Weekly electronic reports of patients who met eligibility criteria for LCS were generated, and EHR review excluded patients who had had chest computed tomography (CT) within 12 months or who were undergoing cancer treatment. We then contacted eligible patients to review eligibility for LCS and communicated with the primary treating physician regarding the plan for LCS. During the first 3 months of the program, 4 patients were enrolled, 2 by physician referral and 2 by self-referral. We then began to use the tobacco screen reports and identified 418 patients potentially eligible for LCS. Over the next 7 months, we enrolled a total of 110 patients. Fifty-eight (53%) were identified from the tobacco screen, 32 (29%) were self-referred, and 20 (18%) were physician referrals. Three stage I lung cancers were detected and treated. The tobacco screen was easily implemented by clinic staff and took a median time of 2 minutes to enter for current and former smokers. Lack of response to attempts at telephone contact and objection to paying out

  14. Novel Mycosin Protease MycP1 Inhibitors Identified by Virtual Screening and 4D Fingerprints

    PubMed Central

    2015-01-01

    The rise of drug-resistant Mycobacterium tuberculosis lends urgency to the need for new drugs for the treatment of tuberculosis (TB). The identification of a serine protease, mycosin protease-1 (MycP1), as the crucial agent in hydrolyzing the virulence factor, ESX-secretion-associated protein B (EspB), potentially opens the door to new tuberculosis treatment options. Using the crystal structure of mycobacterial MycP1 in the apo form, we performed an iterative ligand- and structure-based virtual screening (VS) strategy to identify novel, nonpeptide, small-molecule inhibitors against MycP1 protease. Screening of ∼485 000 ligands from databases at the Genomics Research Institute (GRI) at the University of Cincinnati and the National Cancer Institute (NCI) using our VS approach, which integrated a pharmacophore model and consensus molecular shape patterns of active ligands (4D fingerprints), identified 81 putative inhibitors, and in vitro testing subsequently confirmed two of them as active inhibitors. Thereafter, the lead structures of each VS round were used to generate a new 4D fingerprint that enabled virtual rescreening of the chemical libraries. Finally, the iterative process identified a number of diverse scaffolds as lead compounds that were tested and found to have micromolar IC50 values against the MycP1 target. This study validated the efficiency of the SABRE 4D fingerprints as a means of identifying novel lead compounds in each screening round of the databases. Together, these results underscored the value of using a combination of in silico iterative ligand- and structure-based virtual screening of chemical libraries with experimental validation for the identification of promising structural scaffolds, such as the MycP1 inhibitors. PMID:24628123

  15. Identifying Patients with Undiagnosed COPD in Primary Care Settings: Insight from Screening Tools and Epidemiologic Studies

    PubMed Central

    Han, MeiLan K.; Steenrod, Anna W.; Bacci, Elizabeth D.; Leidy, Nancy K.; Mannino, David M.; Thomashow, Byron M.; Barr, R.G.; Make, Barry J.; Bowler, Russ P.; Rennard, Stephen I.; Houfek, Julia F.; Yawn, Barbara P.; Meldrum, Catherine A.; Walsh, John W.; Martinez, Fernando J.

    2015-01-01

    Objective: Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality, yet research suggests this disease is greatly underdiagnosed. This literature review sought to summarize the most common and significant variables associated with case-finding or missed cases of COPD to inform more effective and efficient detection of high-risk COPD patients in primary care. Methods: PubMed and EMBASE were searched for articles describing case-finding and epidemiologic research to detect or characterize new cases of COPD. International studies in primary and non-primary care settings, published in English from 2002–2014, were eligible for inclusion. Studies related to risk factors for development of COPD were excluded. Results: Of the 33 studies identified and reviewed, 21 were case-finding or screening and 12 were epidemiological, including cross-sectional, longitudinal, and retrospective designs. A range of variables were identified within and across studies. Variables common to both screening and epidemiological studies included age, smoking status, and respiratory symptoms. Seven significant predictors from epidemiologic studies did not appear in screening tools. No studies targeted discovery of higher risk patients such as those with reduced lung function or risks for exacerbations. Conclusion: Variables used to identify new cases of COPD or differentiate COPD cases and non-cases are wide-ranging, (from sociodemographic to self-reported health or health history variables), providing insight into important factors for case identification. Further research is underway to develop and test the best, smallest variable set that can be used as a screening tool to identify people with undiagnosed, high-risk COPD in primary care. PMID:26236776

  16. Genome-Wide RNAi Screens in C. elegans to Identify Genes Influencing Lifespan and Innate Immunity.

    PubMed

    Sinha, Amit; Rae, Robbie

    2016-01-01

    RNA interference is a rapid, inexpensive, and highly effective tool used to inhibit gene function. In C. elegans, whole genome screens have been used to identify genes involved with numerous traits including aging and innate immunity. RNAi in C. elegans can be carried out via feeding, soaking, or injection. Here we outline protocols used to maintain, grow, and carry out RNAi via feeding in C. elegans and determine whether the inhibited genes are essential for lifespan or innate immunity.

  17. A Short Screening Tool to Identify Victims of Child Sex Trafficking in the Health Care Setting.

    PubMed

    Greenbaum, V Jordan; Dodd, Martha; McCracken, Courtney

    2015-11-23

    The aim of this study was to describe characteristics of commercial sexual exploitation of children/child sex trafficking (CSEC/CST) victims and to develop a screening tool to identify victims among a high-risk adolescent population. In this cross-sectional study, patients aged 12 to 18 years who presented to 1 of 3 metropolitan pediatric emergency departments or 1 child protection clinic and who were identified as victims of CSEC/CST were compared with similar-aged patients with allegations of acute sexual assault/sexual abuse (ASA) without evidence of CSEC/CST. The 2 groups were compared on variables related to medical and reproductive history, high-risk behavior, mental health symptoms, and injury history. After univariate analysis, a subset of candidate variables was subjected to multivariable logistic regression to identify an optimum set of 5 to 7 screening items. Of 108 study participants, 25 comprised the CSEC/CST group, and 83 comprised the ASA group. Average (SD) age was 15.4 (1.8) years for CSEC/CST patients and 14.8 (1.6) years for ASA patients; 100% of the CSEC/CST and 95% of the ASA patients were female. The 2 groups differed significantly on 16 variables involving reproductive history, high-risk behavior, sexually transmitted infections, and previous experience with violence. A 6-item screen was constructed, and a cutoff score of 2 positive answers had a sensitivity of 92%, specificity of 73%, positive predictive value of 51%, and negative predictive value of 97%. Adolescent CSEC/CST victims differ from ASA victims without evidence of CSEC/CST across several domains. A 6-item screen effectively identifies CSEC/CST victims in a high-risk adolescent population.

  18. Changing epidemiology of colorectal cancer makes screening sigmoidoscopy less useful for identifying carriers of colorectal neoplasms.

    PubMed

    Rozen, Paul; Liphshitz, Irena; Barchana, Micha

    2012-08-01

    There is renewed interest in flexible sigmoidoscopy (FS) colorectal cancer (CRC) screening following trials showing significantly reduced CRC incidence and mortality. To evaluate the potential usefulness of FS screening in our population. We examined rectosigmoid (RS) cancer epidemiology in our Jewish population using Israel National Cancer Registry data, computed by CRC site, age groups, and gender. We also reviewed endoscopy-screening publications for prevalence of RS and proximal advanced adenomas (AAP) and having both or either. During 1980-2008, there were 64,559 CRCs registered; 31.6 % were RS cancer which has now decreased to 29 % of men's and 26 % of women's CRC (both P < 0.01). In <50 year olds, RS cancer occurred in 42 % of males' and 35 % of females' CRC, and in the last 2 decades this ratio is unchanged. In 50-74 year olds, RS cancer decreased to stable levels of 32 % of males' and 29 % females' CRC (both P < 0.01). In ≥75 year olds, RS cancer progressively decreased to 24 % of males' and 22 % females' CRC (both P < 0.001). From endoscopy screening reports in 40-79 year olds, RS AAPs occurred in 2.0-5.8 %, being least in women, most in men, and not increased with aging. Some 50-57 % of screenees had both RS and proximal AAPs, least when aged 40-49 years at 25 %, women were 35 %, and with aging 40 %, but most in men at 70 %. With the changing CRC epidemiology, having fewer RS neoplasms but more proximal cancer, the effectiveness of FS screening for identifying significant neoplasms decreases with screenees' age and especially in females. These make FS screening less suitable for our aging and increasingly female population.

  19. A BSL-4 high-throughput screen identifies sulfonamide inhibitors of Nipah virus.

    PubMed

    Tigabu, Bersabeh; Rasmussen, Lynn; White, E Lucile; Tower, Nichole; Saeed, Mohammad; Bukreyev, Alexander; Rockx, Barry; LeDuc, James W; Noah, James W

    2014-04-01

    Nipah virus is a biosafety level 4 (BSL-4) pathogen that causes severe respiratory illness and encephalitis in humans. To identify novel small molecules that target Nipah virus replication as potential therapeutics, Southern Research Institute and Galveston National Laboratory jointly developed an automated high-throughput screening platform that is capable of testing 10,000 compounds per day within BSL-4 biocontainment. Using this platform, we screened a 10,080-compound library using a cell-based, high-throughput screen for compounds that inhibited the virus-induced cytopathic effect. From this pilot effort, 23 compounds were identified with EC50 values ranging from 3.9 to 20.0 μM and selectivities >10. Three sulfonamide compounds with EC50 values <12 μM were further characterized for their point of intervention in the viral replication cycle and for broad antiviral efficacy. Development of HTS capability under BSL-4 containment changes the paradigm for drug discovery for highly pathogenic agents because this platform can be readily modified to identify prophylactic and postexposure therapeutic candidates against other BSL-4 pathogens, particularly Ebola, Marburg, and Lassa viruses.

  20. A BSL-4 High-Throughput Screen Identifies Sulfonamide Inhibitors of Nipah Virus

    PubMed Central

    Tigabu, Bersabeh; Rasmussen, Lynn; White, E. Lucile; Tower, Nichole; Saeed, Mohammad; Bukreyev, Alexander; Rockx, Barry; LeDuc, James W.

    2014-01-01

    Abstract Nipah virus is a biosafety level 4 (BSL-4) pathogen that causes severe respiratory illness and encephalitis in humans. To identify novel small molecules that target Nipah virus replication as potential therapeutics, Southern Research Institute and Galveston National Laboratory jointly developed an automated high-throughput screening platform that is capable of testing 10,000 compounds per day within BSL-4 biocontainment. Using this platform, we screened a 10,080-compound library using a cell-based, high-throughput screen for compounds that inhibited the virus-induced cytopathic effect. From this pilot effort, 23 compounds were identified with EC50 values ranging from 3.9 to 20.0 μM and selectivities >10. Three sulfonamide compounds with EC50 values <12 μM were further characterized for their point of intervention in the viral replication cycle and for broad antiviral efficacy. Development of HTS capability under BSL-4 containment changes the paradigm for drug discovery for highly pathogenic agents because this platform can be readily modified to identify prophylactic and postexposure therapeutic candidates against other BSL-4 pathogens, particularly Ebola, Marburg, and Lassa viruses. PMID:24735442

  1. High-content screen using zebrafish (Danio rerio) embryos identifies a novel kinase activator and inhibitor.

    PubMed

    Geldenhuys, Werner J; Bergeron, Sadie A; Mullins, Jackie E; Aljammal, Rowaa; Gaasch, Briah L; Chen, Wei-Chi; Yun, June; Hazlehurst, Lori A

    2017-02-28

    In this report we utilized zebrafish (Danio rerio) embryos in a phenotypical high-content screen (HCS) to identify novel leads in a cancer drug discovery program. We initially validated our HCS model using the flavin adenosine dinucleotide (FAD) containing endoplasmic reticulum (ER) enzyme, endoplasmic reticulum oxidoreductase (ERO1) inhibitor EN460. EN460 showed a dose response effect on the embryos with a dose of 10μM being significantly lethal during early embryonic development. The HCS campaign which employed a small library identified a promising lead compound, a naphthyl-benzoic acid derivative coined compound 1 which had significant dosage and temporally dependent effects on notochord and muscle development in zebrafish embryos. Screening a 369 kinase member panel we show that compound 1 is a PIM3 kinase inhibitor (IC50=4.078μM) and surprisingly a DAPK1 kinase agonist/activator (EC50=39.525μM). To our knowledge this is the first example of a small molecule activating DAPK1 kinase. We provide a putative model for increased phosphate transfer in the ATP binding domain when compound 1 is virtually docked with DAPK1. Our data indicate that observable phenotypical changes can be used in future zebrafish screens to identify compounds acting via similar molecular signaling pathways.

  2. Functional genomics screening utilizing mutant mouse embryonic stem cells identifies novel radiation-response genes.

    PubMed

    Loesch, Kimberly; Galaviz, Stacy; Hamoui, Zaher; Clanton, Ryan; Akabani, Gamal; Deveau, Michael; DeJesus, Michael; Ioerger, Thomas; Sacchettini, James C; Wallis, Deeann

    2015-01-01

    Elucidating the genetic determinants of radiation response is crucial to optimizing and individualizing radiotherapy for cancer patients. In order to identify genes that are involved in enhanced sensitivity or resistance to radiation, a library of stable mutant murine embryonic stem cells (ESCs), each with a defined mutation, was screened for cell viability and gene expression in response to radiation exposure. We focused on a cancer-relevant subset of over 500 mutant ESC lines. We identified 13 genes; 7 genes that have been previously implicated in radiation response and 6 other genes that have never been implicated in radiation response. After screening, proteomic analysis showed enrichment for genes involved in cellular component disassembly (e.g. Dstn and Pex14) and regulation of growth (e.g. Adnp2, Epc1, and Ing4). Overall, the best targets with the highest potential for sensitizing cancer cells to radiation were Dstn and Map2k6, and the best targets for enhancing resistance to radiation were Iqgap and Vcan. Hence, we provide compelling evidence that screening mutant ESCs is a powerful approach to identify genes that alter radiation response. Ultimately, this knowledge can be used to define genetic variants or therapeutic targets that will enhance clinical therapy.

  3. Crystallization Optimum Solubility Screening: using crystallization results to identify the optimal buffer for protein crystal formation

    SciTech Connect

    Collins, Bernard; Stevens, Raymond C.; Page, Rebecca

    2005-12-01

    It is shown how protein crystallization results can be used to identify buffers that improve protein solubility and, in turn, crystallization success. An optimal solubility screen is described that uses the results of crystallization trials to identify buffers that improve protein solubility and, in turn, crystallization success. This screen is useful not only for standard crystallization experiments, but also can easily be implemented into any high-throughput structure-determination pipeline. As a proof of principle, the predicted novel-fold protein AF2059 from Archaeoglobus fulgidus, which was known to precipitate in most buffers and particularly during concentration experiments, was selected. Using the crystallization results of 192 independent crystallization trials, it was possible to identify a buffer containing 100 mM CHES pH 9.25 that significantly improves its solubility. After transferring AF2059 into this ‘optimum-solubility’ buffer, the protein was rescreened for crystal formation against these same 192 conditions. Instead of extensive precipitation, as observed initially, it was found that 24 separate conditions produced crystals and the exchange of AF2059 into CHES buffer significantly improved crystallization success. Fine-screen optimization of these conditions led to the production of a crystal suitable for high-resolution (2.2 Å) structure determination.

  4. In silico and in vitro drug screening identifies new therapeutic approaches for Ewing sarcoma

    PubMed Central

    Pessetto, Ziyan Y.; Chen, Bin; Alturkmani, Hani; Hyter, Stephen; Flynn, Colleen A.; Baltezor, Michael; Ma, Yan; Rosenthal, Howard G.; Neville, Kathleen A.; Weir, Scott J.; Butte, Atul J.; Godwin, Andrew K.

    2017-01-01

    The long-term overall survival of Ewing sarcoma (EWS) patients remains poor; less than 30% of patients with metastatic or recurrent disease survive despite aggressive combinations of chemotherapy, radiation and surgery. To identify new therapeutic options, we employed a multi-pronged approach using in silico predictions of drug activity via an integrated bioinformatics approach in parallel with an in vitro screen of FDA-approved drugs. Twenty-seven drugs and forty-six drugs were identified, respectively, to have anti-proliferative effects for EWS, including several classes of drugs in both screening approaches. Among these drugs, 30 were extensively validated as mono-therapeutic agents and 9 in 14 various combinations in vitro. Two drugs, auranofin, a thioredoxin reductase inhibitor, and ganetespib, an HSP90 inhibitor, were predicted to have anti-cancer activities in silico and were confirmed active across a panel of genetically diverse EWS cells. When given in combination, the survival rate in vivo was superior compared to auranofin or ganetespib alone. Importantly, extensive formulations, dose tolerance, and pharmacokinetics studies demonstrated that auranofin requires alternative delivery routes to achieve therapeutically effective levels of the gold compound. These combined screening approaches provide a rapid means to identify new treatment options for patients with a rare and often-fatal disease. PMID:27863422

  5. A misexpression screen identifies genes that can modulate RAS1 pathway signaling in Drosophila melanogaster.

    PubMed Central

    Huang, A M; Rubin, G M

    2000-01-01

    Differentiation of the R7 photoreceptor cell is dependent on the Sevenless receptor tyrosine kinase, which activates the RAS1/mitogen-activated protein kinase signaling cascade. Kinase suppressor of Ras (KSR) functions genetically downstream of RAS1 in this signal transduction cascade. Expression of dominant-negative KSR (KDN) in the developing eye blocks RAS pathway signaling, prevents R7 cell differentiation, and causes a rough eye phenotype. To identify genes that modulate RAS signaling, we screened for genes that alter RAS1/KSR signaling efficiency when misexpressed. In this screen, we recovered three known genes, Lk6, misshapen, and Akap200. We also identified seven previously undescribed genes; one encodes a novel rel domain member of the NFAT family, and six encode novel proteins. These genes may represent new components of the RAS pathway or components of other signaling pathways that can modulate signaling by RAS. We discuss the utility of gain-of-function screens in identifying new components of signaling pathways in Drosophila. PMID:11063696

  6. Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62

    PubMed Central

    DeJesus, Rowena; Moretti, Francesca; McAllister, Gregory; Wang, Zuncai; Bergman, Phil; Liu, Shanming; Frias, Elizabeth; Alford, John; Reece-Hoyes, John S; Lindeman, Alicia; Kelliher, Jennifer; Russ, Carsten; Knehr, Judith; Carbone, Walter; Beibel, Martin; Roma, Guglielmo; Ng, Aylwin; Tallarico, John A; Porter, Jeffery A; Xavier, Ramnik J; Mickanin, Craig; Murphy, Leon O; Hoffman, Gregory R; Nyfeler, Beat

    2016-01-01

    SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein. DOI: http://dx.doi.org/10.7554/eLife.17290.001 PMID:27351204

  7. High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress

    PubMed Central

    Malik, Nasir; Shah, Sonia; Zhao, Jean; Class, Bradley; Aguisanda, Francis; Southall, Noel; Xia, Menghang; McKew, John C.; Rao, Mahendra

    2016-01-01

    Astrocytes are the predominant cell type in the nervous system and play a significant role in maintaining neuronal health and homeostasis. Recently, astrocyte dysfunction has been implicated in the pathogenesis of many neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. Astrocytes are thus an attractive new target for drug discovery for neurological disorders. Using astrocytes differentiated from human embryonic stem cells, we have developed an assay to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. This phenotypic oxidative stress assay has been optimized for high-throughput screening in a 1,536-well plate format. From a screen of approximately 4,100 bioactive tool compounds and approved drugs, we identified a set of 22 that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress. Nine of these compounds were also found to be protective of induced pluripotent stem cell-differentiated astrocytes in a related assay. These compounds are thought to confer protection through hormesis, activating stress-response pathways and preconditioning astrocytes to handle subsequent exposure to hydrogen peroxide. In fact, four of these compounds were found to activate the antioxidant response element/nuclear factor-E2-related factor 2 pathway, a protective pathway induced by toxic insults. Our results demonstrate the relevancy and utility of using astrocytes differentiated from human stem cells as a disease model for drug discovery and development. Significance Astrocytes play a key role in neurological diseases. Drug discovery efforts that target astrocytes can identify novel therapeutics. Human astrocytes are difficult to obtain and thus are challenging to use for high-throughput screening, which requires large numbers of cells. Using human embryonic stem cell

  8. Screen and clean: a tool for identifying interactions in genome-wide association studies.

    PubMed

    Wu, Jing; Devlin, Bernie; Ringquist, Steven; Trucco, Massimo; Roeder, Kathryn

    2010-04-01

    Epistasis could be an important source of risk for disease. How interacting loci might be discovered is an open question for genome-wide association studies (GWAS). Most researchers limit their statistical analyses to testing individual pairwise interactions (i.e., marginal tests for association). A more effective means of identifying important predictors is to fit models that include many predictors simultaneously (i.e., higher-dimensional models). We explore a procedure called screen and clean (SC) for identifying liability loci, including interactions, by using the lasso procedure, which is a model selection tool for high-dimensional regression. We approach the problem by using a varying dictionary consisting of terms to include in the model. In the first step the lasso dictionary includes only main effects. The most promising single-nucleotide polymorphisms (SNPs) are identified using a screening procedure. Next the lasso dictionary is adjusted to include these main effects and the corresponding interaction terms. Again, promising terms are identified using lasso screening. Then significant terms are identified through the cleaning process. Implementation of SC for GWAS requires algorithms to explore the complex model space induced by the many SNPs genotyped and their interactions. We propose and explore a set of algorithms and find that SC successfully controls Type I error while yielding good power to identify risk loci and their interactions. When the method is applied to data obtained from the Wellcome Trust Case Control Consortium study of Type 1 Diabetes it uncovers evidence supporting interaction within the HLA class II region as well as within Chromosome 12q24.

  9. Screen and Clean: a tool for identifying interactions in genome-wide association studies

    PubMed Central

    Wu, Jing; Devlin, Bernie; Ringquist, Steven; Trucco, Massimo; Roeder, Kathryn

    2010-01-01

    Epistasis could be an important source of risk for disease. How interacting loci might be discovered is an open question for genome-wide association studies (GWAS). Most researchers limit their statistical analyses to testing individual pairwise interactions (i.e., marginal tests for association). A more effective means of identifying important predictors is to fit models that include many predictors simultaneously (i.e., higher dimensional models). We explore a procedure called screen and clean (SC) for identifying liability loci, including interactions, by using the lasso procedure, which is a model selection tool for high dimensional regression. We approach the problem by using a varying dictionary consisting of terms to include in the model. In the first step the lasso dictionary includes only main effects. The most promising SNPs are identified using a screening procedure. Next the lasso dictionary is adjusted to include these main effects and the corresponding interaction terms. Again, promising terms are identified using lasso screening. Then significant terms are identified through the cleaning process. Implementation of SC for GWAS requires algorithms to explore the complex model space induced by the many SNPs genotyped and their interactions. We propose and explore a set of algorithms and find that SC successfully controls Type I error while yielding good power to identify risk loci and their interactions. When the method is applied to data obtained from the Wellcome Trust Case Control Consortium study of Type 1 Diabetes it uncovers evidence supporting interaction within the HLA class II region as well as within Chromosome 12q24. PMID:20088021

  10. Yeast-based automated high-throughput screens to identify anti-parasitic lead compounds

    PubMed Central

    Bilsland, Elizabeth; Sparkes, Andrew; Williams, Kevin; Moss, Harry J.; de Clare, Michaela; Pir, Pınar; Rowland, Jem; Aubrey, Wayne; Pateman, Ron; Young, Mike; Carrington, Mark; King, Ross D.; Oliver, Stephen G.

    2013-01-01

    We have developed a robust, fully automated anti-parasitic drug-screening method that selects compounds specifically targeting parasite enzymes and not their host counterparts, thus allowing the early elimination of compounds with potential side effects. Our yeast system permits multiple parasite targets to be assayed in parallel owing to the strains’ expression of different fluorescent proteins. A strain expressing the human target is included in the multiplexed screen to exclude compounds that do not discriminate between host and parasite enzymes. This form of assay has the advantages of using known targets and not requiring the in vitro culture of parasites. We performed automated screens for inhibitors of parasite dihydrofolate reductases, N-myristoyltransferases and phosphoglycerate kinases, finding specific inhibitors of parasite targets. We found that our ‘hits’ have significant structural similarities to compounds with in vitro anti-parasitic activity, validating our screens and suggesting targets for hits identified in parasite-based assays. Finally, we demonstrate a 60 per cent success rate for our hit compounds in killing or severely inhibiting the growth of Trypanosoma brucei, the causative agent of African sleeping sickness. PMID:23446112

  11. Yeast-based automated high-throughput screens to identify anti-parasitic lead compounds.

    PubMed

    Bilsland, Elizabeth; Sparkes, Andrew; Williams, Kevin; Moss, Harry J; de Clare, Michaela; Pir, Pinar; Rowland, Jem; Aubrey, Wayne; Pateman, Ron; Young, Mike; Carrington, Mark; King, Ross D; Oliver, Stephen G

    2013-02-27

    We have developed a robust, fully automated anti-parasitic drug-screening method that selects compounds specifically targeting parasite enzymes and not their host counterparts, thus allowing the early elimination of compounds with potential side effects. Our yeast system permits multiple parasite targets to be assayed in parallel owing to the strains' expression of different fluorescent proteins. A strain expressing the human target is included in the multiplexed screen to exclude compounds that do not discriminate between host and parasite enzymes. This form of assay has the advantages of using known targets and not requiring the in vitro culture of parasites. We performed automated screens for inhibitors of parasite dihydrofolate reductases, N-myristoyltransferases and phosphoglycerate kinases, finding specific inhibitors of parasite targets. We found that our 'hits' have significant structural similarities to compounds with in vitro anti-parasitic activity, validating our screens and suggesting targets for hits identified in parasite-based assays. Finally, we demonstrate a 60 per cent success rate for our hit compounds in killing or severely inhibiting the growth of Trypanosoma brucei, the causative agent of African sleeping sickness.

  12. An in vivo zebrafish screen identifies organophosphate antidotes with diverse mechanisms of action.

    PubMed

    Jin, Shan; Sarkar, Kumar S; Jin, Youngnam N; Liu, Yan; Kokel, David; Van Ham, Tjakko J; Roberts, Lee D; Gerszten, Robert E; Macrae, Calum A; Peterson, Randall T

    2013-01-01

    Organophosphates are a class of highly toxic chemicals that includes many pesticides and chemical weapons. Exposure to organophosphates, either through accidents or acts of terrorism, poses a significant risk to human health and safety. Existing antidotes, in use for over 50 years, have modest efficacy and undesirable toxicities. Therefore, discovering new organophosphate antidotes is a high priority. Early life stage zebrafish exposed to organophosphates exhibit several phenotypes that parallel the human response to organophosphates, including behavioral deficits, paralysis, and eventual death. Here, we have developed a high-throughput zebrafish screen in a 96-well plate format to find new antidotes that counteract organophosphate-induced lethality. In a pilot screen of 1200 known drugs, we identified 16 compounds that suppress organophosphate toxicity in zebrafish. Several in vitro assays coupled with liquid chromatography/tandem mass spectrometry-based metabolite profiling enabled determination of mechanisms of action for several of the antidotes, including reversible acetylcholinesterase inhibition, cholinergic receptor antagonism, and inhibition of bioactivation. Therefore, the in vivo screen is capable of discovering organophosphate antidotes that intervene in distinct pathways. These findings suggest that zebrafish screens might be a broadly applicable approach for discovering compounds that counteract the toxic effects of accidental or malicious poisonous exposures.

  13. An In Vivo Zebrafish Screen Identifies Organophosphate Antidotes with Diverse Mechanisms of Action

    PubMed Central

    Jin, Shan; Sarkar, Kumar S.; Jin, Youngnam N.; Liu, Yan; Kokel, David; Van Ham, Tjakko J.; Roberts, Lee D.; Gerszten, Robert E.; MacRae, Calum A.; Peterson, Randall T.

    2014-01-01

    Organophosphates are a class of highly toxic chemicals that includes many pesticides and chemical weapons. Exposure to organophosphates, either through accidents or acts of terrorism, poses a significant risk to human health and safety. Existing antidotes, in use for over 50 years, have modest efficacy and undesirable toxicities. Therefore, discovering new organophosphate antidotes is a high priority. Early life stage zebrafish exposed to organophosphates exhibit several phenotypes that parallel the human response to organophosphates, including behavioral deficits, paralysis, and eventual death. Here, we have developed a high-throughput zebrafish screen in a 96-well plate format to find new antidotes that counteract organophosphate-induced lethality. In a pilot screen of 1200 known drugs, we identified 16 compounds that suppress organophosphate toxicity in zebrafish. Several in vitro assays coupled with liquid chromatography/tandem mass spectrometry–based metabolite profiling enabled determination of mechanisms of action for several of the antidotes, including reversible acetylcholinesterase inhibition, cholinergic receptor antagonism, and inhibition of bioactivation. Therefore, the in vivo screen is capable of discovering organophosphate antidotes that intervene in distinct pathways. These findings suggest that zebrafish screens might be a broadly applicable approach for discovering compounds that counteract the toxic effects of accidental or malicious poisonous exposures. PMID:22960781

  14. siRNA screen identifies QPCT as a druggable target for Huntington's disease.

    PubMed

    Jimenez-Sanchez, Maria; Lam, Wun; Hannus, Michael; Sönnichsen, Birte; Imarisio, Sara; Fleming, Angeleen; Tarditi, Alessia; Menzies, Fiona; Ed Dami, Teresa; Xu, Catherine; Gonzalez-Couto, Eduardo; Lazzeroni, Giulia; Heitz, Freddy; Diamanti, Daniela; Massai, Luisa; Satagopam, Venkata P; Marconi, Guido; Caramelli, Chiara; Nencini, Arianna; Andreini, Matteo; Sardone, Gian Luca; Caradonna, Nicola P; Porcari, Valentina; Scali, Carla; Schneider, Reinhard; Pollio, Giuseppe; O'Kane, Cahir J; Caricasole, Andrea; Rubinsztein, David C

    2015-05-01

    Huntington's disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified new modifiers of mutant HTT toxicity by performing a large-scale 'druggable genome' siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen and also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone αB-crystallin and reduced the aggregation of diverse proteins. We generated new QPCT inhibitors using in silico methods followed by in vitro screening, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a new HD druggable target affecting mutant HTT aggregation and provide proof of principle for a discovery pipeline from druggable genome screen to drug development.

  15. siRNA screen identifies QPCT as a druggable target for Huntington’s disease

    PubMed Central

    Jimenez-Sanchez, Maria; Lam, Wun; Tarditi, Alessia; Menzies, Fiona; Dami, Teresa Ed; Xu, Catherine; Gonzalez-Couto, Eduardo; Lazzeroni, Giulia; Heitz, Freddy; Diamanti, Daniela; Massai, Luisa; Satagopam, Venkata P.; Marconi, Guido; Caramelli, Chiara; Nencini, Arianna; Andreini, Matteo; Sardone, Gian Luca; Caradonna, Nicola P.; Porcari, Valentina; Scali, Carla; Schneider, Reinhard; Pollio, Giuseppe; O’Kane, Cahir J.; Caricasole, Andrea; Rubinsztein, David C.

    2015-01-01

    Huntington’s disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified novel modifiers of mutant HTT toxicity by performing a large-scale “druggable genome” siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen, and which also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone alpha B-crystallin and reduced the aggregation of diverse proteins. We generated novel QPCT inhibitors using in silico methods followed by in vitro screens, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a novel HD druggable target affecting mutant huntingtin aggregation, and provide proof-of-principle for a discovery pipeline from druggable genome screen to drug development. PMID:25848931

  16. Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells.

    PubMed

    Guo, Ge; Wang, Wei; Bradley, Allan

    2004-06-24

    Phenotype-driven recessive genetic screens in diploid organisms require a strategy to render the mutation homozygous. Although homozygous mutant mice can be generated by breeding, a reliable method to make homozygous mutations in cultured cells has not been available, limiting recessive screens in culture. Cultured embryonic stem (ES) cells provide access to all of the genes required to elaborate the fundamental components and physiological systems of a mammalian cell. Here we have exploited the high rate of mitotic recombination in Bloom's syndrome protein (Blm)-deficient ES cells to generate a genome-wide library of homozygous mutant cells from heterozygous mutations induced with a revertible gene trap retrovirus. We have screened this library for cells with defects in DNA mismatch repair (MMR), a system that detects and repairs base-base mismatches. We demonstrate the recovery of cells with homozygous mutations in known and novel MMR genes. We identified Dnmt1(ref. 5) as a novel MMR gene and confirmed that Dnmt1-deficient ES cells exhibit micro-satellite instability, providing a mechanistic explanation for the role of Dnmt1 in cancer. The combination of insertional mutagenesis in Blm-deficient ES cells establishes a new approach for phenotype-based recessive genetic screens in ES cells.

  17. Expression-based screening identifies the combination of histone deacetylase inhibitors and retinoids for neuroblastoma differentiation

    PubMed Central

    Hahn, Cynthia K.; Ross, Kenneth N.; Warrington, Ian M.; Mazitschek, Ralph; Kanegai, Cindy M.; Wright, Renee D.; Kung, Andrew L.; Golub, Todd R.; Stegmaier, Kimberly

    2008-01-01

    The discovery of new small molecules and their testing in rational combination poses an ongoing problem for rare diseases, in particular, for pediatric cancers such as neuroblastoma. Despite maximal cytotoxic therapy with double autologous stem cell transplantation, outcome remains poor for children with high-stage disease. Because differentiation is aberrant in this malignancy, compounds that modulate transcription, such as histone deacetylase (HDAC) inhibitors, are of particular interest. However, as single agents, HDAC inhibitors have had limited efficacy. In the present study, we use an HDAC inhibitor as an enhancer to screen a small-molecule library for compounds inducing neuroblastoma maturation. To quantify differentiation, we use an enabling gene expression-based screening strategy. The top hit identified in the screen was all-trans-retinoic acid. Secondary assays confirmed greater neuroblastoma differentiation with the combination of an HDAC inhibitor and a retinoid versus either alone. Furthermore, effects of combination therapy were synergistic with respect to inhibition of cellular viability and induction of apoptosis. In a xenograft model of neuroblastoma, animals treated with combination therapy had the longest survival. This work suggests that testing of an HDAC inhibitor and retinoid in combination is warranted for children with neuroblastoma and demonstrates the success of a signature-based screening approach to prioritize compound combinations for testing in rare diseases. PMID:18607002

  18. Current office-based hearing screening questions fail to identify adolescents at risk for hearing loss.

    PubMed

    Sekhar, Deepa L; Zalewski, Thomas R; King, Tonya S; Paul, Ian M

    2014-12-01

    There is a rising prevalence of hearing loss among adolescents in the United States. Current paediatric preventive care recommendations by the Bright Futures guidelines and the American Academy of Pediatrics suggest that clinicians should ask adolescents ten hearing screening questions to identify those who are at high risk of hearing loss for further objective hearing testing. We assessed the utility of these subjective risk assessment questions to distinguish those adolescents with objectively documented hearing loss. A single public high school in Pennsylvania. We compared results from a prospective study evaluating objective hearing assessments with the use of the ten Bright Futures hearing screening questions plus additional adolescent-specific questions to predict adolescent hearing loss. In relation to the questions used in this study, adolescents who were referred following objective hearing screens were more likely to report "trouble following the conversation when two or more people are talking at the same time" and a "past experience of slight hearing loss". Referrals from sound treated booth testing were more likely to report "trouble hearing over the phone" and have a diagnosis or history of hearing loss. Most Bright Futures questions were not associated with adolescent hearing loss. An objective adolescent hearing screen should be considered in the recommended schedule of preventive care, instead of the current risk-based subjective assessment. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  19. Gene Expression Signature-Based Screening Identifies New Broadly Effective Influenza A Antivirals

    PubMed Central

    Josset, Laurence; Textoris, Julien; Loriod, Béatrice; Ferraris, Olivier; Moules, Vincent; Lina, Bruno; N'Guyen, Catherine; Diaz, Jean-Jacques; Rosa-Calatrava, Manuel

    2010-01-01

    Classical antiviral therapies target viral proteins and are consequently subject to resistance. To counteract this limitation, alternative strategies have been developed that target cellular factors. We hypothesized that such an approach could also be useful to identify broad-spectrum antivirals. The influenza A virus was used as a model for its viral diversity and because of the need to develop therapies against unpredictable viruses as recently underlined by the H1N1 pandemic. We proposed to identify a gene-expression signature associated with infection by different influenza A virus subtypes which would allow the identification of potential antiviral drugs with a broad anti-influenza spectrum of activity. We analyzed the cellular gene expression response to infection with five different human and avian influenza A virus strains and identified 300 genes as differentially expressed between infected and non-infected samples. The most 20 dysregulated genes were used to screen the connectivity map, a database of drug-associated gene expression profiles. Candidate antivirals were then identified by their inverse correlation to the query signature. We hypothesized that such molecules would induce an unfavorable cellular environment for influenza virus replication. Eight potential antivirals including ribavirin were identified and their effects were tested in vitro on five influenza A strains. Six of the molecules inhibited influenza viral growth. The new pandemic H1N1 virus, which was not used to define the gene expression signature of infection, was inhibited by five out of the eight identified molecules, demonstrating that this strategy could contribute to identifying new broad anti-influenza agents acting on cellular gene expression. The identified infection signature genes, the expression of which are modified upon infection, could encode cellular proteins involved in the viral life cycle. This is the first study showing that gene expression-based screening can be

  20. Testing the woman abuse screening tool to identify intimate partner violence in Indonesia.

    PubMed

    Iskandar, Livia; Braun, Kathryn L; Katz, Alan R

    2015-04-01

    Intimate Partner Violence (IPV) is a global public health problem. IPV prevalence in Indonesia has been estimated to be less than 1%, based on reported cases. It is likely that IPV prevalence is underreported in Indonesia, as it is in many other countries. Screening for IPV has been found to increase IPV identification, but no screening tools are in use in Indonesia. The aim of this study was to test the translated Woman Abuse Screening Tool (WAST) for detecting IPV in Indonesia. The WAST was tested against a diagnostic interview by a trained psychologist on 240 women attending two Primary Health Centers in Jakarta. IPV prevalence and the reliability, sensitivity, and specificity of the WAST were estimated. Prevalence of IPV by diagnostic interview was 36.3%, much higher than published estimates. The most common forms of IPV identified were psychological (85%) and physical abuse (24%). Internal reliability of the WAST was high (α = .801). A WAST score of 13 (out of 24) is the recommended cutoff for identifying IPV, but only 17% of the Indonesian sample scored 13 or higher. Test sensitivity of the WAST with a cutoff score of 13 was only 41.9%, with a specificity of 96.8%. With a cutoff score of 10, the sensitivity improved to 84.9%, while the specificity decreased to 61.0%. Use of the WAST with a cutoff score of 10 provides good sensitivity and reasonable specificity and would provide a much-needed screening tool for use in Indonesia. Although a lower cutoff would yield a greater proportion of false positives, most of the true cases would be identified, increasing the possibility that women experiencing abuse would receive needed assistance.

  1. Functional Genetic Screen to Identify Interneurons Governing Behaviorally Distinct Aspects of Drosophila Larval Motor Programs.

    PubMed

    Clark, Matt Q; McCumsey, Stephanie J; Lopez-Darwin, Sereno; Heckscher, Ellie S; Doe, Chris Q

    2016-07-07

    Drosophila larval crawling is an attractive system to study rhythmic motor output at the level of animal behavior. Larval crawling consists of waves of muscle contractions generating forward or reverse locomotion. In addition, larvae undergo additional behaviors, including head casts, turning, and feeding. It is likely that some neurons (e.g., motor neurons) are used in all these behaviors, but the identity (or even existence) of neurons dedicated to specific aspects of behavior is unclear. To identify neurons that regulate specific aspects of larval locomotion, we performed a genetic screen to identify neurons that, when activated, could elicit distinct motor programs. We used 165 Janelia CRM-Gal4 lines-chosen for sparse neuronal expression-to ectopically express the warmth-inducible neuronal activator TrpA1, and screened for locomotor defects. The primary screen measured forward locomotion velocity, and we identified 63 lines that had locomotion velocities significantly slower than controls following TrpA1 activation (28°). A secondary screen was performed on these lines, revealing multiple discrete behavioral phenotypes, including slow forward locomotion, excessive reverse locomotion, excessive turning, excessive feeding, immobile, rigid paralysis, and delayed paralysis. While many of the Gal4 lines had motor, sensory, or muscle expression that may account for some or all of the phenotype, some lines showed specific expression in a sparse pattern of interneurons. Our results show that distinct motor programs utilize distinct subsets of interneurons, and provide an entry point for characterizing interneurons governing different elements of the larval motor program. Copyright © 2016 Clark et al.

  2. Functional Genetic Screen to Identify Interneurons Governing Behaviorally Distinct Aspects of Drosophila Larval Motor Programs

    PubMed Central

    Clark, Matt Q.; McCumsey, Stephanie J.; Lopez-Darwin, Sereno; Heckscher, Ellie S.; Doe, Chris Q.

    2016-01-01

    Drosophila larval crawling is an attractive system to study rhythmic motor output at the level of animal behavior. Larval crawling consists of waves of muscle contractions generating forward or reverse locomotion. In addition, larvae undergo additional behaviors, including head casts, turning, and feeding. It is likely that some neurons (e.g., motor neurons) are used in all these behaviors, but the identity (or even existence) of neurons dedicated to specific aspects of behavior is unclear. To identify neurons that regulate specific aspects of larval locomotion, we performed a genetic screen to identify neurons that, when activated, could elicit distinct motor programs. We used 165 Janelia CRM-Gal4 lines—chosen for sparse neuronal expression—to ectopically express the warmth-inducible neuronal activator TrpA1, and screened for locomotor defects. The primary screen measured forward locomotion velocity, and we identified 63 lines that had locomotion velocities significantly slower than controls following TrpA1 activation (28°). A secondary screen was performed on these lines, revealing multiple discrete behavioral phenotypes, including slow forward locomotion, excessive reverse locomotion, excessive turning, excessive feeding, immobile, rigid paralysis, and delayed paralysis. While many of the Gal4 lines had motor, sensory, or muscle expression that may account for some or all of the phenotype, some lines showed specific expression in a sparse pattern of interneurons. Our results show that distinct motor programs utilize distinct subsets of interneurons, and provide an entry point for characterizing interneurons governing different elements of the larval motor program. PMID:27172197

  3. Testing the Woman Abuse Screening Tool to Identify Intimate Partner Violence in Indonesia

    PubMed Central

    Iskandar, Livia; Braun, Kathryn L.; Katz, Alan R.

    2015-01-01

    Intimate Partner Violence (IPV) is a global public health problem. IPV prevalence in Indonesia has been estimated to be less than 1%, based on reported cases. It is likely that IPV prevalence is underreported in Indonesia, as it is in many other countries. Screening for IPV has been found to increase IPV identification, but no screening tools are in use in Indonesia. The aim of this study was to test the translated Woman Abuse Screening Tool (WAST) for detecting IPV in Indonesia. The WAST was tested against a diagnostic interview by a trained psychologist on 240 women attending two Primary Health Centers in Jakarta. IPV prevalence and the reliability, sensitivity, and specificity of the WAST were estimated. Prevalence of IPV by diagnostic interview was 36.3%, much higher than published estimates. The most common forms of IPV identified were psychological (85%) and physical abuse (24%). Internal reliability of the WAST was high (α = .801). A WAST score of 13 (out of 24) is the recommended cutoff for identifying IPV, but only 17% of the Indonesian sample scored 13 or higher. Test sensitivity of the WAST with a cutoff score of 13 was only 41.9%, with a specificity of 96.8%. With a cutoff score of 10, the sensitivity improved to 84.9%, while the specificity decreased to 61.0%. Use of the WAST with a cutoff score of 10 provides good sensitivity and reasonable specificity and would provide a much-needed screening tool for use in Indonesia. Although a lower cutoff would yield a greater proportion of false positives, most of the true cases would be identified, increasing the possibility that women experiencing abuse would receive needed assistance. PMID:25012952

  4. A small molecule screen identifies a novel compound that induces a homeotic transformation in Hydra

    PubMed Central

    Glauber, Kristine M.; Dana, Catherine E.; Park, Steve S.; Colby, David A.; Noro, Yukihiko; Fujisawa, Toshitaka; Chamberlin, A. Richard; Steele, Robert E.

    2013-01-01

    Developmental processes such as morphogenesis, patterning and differentiation are continuously active in the adult Hydra polyp. We carried out a small molecule screen to identify compounds that affect patterning in Hydra. We identified a novel molecule, DAC-2-25, that causes a homeotic transformation of body column into tentacle zone. This transformation occurs in a progressive and polar fashion, beginning at the oral end of the animal. We have identified several strains that respond to DAC-2-25 and one that does not, and we used chimeras from these strains to identify the ectoderm as the target tissue for DAC-2-25. Using transgenic Hydra that express green fluorescent protein under the control of relevant promoters, we examined how DAC-2-25 affects tentacle patterning. Genes whose expression is associated with the tentacle zone are ectopically expressed upon exposure to DAC-2-25, whereas those associated with body column tissue are turned off as the tentacle zone expands. The expression patterns of the organizer-associated gene HyWnt3 and the hypostome-specific gene HyBra2 are unchanged. Structure-activity relationship studies have identified features of DAC-2-25 that are required for activity and potency. This study shows that small molecule screens in Hydra can be used to dissect patterning processes. PMID:24255098

  5. A small molecule screen identifies a novel compound that induces a homeotic transformation in Hydra.

    PubMed

    Glauber, Kristine M; Dana, Catherine E; Park, Steve S; Colby, David A; Noro, Yukihiko; Fujisawa, Toshitaka; Chamberlin, A Richard; Steele, Robert E

    2013-12-01

    Developmental processes such as morphogenesis, patterning and differentiation are continuously active in the adult Hydra polyp. We carried out a small molecule screen to identify compounds that affect patterning in Hydra. We identified a novel molecule, DAC-2-25, that causes a homeotic transformation of body column into tentacle zone. This transformation occurs in a progressive and polar fashion, beginning at the oral end of the animal. We have identified several strains that respond to DAC-2-25 and one that does not, and we used chimeras from these strains to identify the ectoderm as the target tissue for DAC-2-25. Using transgenic Hydra that express green fluorescent protein under the control of relevant promoters, we examined how DAC-2-25 affects tentacle patterning. Genes whose expression is associated with the tentacle zone are ectopically expressed upon exposure to DAC-2-25, whereas those associated with body column tissue are turned off as the tentacle zone expands. The expression patterns of the organizer-associated gene HyWnt3 and the hypostome-specific gene HyBra2 are unchanged. Structure-activity relationship studies have identified features of DAC-2-25 that are required for activity and potency. This study shows that small molecule screens in Hydra can be used to dissect patterning processes.

  6. Evaluation of non-invasive screening measures to identify individuals with prediabetes

    PubMed Central

    Vanderwood, Karl K; Kramer, Mary Kaye; Miller, Rachel G.; Arena, Vincent C.; Kriska, Andrea M.

    2016-01-01

    Aims Because blood-based screening to identify those with prediabetes to take part in Diabetes Prevention Program (DPP) translation efforts can be costly and time-consuming, non-invasive methods are needed. The aims of this paper are to evaluate the ability of the American Diabetes Association (ADA) risk test in identifying individuals with prediabetes, as well as the use of body composition measures for this purpose. In addition the utility of these alternate methods to ascertain the presence of the metabolic syndrome was assessed. Methods Potential participants were recruited from a worksite and three community centers to take part in a DPP translation study. Participants completed onsite screening where anthropometric measures, fasting lipids and glucose, and hemoglobin A1c were assessed. Those with a BMI ≥24kg/m2 and prediabetes and/or the metabolic syndrome were eligible to participate. Non-invasive screening methods were evaluated for their ability to identify those with prediabetes and the metabolic syndrome based on clinically measured values. Results All non-invasive methods were highly sensitive (68.9% to 98.5%) in the detection of prediabetes, but specificity was low (6.7% to 44.5%). None of the alternatives evaluated achieved acceptable discrimination levels in ROC analysis. Similar results were noted in identifying the metabolic syndrome. Conclusions The non-invasive methods evaluated in this study effectively identify participants with prediabetes, but would also allow for enrollment of a large number of individuals who do not have prediabetes. Deciding whether to use these alternatives, blood-based measures, or a combination of both will ultimately depend on the purpose of the program and the level of flexibility regarding participant eligibility. PMID:25441924

  7. Inhibitors of neutrophil recruitment identified using transgenic zebrafish to screen a natural product library.

    PubMed

    Wang, Xingang; Robertson, Anne L; Li, Jingyu; Chai, Ruth Jinfen; Haishan, Wang; Sadiku, Pranvera; Ogryzko, Nikolay V; Everett, Martin; Yoganathan, Kanagasundaram; Luo, Hongbo Robert; Renshaw, Stephen A; Ingham, Philip W

    2014-01-01

    Cell migration is fundamental to the inflammatory response, but uncontrolled cell migration and excess recruitment of neutrophils and other leukocytes can cause damage to the tissue. Here we describe the use of an in vivo model - the Tg(mpx:GFP)(i114) zebrafish line, in which neutrophils are labelled by green fluorescent protein (GFP) - to screen a natural product library for compounds that can affect neutrophil migratory behaviour. Among 1040 fungal extracts screened, two were found to inhibit neutrophil migration completely. Subfractionation of these extracts identified sterigmatocystin and antibiotic PF1052 as the active components. Using the EZ-TAXIScan chemotaxis assay, both compounds were also found to have a dosage-dependent inhibitory effect on murine neutrophil migration. Furthermore, neutrophils treated with PF1052 failed to form pseudopods and appeared round in shape, suggesting a defect in PI3-kinase (PI3K) signalling. We generated a transgenic neutrophil-specific PtdIns(3,4,5)P3 (PIP3) reporter zebrafish line, which revealed that PF1052 does not affect the activation of PI3K at the plasma membrane. In human neutrophils, PF1052 neither induced apoptosis nor blocked AKT phosphorylation. In conclusion, we have identified an antibiotic from a natural product library with potent anti-inflammatory properties, and have established the utility of the mpx:GFP transgenic zebrafish for high-throughput in vivo screens for novel inhibitors of neutrophil migration.

  8. Inhibitors of neutrophil recruitment identified using transgenic zebrafish to screen a natural product library

    PubMed Central

    Wang, Xingang; Robertson, Anne L.; Li, Jingyu; Chai, Ruth Jinfen; Haishan, Wang; Sadiku, Pranvera; Ogryzko, Nikolay V.; Everett, Martin; Yoganathan, Kanagasundaram; Luo, Hongbo Robert; Renshaw, Stephen A.; Ingham, Philip W.

    2014-01-01

    Cell migration is fundamental to the inflammatory response, but uncontrolled cell migration and excess recruitment of neutrophils and other leukocytes can cause damage to the tissue. Here we describe the use of an in vivo model – the Tg(mpx:GFP)i114 zebrafish line, in which neutrophils are labelled by green fluorescent protein (GFP) – to screen a natural product library for compounds that can affect neutrophil migratory behaviour. Among 1040 fungal extracts screened, two were found to inhibit neutrophil migration completely. Subfractionation of these extracts identified sterigmatocystin and antibiotic PF1052 as the active components. Using the EZ-TAXIScan chemotaxis assay, both compounds were also found to have a dosage-dependent inhibitory effect on murine neutrophil migration. Furthermore, neutrophils treated with PF1052 failed to form pseudopods and appeared round in shape, suggesting a defect in PI3-kinase (PI3K) signalling. We generated a transgenic neutrophil-specific PtdIns(3,4,5)P3 (PIP3) reporter zebrafish line, which revealed that PF1052 does not affect the activation of PI3K at the plasma membrane. In human neutrophils, PF1052 neither induced apoptosis nor blocked AKT phosphorylation. In conclusion, we have identified an antibiotic from a natural product library with potent anti-inflammatory properties, and have established the utility of the mpx:GFP transgenic zebrafish for high-throughput in vivo screens for novel inhibitors of neutrophil migration. PMID:24291762

  9. Microglia-Based Phenotypic Screening Identifies a Novel Inhibitor of Neuroinflammation Effective in Alzheimer's Disease Models.

    PubMed

    Zhou, Wei; Zhong, Guifa; Fu, Sihai; Xie, Hui; Chi, Tianyan; Li, Luyi; Rao, Xiurong; Zeng, Shaogao; Xu, Dengfeng; Wang, Hao; Sheng, Guoqing; Ji, Xing; Liu, Xiaorong; Ji, Xuefei; Wu, Donghai; Zou, Libo; Tortorella, Micky; Zhang, Kejian; Hu, Wenhui

    2016-11-16

    Currently, anti-AD drug discovery using target-based approaches is extremely challenging due to unclear etiology of AD and absence of validated therapeutic protein targets. Neuronal death, regardless of causes, plays a key role in AD progression, and it is directly linked to neuroinflammation. Meanwhile, phenotypic screening is making a resurgence in drug discovery process as an alternative to target-focused approaches. Herein, we employed microglia-based phenotypic screenings to search for small molecules that modulate the release of detrimental proinflammatory cytokines. The identified novel pharmacological inhibitor of neuroinflammation (named GIBH-130) was validated to alter phenotypes of neuroinflammation in AD brains. Notably, this molecule exhibited comparable in vivo efficacy of cognitive impairment relief to donepezil and memantine respectively in both β amyloid-induced and APP/PS1 double transgenic Alzheimer's murine models at a substantially lower dose (0.25 mg/kg). Therefore, GIBH-130 constitutes a unique chemical probe for pathogenesis research and drug development of AD, and it also suggests microglia-based phenotypic screenings that target neuroinflammation as an effective and feasible strategy to identify novel anti-AD agents.

  10. A novel yeast cell-based screen identifies flavone as a tankyrase inhibitor

    SciTech Connect

    Yashiroda, Yoko; Hatsugai, Kaori; Takemoto, Yasushi; Saito, Tamio; Sugimoto, Yoshikazu; Osada, Hiroyuki; Yoshida, Minoru

    2010-04-09

    The telomere-associated protein tankyrase 1 is a poly(ADP-ribose) polymerase and is considered to be a promising target for cancer therapy, especially for BRCA-associated cancers. However, an efficient assay system for inhibitor screening has not been established, mainly due to the difficulty of efficient preparation of the enzyme and its substrate. Here, we report a cell-based assay system for detecting inhibitory activity against tankyrase 1. We found that overexpression of the human tankyrase 1 gene causes a growth defect in the fission yeast Schizosaccharomyces pombe. Chemicals that restore the growth defect phenotype can be identified as potential tankyrase 1 inhibitors. We performed a high-throughput screen using this system, and identified flavone as a compound that restores the growth of yeast cells overexpressing tankyrase 1. Indeed, flavone inhibited poly(ADP-ribosyl)ation of proteins caused by overexpression of tankyrase 1 in yeast cells. This system allows rapid identification of inhibitory activity against tankyrase 1 and is amenable to high-throughput screening using robotics.

  11. The incidence and visual acuity outcomes of children identified with ametropic amblyopia by vision screening.

    PubMed

    Maqsud, Mohammed Aftab; Arblaster, Gemma E

    2015-04-01

    To determine the incidence of ametropic amblyopia within a vision screening program's population and report the visual acuity outcomes of children identified with the condition. The medical records of children who underwent vision screening as their first assessment at 4-5 years of age between September 1, 2005 and August 31, 2006, were retrospectively reviewed. Children referred with ≤0.30 logMAR in each eye with at least 1 year of follow-up had their hospital notes reviewed and data on final visual acuity, refractive error, and follow-up period collected. A total of 33 children identified as having ametropic amblyopia with a follow-up of at least 1 year. The incidence of ametropic amblyopia was 2%-3.2%, depending on the definition used. The mean visual acuity achieved after treatment was 0.12 logMAR, which is significantly less than the age-appropriate mean of 0.00 logMAR (P < 0.01). Ametropic amblyopia responds to treatment, but most children demonstrate persistent reduced visual acuity at age 7 years. The incidence of ametropic amblyopia within a routine vision screening population shows that significant numbers fail to self-present. Copyright © 2015 American Association for Pediatric Ophthalmology and Strabismus. Published by Elsevier Inc. All rights reserved.

  12. A new screening pathway for identifying asymptomatic patients using dental panoramic radiographs

    NASA Astrophysics Data System (ADS)

    Hayashi, Tatsuro; Matsumoto, Takuya; Sawagashira, Tsuyoshi; Tagami, Motoki; Katsumata, Akitoshi; Hayashi, Yoshinori; Muramatsu, Chisako; Zhou, Xiangrong; Iida, Yukihiro; Matsuoka, Masato; Katagi, Kiyoji; Fujita, Hiroshi

    2012-03-01

    To identify asymptomatic patients is the challenging task and the essential first step in diagnosis. Findings of dental panoramic radiographs include not only dental conditions but also radiographic signs that are suggestive of possible systemic diseases such as osteoporosis, arteriosclerosis, and maxillary sinusitis. Detection of such signs on panoramic radiographs has a potential to provide supplemental benefits for patients. However, it is not easy for general dental practitioners to pay careful attention to such signs. We addressed the development of a computer-aided detection (CAD) system that detects radiographic signs of pathology on panoramic images, and the design of the framework of new screening pathway by cooperation of dentists and our CAD system. The performance evaluation of our CAD system showed the sensitivity and specificity in the identification of osteoporotic patients were 92.6 % and 100 %, respectively, and those of the maxillary sinus abnormality were 89.6 % and 73.6 %, respectively. The detection rate of carotid artery calcifications that suggests the need for further medical evaluation was approximately 93.6 % with 4.4 false-positives per image. To validate the utility of the new screening pathway, preliminary clinical trials by using our CAD system were conducted. To date, 223 panoramic images were processed and 4 asymptomatic patients with suspected osteoporosis, 7 asymptomatic patients with suspected calcifications, and 40 asymptomatic patients with suspected maxillary sinusitis were detected in our initial trial. It was suggested that our new screening pathway could be useful to identify asymptomatic patients with systemic diseases.

  13. RNAi screens identify CHD4 as an essential gene in breast cancer growth

    PubMed Central

    Cicalese, Angelo; Fornasari, Lorenzo; Furia, Laura; Riva, Laura; Carugo, Alessandro; Curigliano, Giuseppe; Criscitiello, Carmen; Pruneri, Giancarlo; Pelicci, Pier Giuseppe; Faretta, Mario; Bossi, Daniela; Lanfrancone, Luisa

    2016-01-01

    Epigenetic regulation plays an essential role in tumor development and epigenetic modifiers are considered optimal potential druggable candidates. In order to identify new breast cancer vulnerabilities and improve therapeutic chances for patients, we performed in vivo and in vitro shRNA screens in a human breast cancer cell model (MCF10DCIS.com cell line) using epigenetic libraries. Among the genes identified in our screening, we deeply investigated the role of Chromodomain Helicase DNA binding Protein 4 (CHD4) in breast cancer tumorigenesis. CHD4 silencing significantly reduced tumor growth in vivo and proliferation in vitro of MCF10DCIS.com cells. Similarly, in vivo breast cancer growth was decreased in a spontaneous mouse model of breast carcinoma (MMTV-NeuT system) and in metastatic patient-derived xenograft models. Conversely, no reduction in proliferative ability of non-transformed mammary epithelial cells (MCF10A) was detected. Moreover, we showed that CHD4 depletion arrests proliferation by inducing a G0/G1 block of cell cycle associated with up-regulation of CDKN1A (p21). These results highlight the relevance of genetic screens in the identification of tumor frailties and the role of CHD4 as a potential pharmacological target to inhibit breast cancer growth. PMID:27779108

  14. High-Throughput Phenotypic Screening of Kinase Inhibitors to Identify Drug Targets for Polycystic Kidney Disease

    PubMed Central

    Booij, Tijmen H.; Bange, Hester; Leonhard, Wouter N.; Yan, Kuan; Fokkelman, Michiel; Kunnen, Steven J.; Dauwerse, Johannes G.; Qin, Yu; van de Water, Bob; van Westen, Gerard J. P.; Peters, Dorien J. M.; Price, Leo S.

    2017-01-01

    Polycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most interventions still focus on alleviating PKD-associated symptoms. The mechanistic complexity of the disease, as well as the lack of functional in vitro assays for compound testing, has made drug discovery for PKD challenging. To identify modulators of PKD, Pkd1–/– kidney tubule epithelial cells were applied to a scalable and automated 3D cyst culture model for compound screening, followed by phenotypic profiling to determine compound efficacy. We used this screening platform to screen a library of 273 kinase inhibitors to probe various signaling pathways involved in cyst growth. We show that inhibition of several targets, including aurora kinase, CDK, Chk, IGF-1R, Syk, and mTOR, but, surprisingly, not PI3K, prevented forskolin-induced cyst swelling. Additionally, we show that multiparametric phenotypic classification discriminated potentially undesirable (i.e., cytotoxic) compounds from molecules inducing the desired phenotypic change, greatly facilitating hit selection and validation. Our findings show that a pathophysiologically relevant 3D cyst culture model of PKD coupled to phenotypic profiling can be used to identify potentially therapeutic compounds and predict and validate molecular targets for PKD. PMID:28644734

  15. Optical pre-screening in breast screening programs: Can we identify women who benefit most from limited mammography resources?

    NASA Astrophysics Data System (ADS)

    Walter, Jane; Loshchenov, Maxim; Zhilkin, Vladimir; Peake, Rachel; Stone, Jennifer; Lilge, Lothar

    2017-04-01

    Background: In excess of 60% of all cancers are detected in low and middle-income countries, with breast cancer (BC) the dominant malignancy for women. Incidence rates continue to climb, most noticeably in the less than 50-year-old population. Expansion of mammography infrastructure and resources is lacking, resulting in over 60% of women diagnosed with stage III/IV BC in the majority of these countries. Optical Breast Spectroscopy (OBS) was shown to correlate well with mammographic breast density (MBD). OBS could aid breast screening programs in low- and middle-income countries by lowering the number of mammographs required for complete population coverage. However, its performance needs to be tested in large population trails to ensure high sensitivity and acceptable specificity. Methods: For the planned studies in low- and middle-income countries in different continents, online methods need to be implemented to monitor the performance and data collection by these devices, operated by trained nurses. Based on existing datasets, procedures were developed to validate an individual woman's data integrity and to identify operator errors versus system malfunctions. Results: Using a dataset comprising spectra from 360 women collected by 2 instruments in different locations and with 3 different trained operators, automated methods were developed to identify 100% of the source or photodetector malfunctions as well as incorrect calibrations and 96% of instances of insufficient tissue contact. Conclusions: Implementing the dataset validation locally in each instrument and tethered to a cloud database will allow the planned clinical trials to proceed.

  16. An open-access endoscopy screen correctly and safely identifies patients for conscious sedation.

    PubMed

    Kothari, Darshan; Feuerstein, Joseph D; Moss, Laureen; D'Souza, Julie; Montanaro, Kerri; Leffler, Daniel A; Sheth, Sunil G

    2016-11-01

    Open-access scheduling is highly utilized for facilitating generally low-risk endoscopies. Preprocedural screening addresses sedation requirements; however, procedural safety may be compromised if screening is inaccurate. We sought to determine the reliability of our open-access scheduling system for appropriate use of conscious sedation. We prospectively and consecutively enrolled outpatient procedures booked at an academic center by open-access using screening after in-office gastroenterology (GI) consultation. We collected the cases inappropriately booked for conscious sedation and compared the characteristics for significant differences. A total of 8063 outpatients were scheduled for procedures with conscious sedation, and 5959 were booked with open-access. Only 78 patients (0.97%, 78/8063) were identified as subsequently needing anesthesiologist-assisted sedation; 44 (56.4%, 44/78) were booked through open-access, of which chronic opioid (47.7%, 21/44) or benzodiazepine use (34.1%, 15/44) were the most common reasons for needing anesthesiologist-assisted sedation. Patients on chronic benzodiazepines required more midazolam than those not on chronic benzodiazepines (P = .03) of those patients who underwent conscious sedation. Similarly, patients with chronic opioid use required more fentanyl than those without chronic opioid use (P = .04). Advanced liver disease and alcohol use were common reasons for patients being booked after in-office consultation and were significantly higher than those booked with open-access (both P < .01). We observed that the majority of patients can be triaged for conscious sedation using a multi-tiered screening process. Importantly, few patients (<1.0%) were inappropriately booked for conscious sedation. The most common reasons for considering anesthesiologist-assisted sedation were chronic opioid, benzodiazepine and/or alcohol use and advanced liver disease. This suggests that these entities could be included in screening processes

  17. Genome-wide Linkage Screen in Familial Parkinson Disease Identifies Loci on Chromosomes 3 and 18

    PubMed Central

    Gao, Xiaoyi; Martin, Eden R.; Liu, Yutao; Mayhew, Gregory; Vance, Jeffery M.; Scott, William K.

    2009-01-01

    Parkinson disease (PD) is a complex, multifactorial neurodegenerative disease with substantial evidence for genetic risk factors. We conducted a genome-wide linkage screen of 5824 single-nucleotide polymorphisms in 278 families of European, non-Hispanic descent to localize regions that harbor susceptibility loci for PD. By using parametric and nonparametric linkage analyses and allowing for genetic heterogeneity among families, we found two loci for PD. Significant evidence for linkage was detected on chromosome 18q11 (maximum lod score [MLOD] = 4.1) and suggestive evidence for linkage was obtained on chromosome 3q25 (MLOD = 2.5). These results were strongest in families not previously screened for linkage, and simulation studies suggest that these findings are likely due to locus heterogeneity rather than random statistical error. The finding of two loci (one highly statistically significant) suggests that additional PD susceptibility genes might be identified through targeted candidate gene studies in these regions. PMID:19327735

  18. Novel inhibitors to Taenia solium Cu/Zn superoxide dismutase identified by virtual screening

    NASA Astrophysics Data System (ADS)

    García-Gutiérrez, P.; Landa-Piedra, A.; Rodríguez-Romero, A.; Parra-Unda, R.; Rojo-Domínguez, A.

    2011-12-01

    We describe in this work a successful virtual screening and experimental testing aimed to the identification of novel inhibitors of superoxide dismutase of the worm Taenia solium ( TsCu/Zn-SOD), a human parasite. Conformers from LeadQuest® database of drug-like compounds were selected and then docked on the surface of TsCu/Zn-SOD. Results were screened looking for ligand contacts with receptor side-chains not conserved in the human homologue, with a subsequent development of a score optimization by a set of energy minimization steps, aimed to identify lead compounds for in vitro experiments. Six out of fifty experimentally tested compounds showed μM inhibitory activity toward TsCu/Zn-SOD. Two of them showed species selectivity since did not inhibit the homologous human enzyme when assayed in vitro.

  19. Novel inhibitors to Taenia solium Cu/Zn superoxide dismutase identified by virtual screening.

    PubMed

    García-Gutiérrez, P; Landa-Piedra, A; Rodríguez-Romero, A; Parra-Unda, R; Rojo-Domínguez, A

    2011-12-01

    We describe in this work a successful virtual screening and experimental testing aimed to the identification of novel inhibitors of superoxide dismutase of the worm Taenia solium (TsCu/Zn-SOD), a human parasite. Conformers from LeadQuest(®) database of drug-like compounds were selected and then docked on the surface of TsCu/Zn-SOD. Results were screened looking for ligand contacts with receptor side-chains not conserved in the human homologue, with a subsequent development of a score optimization by a set of energy minimization steps, aimed to identify lead compounds for in vitro experiments. Six out of fifty experimentally tested compounds showed μM inhibitory activity toward TsCu/Zn-SOD. Two of them showed species selectivity since did not inhibit the homologous human enzyme when assayed in vitro.

  20. A Chemical Screening Approach to Identify Novel Key Mediators of Erythroid Enucleation.

    PubMed

    Wölwer, Christina B; Pase, Luke B; Pearson, Helen B; Gödde, Nathan J; Lackovic, Kurt; Huang, David C S; Russell, Sarah M; Humbert, Patrick O

    2015-01-01

    Erythroid enucleation is critical for terminal differentiation of red blood cells, and involves extrusion of the nucleus by orthochromatic erythroblasts to produce reticulocytes. Due to the difficulty of synchronizing erythroblasts, the molecular mechanisms underlying the enucleation process remain poorly understood. To elucidate the cellular program governing enucleation, we utilized a novel chemical screening approach whereby orthochromatic cells primed for enucleation were enriched ex vivo and subjected to a functional drug screen using a 324 compound library consisting of structurally diverse, medicinally active and cell permeable drugs. Using this approach, we have confirmed the role of HDACs, proteasomal regulators and MAPK in erythroid enucleation and introduce a new role for Cyclin-dependent kinases, in particular CDK9, in this process. Importantly, we demonstrate that when coupled with imaging analysis, this approach provides a powerful means to identify and characterize rate limiting steps involved in the erythroid enucleation process.

  1. Tetrandrine identified in a small molecule screen to activate mesenchymal stem cells for enhanced immunomodulation.

    PubMed

    Yang, Zijiang; Concannon, John; Ng, Kelvin S; Seyb, Kathleen; Mortensen, Luke J; Ranganath, Sudhir; Gu, Fangqi; Levy, Oren; Tong, Zhixiang; Martyn, Keir; Zhao, Weian; Lin, Charles P; Glicksman, Marcie A; Karp, Jeffrey M

    2016-07-26

    Pre-treatment or priming of mesenchymal stem cells (MSC) prior to transplantation can significantly augment the immunosuppressive effect of MSC-based therapies. In this study, we screened a library of 1402 FDA-approved bioactive compounds to prime MSC. We identified tetrandrine as a potential hit that activates the secretion of prostaglandin E2 (PGE2), a potent immunosuppressive agent, by MSC. Tetrandrine increased MSC PGE2 secretion through the NF-κB/COX-2 signaling pathway. When co-cultured with mouse macrophages (RAW264.7), tetrandrine-primed MSC attenuated the level of TNF-α secreted by RAW264.7. Furthermore, systemic transplantation of primed MSC into a mouse ear skin inflammation model significantly reduced the level of TNF-α in the inflamed ear, compared to unprimed cells. Screening of small molecules to pre-condition cells prior to transplantation represents a promising strategy to boost the therapeutic potential of cell therapy.

  2. A forward genetic screen identifies erythrocyte CD55 as essential for Plasmodium falciparum invasion **

    PubMed Central

    Egan, Elizabeth S.; Jiang, Rays H.Y.; Moechtar, Mischka A.; Barteneva, Natasha S.; Weekes, Michael P.; Nobre, Luis V.; Gygi, Steven P.; Paulo, Joao A.; Frantzreb, Charles; Tani, Yoshihiko; Takahashi, Junko; Watanabe, Seishi; Goldberg, Jonathan; Paul, Aditya S.; Brugnara, Carlo; Root, David E.; Wiegand, Roger C.; Doench, John G.; Duraisingh, Manoj T.

    2015-01-01

    Efforts to identify host determinants for malaria have been hindered by the absence of a nucleus in erythrocytes, precluding genetic manipulation in the cell where the parasite replicates. We used cultured red blood cells derived from hematopoietic stem cells to carry out a forward genetic screen for Plasmodium falciparum host determinants. We found that CD55 is an essential host factor for P. falciparum invasion. CD55-null erythrocytes were refractory to invasion by all isolates of P. falciparum because parasites failed to attach properly to the erythrocyte surface. Thus, CD55 is an attractive target for the development of malaria therapeutics. Hematopoietic stem cell-based forward genetic screens may be valuable for the identification of additional host determinants of malaria pathogenesis. PMID:25954012

  3. A genetic screen for zebrafish mutants with hepatic steatosis identifies a locus required for larval growth.

    PubMed

    Hugo, Sarah E; Schlegel, Amnon

    2017-03-01

    In a screen for zebrafish larval mutants with excessive liver lipid accumulation (hepatic steatosis), we identified harvest moon (hmn). Cytoplasmic lipid droplets, surrounded by multivesicular structures and mitochondria whose cristae appeared swollen, are seen in hmn mutant hepatocytes. Whole body triacylglycerol is increased in hmn mutant larvae. When we attempted to raise mutants, which were morphologically normal at the developmental stage that the screen was conducted, to adulthood, we observed that most hmn mutants do not survive to the juvenile period when raised. An arrest in growth occurs in the late larval period without obvious organ defects. Maternal zygotic mutants have no additional defects, suggesting that the mutation affects a late developmental process. The developmental window between embryogenesis and the metamorphosis remains under-studied, and hmn mutants might be useful for exploring the molecular and anatomic processes occurring during this transition period.

  4. Tetrandrine identified in a small molecule screen to activate mesenchymal stem cells for enhanced immunomodulation

    PubMed Central

    Yang, Zijiang; Concannon, John; Ng, Kelvin S.; Seyb, Kathleen; Mortensen, Luke J.; Ranganath, Sudhir; Gu, Fangqi; Levy, Oren; Tong, Zhixiang; Martyn, Keir; Zhao, Weian; Lin, Charles P.; Glicksman, Marcie A.; Karp, Jeffrey M.

    2016-01-01

    Pre-treatment or priming of mesenchymal stem cells (MSC) prior to transplantation can significantly augment the immunosuppressive effect of MSC-based therapies. In this study, we screened a library of 1402 FDA-approved bioactive compounds to prime MSC. We identified tetrandrine as a potential hit that activates the secretion of prostaglandin E2 (PGE2), a potent immunosuppressive agent, by MSC. Tetrandrine increased MSC PGE2 secretion through the NF-κB/COX-2 signaling pathway. When co-cultured with mouse macrophages (RAW264.7), tetrandrine-primed MSC attenuated the level of TNF-α secreted by RAW264.7. Furthermore, systemic transplantation of primed MSC into a mouse ear skin inflammation model significantly reduced the level of TNF-α in the inflamed ear, compared to unprimed cells. Screening of small molecules to pre-condition cells prior to transplantation represents a promising strategy to boost the therapeutic potential of cell therapy. PMID:27457881

  5. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

    NASA Astrophysics Data System (ADS)

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-03-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery.

  6. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

    PubMed Central

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-01-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery. PMID:27010513

  7. Combinatorial Library Screening Coupled to Mass Spectrometry to Identify Valuable Cyclic Peptides.

    PubMed

    Camperi, Silvia A; Giudicessi, Silvana L; Martínez-Ceron, María C; Gurevich-Messina, Juan M; Saavedra, Soledad L; Acosta, Gerardo; Cascone, Osvaldo; Erra-Balsells, Rosa; Albericio, Fernando

    2016-06-02

    Combinatorial library screening coupled to mass spectrometry (MS) analysis is a practical approach to identify useful peptides. Cyclic peptides can have high biological activity, selectivity, and affinity for target proteins, and high stability against proteolytic degradation. Here we describe two strategies to prepare combinatorial libraries suitable for MS analysis to accelerate the discovery of cyclic peptide structures. Both approaches use ChemMatrix resin and the linker 4-hydroxymethylbenzoic acid. One strategy involves the synthesis of a one-bead-two-peptides library in which each bead contains both the cyclic peptide and its linear counterpart to facilitate MS analysis. The other protocol is based on the synthesis of a cyclic depsipeptide library in which a glycolamidic ester group is incorporated by adding glycolic acid. After library screening, the ring is opened and the peptide is released simultaneously for subsequent MS analysis. © 2016 by John Wiley & Sons, Inc.

  8. High Throughput Screen Identifies Small Molecule Inhibitors Specific for Mycobacterium tuberculosis Phosphoserine Phosphatase*

    PubMed Central

    Arora, Garima; Tiwari, Prabhakar; Mandal, Rahul Shubhra; Gupta, Arpit; Sharma, Deepak; Saha, Sudipto; Singh, Ramandeep

    2014-01-01

    The emergence of drug-resistant strains of Mycobacterium tuberculosis makes identification and validation of newer drug targets a global priority. Phosphoserine phosphatase (PSP), a key essential metabolic enzyme involved in conversion of O-phospho-l-serine to l-serine, was characterized in this study. The M. tuberculosis genome harbors all enzymes involved in l-serine biosynthesis including two PSP homologs: Rv0505c (SerB1) and Rv3042c (SerB2). In the present study, we have biochemically characterized SerB2 enzyme and developed malachite green-based high throughput assay system to identify SerB2 inhibitors. We have identified 10 compounds that were structurally different from known PSP inhibitors, and few of these scaffolds were highly specific in their ability to inhibit SerB2 enzyme, were noncytotoxic against mammalian cell lines, and inhibited M. tuberculosis growth in vitro. Surface plasmon resonance experiments demonstrated the relative binding for these inhibitors. The two best hits identified in our screen, clorobiocin and rosaniline, were bactericidal in activity and killed intracellular bacteria in a dose-dependent manner. We have also identified amino acid residues critical for these SerB2-small molecule interactions. This is the first study where we validate that M. tuberculosis SerB2 is a druggable and suitable target to pursue for further high throughput assay system screening. PMID:25037224

  9. Crizotinib-Resistant Mutants of EML4-ALK Identified Through an Accelerated Mutagenesis Screen

    PubMed Central

    Zhang, Sen; Wang, Frank; Keats, Jeffrey; Zhu, Xiaotian; Ning, Yaoyu; Wardwell, Scott D; Moran, Lauren; Mohemmad, Qurish K; Anjum, Rana; Wang, Yihan; Narasimhan, Narayana I; Dalgarno, David; Shakespeare, William C; Miret, Juan J; Clackson, Tim; Rivera, Victor M

    2011-01-01

    Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance. PMID:22034911

  10. Modulators of Prostate Cancer Cell Proliferation and Viability Identified by Short-Hairpin RNA Library Screening

    PubMed Central

    Dahlman, Kimberly Brown; Parker, Joel S.; Shamu, Tambudzai; Hieronymus, Haley; Chapinski, Caren; Carver, Brett; Chang, Kenneth; Hannon, Gregory J.; Sawyers, Charles L.

    2012-01-01

    There is significant need to identify novel prostate cancer drug targets because current hormone therapies eventually fail, leading to a drug-resistant and fatal disease termed castration-resistant prostate cancer. To functionally identify genes that, when silenced, decrease prostate cancer cell proliferation or induce cell death in combination with antiandrogens, we employed an RNA interference-based short hairpin RNA barcode screen in LNCaP human prostate cancer cells. We identified and validated four candidate genes (AKT1, PSMC1, STRADA, and TTK) that impaired growth when silenced in androgen receptor positive prostate cancer cells and enhanced the antiproliferative effects of antiandrogens. Inhibition of AKT with a pharmacologic inhibitor also induced apoptosis when combined with antiandrogens, consistent with recent evidence for PI3K and AR pathway crosstalk in prostate cancer cells. Recovery of hairpins targeting a known prostate cancer pathway validates the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets. PMID:22509301

  11. High throughput screen identifies small molecule inhibitors specific for Mycobacterium tuberculosis phosphoserine phosphatase.

    PubMed

    Arora, Garima; Tiwari, Prabhakar; Mandal, Rahul Shubhra; Gupta, Arpit; Sharma, Deepak; Saha, Sudipto; Singh, Ramandeep

    2014-09-05

    The emergence of drug-resistant strains of Mycobacterium tuberculosis makes identification and validation of newer drug targets a global priority. Phosphoserine phosphatase (PSP), a key essential metabolic enzyme involved in conversion of O-phospho-l-serine to l-serine, was characterized in this study. The M. tuberculosis genome harbors all enzymes involved in l-serine biosynthesis including two PSP homologs: Rv0505c (SerB1) and Rv3042c (SerB2). In the present study, we have biochemically characterized SerB2 enzyme and developed malachite green-based high throughput assay system to identify SerB2 inhibitors. We have identified 10 compounds that were structurally different from known PSP inhibitors, and few of these scaffolds were highly specific in their ability to inhibit SerB2 enzyme, were noncytotoxic against mammalian cell lines, and inhibited M. tuberculosis growth in vitro. Surface plasmon resonance experiments demonstrated the relative binding for these inhibitors. The two best hits identified in our screen, clorobiocin and rosaniline, were bactericidal in activity and killed intracellular bacteria in a dose-dependent manner. We have also identified amino acid residues critical for these SerB2-small molecule interactions. This is the first study where we validate that M. tuberculosis SerB2 is a druggable and suitable target to pursue for further high throughput assay system screening.

  12. Modulators of prostate cancer cell proliferation and viability identified by short-hairpin RNA library screening.

    PubMed

    Dahlman, Kimberly Brown; Parker, Joel S; Shamu, Tambudzai; Hieronymus, Haley; Chapinski, Caren; Carver, Brett; Chang, Kenneth; Hannon, Gregory J; Sawyers, Charles L

    2012-01-01

    There is significant need to identify novel prostate cancer drug targets because current hormone therapies eventually fail, leading to a drug-resistant and fatal disease termed castration-resistant prostate cancer. To functionally identify genes that, when silenced, decrease prostate cancer cell proliferation or induce cell death in combination with antiandrogens, we employed an RNA interference-based short hairpin RNA barcode screen in LNCaP human prostate cancer cells. We identified and validated four candidate genes (AKT1, PSMC1, STRADA, and TTK) that impaired growth when silenced in androgen receptor positive prostate cancer cells and enhanced the antiproliferative effects of antiandrogens. Inhibition of AKT with a pharmacologic inhibitor also induced apoptosis when combined with antiandrogens, consistent with recent evidence for PI3K and AR pathway crosstalk in prostate cancer cells. Recovery of hairpins targeting a known prostate cancer pathway validates the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets.

  13. Duplex High Throughput Flow Cytometry Screen Identifies Two Novel Formylpeptide Receptor Family Probes1

    PubMed Central

    Young, Susan M.; Bologa, Cristian M.; Fara, Dan; Bryant, Bj K.; Strouse, J. Jacob; Arterburn, Jeffrey B.; Ye, Richard D.; Oprea, Tudor I.; Prossnitz, Eric R.; Sklar, Larry A.; Edwards, Bruce S.

    2008-01-01

    Introduction Of recent clinical interest have been two related human G-protein coupled receptors: formylpeptide receptor (FPR), linked to anti-bacterial inflammation and malignant glioma cell metastasis; and formylpeptide receptor 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. Methods 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 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 ∼4000 cells (∼2000/receptor) in a 2 μL aspirate from each well. Results/Conclusions 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

  14. Duplex high-throughput flow cytometry screen identifies two novel formylpeptide receptor family probes.

    PubMed

    Young, Susan M; Bologa, Cristian M; Fara, Dan; Bryant, Bj K; Strouse, Juan Jacob; Arterburn, Jeffrey B; Ye, Richard D; Oprea, Tudor I; Prossnitz, Eric R; Sklar, Larry A; Edwards, Bruce S

    2009-03-01

    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 microl assay volumes in 384-well plates. Throughput averaged approximately 11 min per plate to analyze approximately 4,000 cells ( approximately 2,000/receptor) in a 2 microl 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 (K(i)) < or = 4 microM (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, K(i) = 95 +/- 10 nM) and FPRL1 (BB-V-115, K(i) = 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

  15. An RNAi Screen To Identify Protein Phosphatases That Function Within the Drosophila Circadian Clock.

    PubMed

    Agrawal, Parul; Hardin, Paul E

    2016-12-07

    Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. A well-characterized example of such a transcriptional feedback loop is in Drosophila, where CLOCK-CYCLE (CLK-CYC) complexes activate transcription of period (per) and timeless (tim) genes, rising levels of PER-TIM complexes feed-back to repress CLK-CYC activity, and degradation of PER and TIM permits the next cycle of CLK-CYC transcription. The timing of CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Previous behavioral screens identified several kinases that control CLK, PER, and TIM levels, subcellular localization, and/or activity, but two phosphatases that function within the clock were identified through the analysis of candidate genes from other pathways or model systems. To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi) knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. This screen identified 19 protein phosphatases that lengthened or shortened the circadian period by ≥1 hr (p ≤ 0.05 compared to controls) or were arrhythmic. Additional RNAi lines, transposon inserts, overexpression, and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. Based on genetic validation and molecular analysis, 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans.

  16. Novel high-throughput screen against Candida albicans identifies antifungal potentiators and agents effective against biofilms

    PubMed Central

    LaFleur, Michael D.; Lucumi, Edinson; Napper, Andrew D.; Diamond, Scott L.; Lewis, Kim

    2011-01-01

    Objectives Microbial adhesion and biofilms have important implications for human health and disease. Candida albicans is an opportunistic pathogen which forms drug-resistant biofilms that contribute to the recalcitrance of disease. We have developed a high-throughput screen for potentiators of clotrimazole, a common therapy for Candida infections, including vaginitis and thrush. The screen was performed against C. albicans biofilms grown in microtitre plates in order to target the most resilient forms of the pathogen. Methods Biofilm growth, in individual wells of 384-well plates, was measured using the metabolic indicator alamarBlue® and found to be very consistent and reproducible. This assay was used to test the effect of more than 120 000 small molecule compounds from the NIH Molecular Libraries Small Molecule Repository, and compounds that enhanced the activity of clotrimazole or acted on the biofilms alone were identified as hits. Results Nineteen compounds (0.016% hit rate) were identified and found to cause more than 30% metabolic inhibition of biofilms compared with clotrimazole alone, which had a modest effect on biofilm viability at the concentration tested. Hits were confirmed for activity against biofilms with dose–response measurements. Several compounds had increased activity in combination with clotrimazole, including a 1,3-benzothiazole scaffold that exhibited a >100-fold improvement against biofilms of three separate C. albicans isolates. Cytotoxicity experiments using human fibroblasts confirmed the presence of lead molecules with favourable antifungal activity relative to cytotoxicity. Conclusions We have validated a novel approach to identify antifungal potentiators and completed a high-throughput screen to identify small molecules with activity against C. albicans biofilms. These small molecules may specifically target the biofilm and make currently available antifungals more effective. PMID:21393183

  17. An RNAi Screen To Identify Protein Phosphatases That Function Within the Drosophila Circadian Clock

    PubMed Central

    Agrawal, Parul; Hardin, Paul E.

    2016-01-01

    Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. A well-characterized example of such a transcriptional feedback loop is in Drosophila, where CLOCK-CYCLE (CLK-CYC) complexes activate transcription of period (per) and timeless (tim) genes, rising levels of PER-TIM complexes feed-back to repress CLK-CYC activity, and degradation of PER and TIM permits the next cycle of CLK-CYC transcription. The timing of CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Previous behavioral screens identified several kinases that control CLK, PER, and TIM levels, subcellular localization, and/or activity, but two phosphatases that function within the clock were identified through the analysis of candidate genes from other pathways or model systems. To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi) knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. This screen identified 19 protein phosphatases that lengthened or shortened the circadian period by ≥1 hr (p ≤ 0.05 compared to controls) or were arrhythmic. Additional RNAi lines, transposon inserts, overexpression, and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. Based on genetic validation and molecular analysis, 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans. PMID:27784754

  18. Quantitative High-Throughput Screen Identifies Inhibitors of the Schistosoma mansoni Redox Cascade

    PubMed Central

    Simeonov, Anton; Jadhav, Ajit; Sayed, Ahmed A.; Wang, Yuhong; Nelson, Michael E.; Thomas, Craig J.; Inglese, James; Williams, David L.; Austin, Christopher P.

    2008-01-01

    Schistosomiasis is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease, and with its increased use the probability of developing drug resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principal components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR) and peroxiredoxin (Prx) and as such these enzymes present attractive new targets for anti-schistosomiasis drug development. Inhibition of TGR/Prx activity was screened in a dual-enzyme format with reducing equivalents being transferred from NADPH to glutathione via a TGR-catalyzed reaction and then to hydrogen peroxide via a Prx-catalyzed step. A fully automated quantitative high-throughput (qHTS) experiment was performed against a collection of 71,028 compounds tested as 7- to 15-point concentration series at 5 µL reaction volume in 1536-well plate format. In order to generate a robust data set and to minimize the effect of compound autofluorescence, apparent reaction rates derived from a kinetic read were utilized instead of end-point measurements. Actives identified from the screen, along with previously untested analogues, were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in secondary assays. Several novel active series were identified which inhibited TGR at a range of potencies, with IC50s ranging from micromolar to the assay response limit (∼25 nM). This is, to our knowledge, the first report of a large-scale HTS to identify lead compounds for a helminthic disease, and provides a paradigm that can be used to jump-start development of novel

  19. Phenotypic screen quantifying differential regulation of cardiac myocyte hypertrophy identifies CITED4 regulation of myocyte elongation

    PubMed Central

    Ryall, Karen A.; Bezzerides, Vassilios J.; Rosenzweig, Anthony; Saucerman, Jeffrey J.

    2014-01-01

    Cardiac hypertrophy is controlled by a highly connected signaling network with many effectors of cardiac myocyte size. Quantification of the contribution of individual pathways to specific changes in shape and transcript abundance is needed to better understand hypertrophy signaling and to improve heart failure therapies. We stimulated cardiac myocytes with 15 hypertrophic agonists and quantitatively characterized differential regulation of 5 shape features using high-throughput microscopy and transcript levels of 12 genes using qPCR. Transcripts measured were associated with phenotypes including fibrosis, cell death, contractility, proliferation, angiogenesis, inflammation, and the fetal cardiac gene program. While hypertrophy pathways are highly connected, the agonist screen revealed distinct hypertrophy phenotypic signatures for the 15 receptor agonists. We then used k-means clustering of inputs and outputs to identify a network map linking input modules to output modules. Five modules were identified within inputs and outputs with many maladaptive outputs grouping together in one module: Bax, C/EBPβ, Serca2a, TNFα, and CTGF. Subsequently, we identified mechanisms underlying two correlations revealed in the agonist screen: correlation between regulators of fibrosis and cell death signaling (CTGF and Bax mRNA) caused by AngII; and myocyte proliferation (CITED4 mRNA) and elongation caused by Nrg1. Follow-up experiments revealed positive regulation of Bax mRNA level by CTGF and an incoherent feedforward loop linking Nrg1, CITED4 and elongation. With this agonist screen, we identified the most influential inputs in the cardiac hypertrophy signaling network for a variety of features related to pathological and protective hypertrophy signaling and shared regulation among cardiac myocyte phenotypes. PMID:24613264

  20. Combined Gene Expression and RNAi Screening to Identify Alkylation Damage Survival Pathways from Fly to Human.

    PubMed

    Zanotto-Filho, Alfeu; Dashnamoorthy, Ravi; Loranc, Eva; de Souza, Luis H T; Moreira, José C F; Suresh, Uthra; Chen, Yidong; Bishop, Alexander J R

    2016-01-01

    Alkylating agents are a key component of cancer chemotherapy. Several cellular mechanisms are known to be important for its survival, particularly DNA repair and xenobiotic detoxification, yet genomic screens indicate that additional cellular components may be involved. Elucidating these components has value in either identifying key processes that can be modulated to improve chemotherapeutic efficacy or may be altered in some cancers to confer chemoresistance. We therefore set out to reevaluate our prior Drosophila RNAi screening data by comparison to gene expression arrays in order to determine if we could identify any novel processes in alkylation damage survival. We noted a consistent conservation of alkylation survival pathways across platforms and species when the analysis was conducted on a pathway/process level rather than at an individual gene level. Better results were obtained when combining gene lists from two datasets (RNAi screen plus microarray) prior to analysis. In addition to previously identified DNA damage responses (p53 signaling and Nucleotide Excision Repair), DNA-mRNA-protein metabolism (transcription/translation) and proteasome machinery, we also noted a highly conserved cross-species requirement for NRF2, glutathione (GSH)-mediated drug detoxification and Endoplasmic Reticulum stress (ER stress)/Unfolded Protein Responses (UPR) in cells exposed to alkylation. The requirement for GSH, NRF2 and UPR in alkylation survival was validated by metabolomics, protein studies and functional cell assays. From this we conclude that RNAi/gene expression fusion is a valid strategy to rapidly identify key processes that may be extendable to other contexts beyond damage survival.

  1. Gene expression profiling identifies Fibronectin 1 and CXCL9 as candidate biomarkers for breast cancer screening

    PubMed Central

    Ruiz-Garcia, E; Scott, V; Machavoine, C; Bidart, J M; Lacroix, L; Delaloge, S; Andre, F

    2010-01-01

    Background: There is a need to develop blood-based bioassays for breast cancer (BC) screening. In this study, differential gene expression between BC samples and benign tumours was used to identify candidate biomarkers for blood-based screening. Methods: We identified two proteins (Fibronectin 1 and CXCL9) from a gene expression data set that included 120 BC samples and 45 benign lesions. These proteins fulfil the following criteria: differential gene expression between cancer and benign lesion, protein released in the extracellular medium and stable in the serum, commercially available ELISA kit, ELISA accuracy in a feasibility study. Protein concentrations were determined by ELISA. Blood samples were from normal volunteers (n=119) and early BC patients (n=133). Results: Seventy-three per cent of patients had cT1-T2 tumour. Patients had higher CXCL9 and Fibronectin 1 concentrations than volunteers. CXCL9 mean concentration was 851 and 635 pg ml−1 for patients and volunteers respectively (P=0.013). CXCL9 concentration was significantly higher in patients with estrogen receptor (ER)-negative compared with volunteers (P=0.003), data consistent with gene expression profile. Fibronectin 1 mean concentration was 190 μg ml−1 for patients and 125 μg ml−1 for volunteers (P<0.001). Areas under the curve for BC diagnosis were 0.78 and 0.62 for Fibronectin 1 and CXCL9 respectively. A combined score including Fibronectin 1 and CXCL9 dosages presented 53% of sensitivity and 98% of specificity. Similar performances were observed for ER-negative tumours. Conclusions: This study suggests that Fibronectin 1/CXCL9 dosage in serum could screen a significant rate of BC, including ER-negative, and that differential gene expression analysis is a good approach to select candidate biomarkers to set up blood assays cancer screening. PMID:20068563

  2. Accuracy of Brief Screening Tools for Identifying Postpartum Depression Among Adolescent Mothers

    PubMed Central

    Venkatesh, Kartik K.; Zlotnick, Caron; Triche, Elizabeth W.; Ware, Crystal

    2014-01-01

    OBJECTIVE: To evaluate the accuracy of the Edinburgh Postnatal Depression Scale (EPDS) and 3 subscales for identifying postpartum depression among primiparous adolescent mothers. METHODS: Mothers enrolled in a randomized controlled trial to prevent postpartum depression completed a psychiatric diagnostic interview and the 10-item EPDS at 6 weeks, 3 months, and 6 months postpartum. Three subscales of the EPDS were assessed as brief screening tools: 3-item anxiety subscale (EPDS-3), 7-item depressive symptoms subscale (EPDS-7), and 2-item subscale (EPDS-2) that resemble the Patient Health Questionnaire-2. Receiver operating characteristic curves and the areas under the curves for each tool were compared to assess accuracy. The sensitivities and specificities of each screening tool were calculated in comparison with diagnostic criteria for a major depressive disorder. Repeated-measures longitudinal analytical techniques were used. RESULTS: A total of 106 women contributed 289 postpartum visits; 18% of the women met criteria for incident postpartum depression by psychiatric diagnostic interview. When used as continuous measures, the full EPDS, EPDS-7, and EPDS-2 performed equally well (area under the curve >0.9). Optimal cutoff scores for a positive depression screen for the EPDS and EPDS-7 were lower (≥9 and ≥7, respectively) than currently recommended cutoff scores (≥10). At optimal cutoff scores, the EPDS and EPDS-7 both had sensitivities of 90% and specificities of >85%. CONCLUSIONS: The EPDS, EPDS-7, and EPDS-2 are highly accurate at identifying postpartum depression among adolescent mothers. In primary care pediatric settings, the EPDS and its shorter subscales have potential for use as effective depression screening tools. PMID:24344102

  3. Identifying Relationships between High-Risk Sexual Behaviors and Screening Positive for Chlamydia and Gonorrhea in School-Wide Screening Events

    ERIC Educational Resources Information Center

    Salerno, Jennifer; Darling-Fisher, Cindy; Hawkins, Nicole M.; Fraker, Elizabeth

    2013-01-01

    Background: This article describes a school-wide sexually transmitted infection (STI) screening to identify adolescent high-risk sexual behaviors, STI history/incidence, and presence of chlamydia and gonorrhea, and examines relationships between high-risk behaviors and screening positive for chlamydia and gonorrhea in an alternative high school…

  4. Identifying Relationships between High-Risk Sexual Behaviors and Screening Positive for Chlamydia and Gonorrhea in School-Wide Screening Events

    ERIC Educational Resources Information Center

    Salerno, Jennifer; Darling-Fisher, Cindy; Hawkins, Nicole M.; Fraker, Elizabeth

    2013-01-01

    Background: This article describes a school-wide sexually transmitted infection (STI) screening to identify adolescent high-risk sexual behaviors, STI history/incidence, and presence of chlamydia and gonorrhea, and examines relationships between high-risk behaviors and screening positive for chlamydia and gonorrhea in an alternative high school…

  5. A Virtual Screening Approach For Identifying Plants with Anti H5N1 Neuraminidase Activity

    PubMed Central

    2016-01-01

    Recent outbreaks of highly pathogenic and occasional drug-resistant influenza strains have highlighted the need to develop novel anti-influenza therapeutics. Here, we report computational and experimental efforts to identify influenza neuraminidase inhibitors from among the 3000 natural compounds in the Malaysian-Plants Natural-Product (NADI) database. These 3000 compounds were first docked into the neuraminidase active site. The five plants with the largest number of top predicted ligands were selected for experimental evaluation. Twelve specific compounds isolated from these five plants were shown to inhibit neuraminidase, including two compounds with IC50 values less than 92 μM. Furthermore, four of the 12 isolated compounds had also been identified in the top 100 compounds from the virtual screen. Together, these results suggest an effective new approach for identifying bioactive plant species that will further the identification of new pharmacologically active compounds from diverse natural-product resources. PMID:25555059

  6. An integrated in vitro and in vivo high throughput screen identifies treatment leads for ependymoma

    PubMed Central

    Atkinson, Jennifer M.; Shelat, Anang A.; Carcaboso, Angel Montero; Kranenburg, Tanya A.; Arnold, Alexander; Boulos, Nidal; Wright, Karen; Johnson, Robert A.; Poppleton, Helen; Mohankumar, Kumarasamypet M.; Feau, Clementine; Phoenix, Timothy; Gibson, Paul; Zhu, Liqin; Tong, Yiai; Eden, Chris; Ellison, David W.; Priebe, Waldemar; Koul, Dimpy; Yung, W. K. Alfred; Gajjar, Amar; Stewart, Clinton F.; Guy, R. Kip; Gilbertson, Richard J.

    2011-01-01

    Summary Using a mouse model of ependymoma—a chemoresistant brain tumor—we combined multi-cell high-throughput screening (HTS), kinome-wide binding assays, and in vivo efficacy studies, to identify potential treatments with predicted toxicity against neural stem cells (NSC). We identified kinases within the insulin signaling pathway and centrosome cycle as regulators of ependymoma cell proliferation, and their corresponding inhibitors as potential therapies. FDA approved drugs not currently used to treat ependymoma were also identified that posses selective toxicity against ependymoma cells relative to normal NSCs both in vitro and in vivo e.g., 5-fluoruracil. Our comprehensive approach advances understanding of the biology and treatment of ependymoma including the discovery of several treatment leads for immediate clinical translation. PMID:21907928

  7. A CRISPR/Cas9 Functional Screen Identifies Rare Tumor Suppressors

    PubMed Central

    Katigbak, Alexandra; Cencic, Regina; Robert, Francis; Sénécha, Patrick; Scuoppo, Claudio; Pelletier, Jerry

    2016-01-01

    An enormous amount of tumor sequencing data has been generated through large scale sequencing efforts. The functional consequences of the majority of mutations identified by such projects remain an open, unexplored question. This problem is particularly complicated in the case of rare mutations where frequency of occurrence alone or prediction of functional consequences are insufficient to distinguish driver from passenger or bystander mutations. We combine genome editing technology with a powerful mouse cancer model to uncover previously unsuspected rare oncogenic mutations in Burkitt’s lymphoma. We identify two candidate tumor suppressors whose loss cooperate with MYC over-expression to accelerate lymphomagenesis. Our results highlight the utility of in vivo CRISPR/Cas9 screens combined with powerful mouse models to identify and validate rare oncogenic modifier events from tumor mutational data. PMID:27982060

  8. Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics

    PubMed Central

    2013-01-01

    Background The development of resistance to chemotherapies represents a significant barrier to successful cancer treatment. Resistance mechanisms are complex, can involve diverse and often unexpected cellular processes, and can vary with both the underlying genetic lesion and the origin or type of tumor. For these reasons developing experimental strategies that could be used to understand, identify and predict mechanisms of resistance in different malignant cells would be a major advance. Methods Here we describe a gain-of-function forward genetic approach for identifying mechanisms of resistance. This approach uses a modified piggyBac transposon to generate libraries of mutagenized cells, each containing transposon insertions that randomly activate nearby gene expression. Genes of interest are identified using next-gen high-throughput sequencing and barcode multiplexing is used to reduce experimental cost. Results Using this approach we successfully identify genes involved in paclitaxel resistance in a variety of cancer cell lines, including the multidrug transporter ABCB1, a previously identified major paclitaxel resistance gene. Analysis of co-occurring transposons integration sites in single cell clone allows for the identification of genes that might act cooperatively to produce drug resistance a level of information not accessible using RNAi or ORF expression screening approaches. Conclusion We have developed a powerful pipeline to systematically discover drug resistance in mammalian cells in vitro. This cost-effective approach can be readily applied to different cell lines, to identify canonical or context specific resistance mechanisms. Its ability to probe complex genetic context and non-coding genomic elements as well as cooperative resistance events makes it a good complement to RNAi or ORF expression based screens. PMID:23442791

  9. Differential nuclear staining assay for high-throughput screening to identify cytotoxic compounds.

    PubMed

    Lema, Carolina; Varela-Ramirez, Armando; Aguilera, Renato J

    As large quantities of novel synthetic molecules continue to be generated there is a challenge to identify therapeutic agents with cytotoxic activity. Here we introduce a Differential Nuclear Staining (DNS) assay adapted to live-cell imaging for high throughput screening (HTS) that utilizes two fluorescent DNA intercalators, Hoechst 33342 and Propidium iodide (PI). Since Hoechst can readily cross cell membranes to stain DNA of living and dead cells, it was used to label the total number of cells. In contrast, PI only enters cells with compromised plasma membranes, thus selectively labeling dead cells. The DNS assay was successfully validated by utilizing well known cytotoxic agents with fast or slow cytotoxic activities. The assay was found to be suitable for HTS with Z' factors ranging from 0.86 to 0.60 for 96 and 384-well formats, respectively. Furthermore, besides plate-to-plate reproducibility, assay quality performance was evaluated by determining ratios of signal-to-noise and signal-to-background, as well as coefficient of variation, which resulted in adequate values and validated the assay for HTS initiatives. As proof of concept, eighty structurally diverse compounds from a small molecule library were screened in a 96-well plate format using the DNS assay. Using this DNS assay, six hits with cytotoxic properties were identified and all of them were also successfully identified by using the commercially available MTS assay (CellTiter 96® Cell Proliferation Assay). In addition, the DNS and a flow cytometry assay were used to validate the activity of the cytotoxic compounds. The DNS assay was also used to generate dose-response curves and to obtain CC50 values. The results indicate that the DNS assay is reliable and robust and suitable for primary and secondary screens of compounds with potential cytotoxic activity.

  10. Differential nuclear staining assay for high-throughput screening to identify cytotoxic compounds

    PubMed Central

    LEMA, Carolina; VARELA-RAMIREZ, Armando; AGUILERA, Renato J.

    2016-01-01

    As large quantities of novel synthetic molecules continue to be generated there is a challenge to identify therapeutic agents with cytotoxic activity. Here we introduce a Differential Nuclear Staining (DNS) assay adapted to live-cell imaging for high throughput screening (HTS) that utilizes two fluorescent DNA intercalators, Hoechst 33342 and Propidium iodide (PI). Since Hoechst can readily cross cell membranes to stain DNA of living and dead cells, it was used to label the total number of cells. In contrast, PI only enters cells with compromised plasma membranes, thus selectively labeling dead cells. The DNS assay was successfully validated by utilizing well known cytotoxic agents with fast or slow cytotoxic activities. The assay was found to be suitable for HTS with Z′ factors ranging from 0.86 to 0.60 for 96 and 384-well formats, respectively. Furthermore, besides plate-to-plate reproducibility, assay quality performance was evaluated by determining ratios of signal-to-noise and signal-to-background, as well as coefficient of variation, which resulted in adequate values and validated the assay for HTS initiatives. As proof of concept, eighty structurally diverse compounds from a small molecule library were screened in a 96-well plate format using the DNS assay. Using this DNS assay, six hits with cytotoxic properties were identified and all of them were also successfully identified by using the commercially available MTS assay (CellTiter 96® Cell Proliferation Assay). In addition, the DNS and a flow cytometry assay were used to validate the activity of the cytotoxic compounds. The DNS assay was also used to generate dose-response curves and to obtain CC50 values. The results indicate that the DNS assay is reliable and robust and suitable for primary and secondary screens of compounds with potential cytotoxic activity. PMID:27042697

  11. A Genome-Wide Screen with Nicotinamide to Identify Sirtuin-Dependent Pathways in Saccharomyces cerevisiae

    PubMed Central

    Choy, John S.; Qadri, Bayan; Henry, Leah; Shroff, Kunal; Bifarin, Olatomiwa; Basrai, Munira A.

    2015-01-01

    Sirtuins are evolutionarily conserved NAD-dependent deacetylases that catalyze the cleavage of NAD+ into nicotinamide (NAM), which can act as a pan-sirtuin inhibitor in unicellular and multicellular organisms. Sirtuins regulate processes such as transcription, DNA damage repair, chromosome segregation, and longevity extension in yeast and metazoans. The founding member of the evolutionarily conserved sirtuin family, SIR2, was first identified in budding yeast. Subsequent studies led to the identification of four yeast SIR2 homologs HST1, HST2, HST3, and HST4. Understanding the downstream physiological consequences of inhibiting sirtuins can be challenging since most studies focus on single or double deletions of sirtuins, and mating defects in SIR2 deletions hamper genome-wide screens. This represents an important gap in our knowledge of how sirtuins function in highly complex biological processes such as aging, metabolism, and chromosome segregation. In this report, we used a genome-wide screen to explore sirtuin-dependent processes in Saccharomyces cerevisiae by identifying deletion mutants that are sensitive to NAM. We identified 55 genes in total, 36 of which have not been previously reported to be dependent on sirtuins. We find that genome stability pathways are particularly vulnerable to loss of sirtuin activity. Here, we provide evidence that defects in sister chromatid cohesion renders cells sensitive to growth in the presence of NAM. The results of our screen provide a broad view of the biological pathways sensitive to inhibition of sirtuins, and advance our understanding of the function of sirtuins and NAD+ biology. PMID:26646153

  12. BRIEF REPORT: Screening Items to Identify Patients with Limited Health Literacy Skills

    PubMed Central

    Wallace, Lorraine S; Rogers, Edwin S; Roskos, Steven E; Holiday, David B; Weiss, Barry D

    2006-01-01

    BACKGROUND Patients with limited literacy skills are routinely encountered in clinical practice, but they are not always identified by clinicians. OBJECTIVE To evaluate 3 candidate questions to determine their accuracy in identifying patients with limited or marginal health literacy skills. METHODS We studied 305 English-speaking adults attending a university-based primary care clinic. Demographic items, health literacy screening questions, and the Rapid Estimate of Adult Literacy in Medicine (REALM) were administered to patients. To determine the accuracy of the candidate questions for identifying limited or marginal health literacy skills, we plotted area under the receiver operating characteristic (AUROC) curves for each item, using REALM scores as a reference standard. RESULTS The mean age of subjects was 49.5; 67.5% were female, 85.2% Caucasian, and 81.3% insured by TennCare and/or Medicare. Fifty-four (17.7%) had limited and 52 (17.0%) had marginal health literacy skills. One screening question, “How confident are you filling out medical forms by yourself?” was accurate in detecting limited (AUROC of 0.82; 95% confidence interval [CI]=0.77 to 0.86) and limited/marginal (AUROC of 0.79; 95% CI=0.74 to 0.83) health literacy skills. This question had significantly greater AUROC than either of the other questions (P<.01) and also a greater AUROC than questions based on demographic characteristics. CONCLUSIONS One screening question may be sufficient for detecting limited and marginal health literacy skills in clinic populations. PMID:16881950

  13. A Differential Fluorescence-Based Genetic Screen Identifies Listeria monocytogenes Determinants Required for Intracellular Replication

    PubMed Central

    Perry, Kyle J.

    2013-01-01

    Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen capable of causing severe invasive disease with high mortality rates in humans. While previous studies have largely elucidated the bacterial and host cell mechanisms necessary for invasion, vacuolar escape, and subsequent cell-to-cell spread, the L. monocytogenes factors required for rapid replication within the restrictive environment of the host cell cytosol are poorly understood. In this report, we describe a differential fluorescence-based genetic screen utilizing fluorescence-activated cell sorting (FACS) and high-throughput microscopy to identify L. monocytogenes mutants defective in optimal intracellular replication. Bacteria harboring deletions within the identified gene menD or pepP were defective for growth in primary murine macrophages and plaque formation in monolayers of L2 fibroblasts, thus validating the ability of the screening method to identify intracellular replication-defective mutants. Genetic complementation of the menD and pepP deletion strains rescued the in vitro intracellular infection defects. Furthermore, the menD deletion strain displayed a general extracellular replication defect that could be complemented by growth under anaerobic conditions, while the intracellular growth defect of this strain could be complemented by the addition of exogenous menaquinone. As prior studies have indicated the importance of aerobic metabolism for L. monocytogenes infection, these findings provide further evidence for the importance of menaquinone and aerobic metabolism for L. monocytogenes pathogenesis. Lastly, both the menD and pepP deletion strains were attenuated during in vivo infection of mice. These findings demonstrate that the differential fluorescence-based screening approach provides a powerful tool for the identification of intracellular replication determinants in multiple bacterial systems. PMID:23687268

  14. Genome-wide RNAi screening identifies host restriction factors critical for in vivo AAV transduction.

    PubMed

    Mano, Miguel; Ippodrino, Rudy; Zentilin, Lorena; Zacchigna, Serena; Giacca, Mauro

    2015-09-08

    Viral vectors based on the adeno-associated virus (AAV) hold great promise for in vivo gene transfer; several unknowns, however, still limit the vectors' broader and more efficient application. Here, we report the results of a high-throughput, whole-genome siRNA screening aimed at identifying cellular factors regulating AAV transduction. We identified 1,483 genes affecting vector efficiency more than 4-fold and up to 50-fold, either negatively or positively. Most of these factors have not previously been associated to AAV infection. The most effective siRNAs were independent from the virus serotype or analyzed cell type and were equally evident for single-stranded and self-complementary AAV vectors. A common characteristic of the most effective siRNAs was the induction of cellular DNA damage and activation of a cell cycle checkpoint. This information can be exploited for the development of more efficient AAV-based gene delivery procedures. Administration of the most effective siRNAs identified by the screening to the liver significantly improved in vivo AAV transduction efficiency.

  15. A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors.

    PubMed

    Park, Ryan J; Wang, Tim; Koundakjian, Dylan; Hultquist, Judd F; Lamothe-Molina, Pedro; Monel, Blandine; Schumann, Kathrin; Yu, Haiyan; Krupzcak, Kevin M; Garcia-Beltran, Wilfredo; Piechocka-Trocha, Alicja; Krogan, Nevan J; Marson, Alexander; Sabatini, David M; Lander, Eric S; Hacohen, Nir; Walker, Bruce D

    2017-02-01

    Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues, facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation, which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4(+) T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention.

  16. A modifier screen identifies DNAJB6 as a cardiomyopathy susceptibility gene

    PubMed Central

    Ding, Yonghe; Long, Pamela A.; Bos, J. Martijn; Shih, Yu-Huan; Ma, Xiao; Sundsbak, Rhianna S.; Chen, Jianhua; Zhao, Liqun; Hu, Xinyang; Wang, Jianan; Shi, Yongyong; Ackerman, Michael J.; Lin, Xueying; Ekker, Stephen C.; Redfield, Margaret M.; Olson, Timothy M.

    2016-01-01

    Mutagenesis screening is a powerful forward genetic approach that has been successfully applied in lower-model organisms to discover genetic factors for biological processes. This phenotype-based approach has yet to be established in vertebrates for probing major human diseases, largely because of the complexity of colony management. Herein, we report a rapid strategy for identifying genetic modifiers of cardiomyopathy (CM). Based on the application of doxorubicin stress to zebrafish insertional cardiac (ZIC) mutants, we identified 4 candidate CM-modifying genes, of which 3 have been linked previously to CM. The long isoform of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b(L)) was identified as a CM susceptibility gene, supported by identification of rare variants in its human ortholog DNAJB6 from CM patients. Mechanistic studies indicated that the deleterious, loss-of-function modifying effects of dnajb6b(L) can be ameliorated by inhibition of ER stress. In contrast, overexpression of dnajb6(L) exerts cardioprotective effects on both fish and mouse CM models. Together, our findings establish a mutagenesis screening strategy that is scalable for systematic identification of genetic modifiers of CM, feasible to suggest therapeutic targets, and expandable to other major human diseases. PMID:27642634

  17. A Chemical Rescue Screen Identifies a Plasmodium falciparum Apicoplast Inhibitor Targeting MEP Isoprenoid Precursor Biosynthesis

    PubMed Central

    Wu, Wesley; Herrera, Zachary; Ebert, Danny; Baska, Katie; Cho, Seok H.

    2014-01-01

    The apicoplast is an essential plastid organelle found in Plasmodium parasites which contains several clinically validated antimalarial-drug targets. A chemical rescue screen identified MMV-08138 from the “Malaria Box” library of growth-inhibitory antimalarial compounds as having specific activity against the apicoplast. MMV-08138 inhibition of blood-stage Plasmodium falciparum growth is stereospecific and potent, with the most active diastereomer demonstrating a 50% effective concentration (EC50) of 110 nM. Whole-genome sequencing of 3 drug-resistant parasite populations from two independent selections revealed E688Q and L244I mutations in P. falciparum IspD, an enzyme in the MEP (methyl-d-erythritol-4-phosphate) isoprenoid precursor biosynthesis pathway in the apicoplast. The active diastereomer of MMV-08138 directly inhibited PfIspD activity in vitro with a 50% inhibitory concentration (IC50) of 7.0 nM. MMV-08138 is the first PfIspD inhibitor to be identified and, together with heterologously expressed PfIspD, provides the foundation for further development of this promising antimalarial drug candidate lead. Furthermore, this report validates the use of the apicoplast chemical rescue screen coupled with target elucidation as a discovery tool to identify specific apicoplast-targeting compounds with new mechanisms of action. PMID:25367906

  18. A genome-wide RNA interference screen identifies two novel components of the metazoan secretory pathway

    PubMed Central

    Wendler, Franz; Gillingham, Alison K; Sinka, Rita; Rosa-Ferreira, Cláudia; Gordon, David E; Franch-Marro, Xavier; Peden, Andrew A; Vincent, Jean-Paul; Munro, Sean

    2010-01-01

    Genetic screens in the yeast Saccharomyces cerevisiae have identified many proteins involved in the secretory pathway, most of which have orthologues in higher eukaryotes. To investigate whether there are additional proteins that are required for secretion in metazoans but are absent from yeast, we used genome-wide RNA interference (RNAi) to look for genes required for secretion of recombinant luciferase from Drosophila S2 cells. This identified two novel components of the secretory pathway that are conserved from humans to plants. Gryzun is distantly related to, but distinct from, the Trs130 subunit of the TRAPP complex but is absent from S. cerevisiae. RNAi of human Gryzun (C4orf41) blocks Golgi exit. Kish is a small membrane protein with a previously uncharacterised orthologue in yeast. The screen also identified Drosophila orthologues of almost 60% of the yeast genes essential for secretion. Given this coverage, the small number of novel components suggests that contrary to previous indications the number of essential core components of the secretory pathway is not much greater in metazoans than in yeasts. PMID:19942856

  19. Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome

    PubMed Central

    Roosing, Susanne; Hofree, Matan; Kim, Sehyun; Scott, Eric; Copeland, Brett; Romani, Marta; Silhavy, Jennifer L; Rosti, Rasim O; Schroth, Jana; Mazza, Tommaso; Miccinilli, Elide; Zaki, Maha S; Swoboda, Kathryn J; Milisa-Drautz, Joanne; Dobyns, William B; Mikati, Mohamed A; İncecik, Faruk; Azam, Matloob; Borgatti, Renato; Romaniello, Romina; Boustany, Rose-Mary; Clericuzio, Carol L; D'Arrigo, Stefano; Strømme, Petter; Boltshauser, Eugen; Stanzial, Franco; Mirabelli-Badenier, Marisol; Moroni, Isabella; Bertini, Enrico; Emma, Francesco; Steinlin, Maja; Hildebrandt, Friedhelm; Johnson, Colin A; Freilinger, Michael; Vaux, Keith K; Gabriel, Stacey B; Aza-Blanc, Pedro; Heynen-Genel, Susanne; Ideker, Trey; Dynlacht, Brian D; Lee, Ji Eun; Valente, Enza Maria; Kim, Joon; Gleeson, Joseph G

    2015-01-01

    Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised-learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and Sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies. DOI: http://dx.doi.org/10.7554/eLife.06602.001 PMID:26026149

  20. Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome.

    PubMed

    Roosing, Susanne; Hofree, Matan; Kim, Sehyun; Scott, Eric; Copeland, Brett; Romani, Marta; Silhavy, Jennifer L; Rosti, Rasim O; Schroth, Jana; Mazza, Tommaso; Miccinilli, Elide; Zaki, Maha S; Swoboda, Kathryn J; Milisa-Drautz, Joanne; Dobyns, William B; Mikati, Mohamed A; İncecik, Faruk; Azam, Matloob; Borgatti, Renato; Romaniello, Romina; Boustany, Rose-Mary; Clericuzio, Carol L; D'Arrigo, Stefano; Strømme, Petter; Boltshauser, Eugen; Stanzial, Franco; Mirabelli-Badenier, Marisol; Moroni, Isabella; Bertini, Enrico; Emma, Francesco; Steinlin, Maja; Hildebrandt, Friedhelm; Johnson, Colin A; Freilinger, Michael; Vaux, Keith K; Gabriel, Stacey B; Aza-Blanc, Pedro; Heynen-Genel, Susanne; Ideker, Trey; Dynlacht, Brian D; Lee, Ji Eun; Valente, Enza Maria; Kim, Joon; Gleeson, Joseph G

    2015-05-30

    Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised-learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and Sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies.

  1. Genome-wide RNAi screening identifies host restriction factors critical for in vivo AAV transduction

    PubMed Central

    Mano, Miguel; Ippodrino, Rudy; Zentilin, Lorena; Zacchigna, Serena; Giacca, Mauro

    2015-01-01

    Viral vectors based on the adeno-associated virus (AAV) hold great promise for in vivo gene transfer; several unknowns, however, still limit the vectors’ broader and more efficient application. Here, we report the results of a high-throughput, whole-genome siRNA screening aimed at identifying cellular factors regulating AAV transduction. We identified 1,483 genes affecting vector efficiency more than 4-fold and up to 50-fold, either negatively or positively. Most of these factors have not previously been associated to AAV infection. The most effective siRNAs were independent from the virus serotype or analyzed cell type and were equally evident for single-stranded and self-complementary AAV vectors. A common characteristic of the most effective siRNAs was the induction of cellular DNA damage and activation of a cell cycle checkpoint. This information can be exploited for the development of more efficient AAV-based gene delivery procedures. Administration of the most effective siRNAs identified by the screening to the liver significantly improved in vivo AAV transduction efficiency. PMID:26305933

  2. A genome-wide screen for identifying all regulators of a target gene

    PubMed Central

    Baptist, Guillaume; Pinel, Corinne; Ranquet, Caroline; Izard, Jérôme; Ropers, Delphine; de Jong, Hidde; Geiselmann, Johannes

    2013-01-01

    We have developed a new screening methodology for identifying all genes that control the expression of a target gene through genetic or metabolic interactions. The screen combines mutant libraries with luciferase reporter constructs, whose expression can be monitored in vivo and over time in different environmental conditions. We apply the method to identify the genes that control the expression of the gene acs, encoding the acetyl coenzyme A synthetase, in Escherichia coli. We confirm most of the known genetic regulators, including CRP–cAMP, IHF and components of the phosphotransferase system. In addition, we identify new regulatory interactions, many of which involve metabolic intermediates or metabolic sensing, such as the genes pgi, pfkA, sucB and lpdA, encoding enzymes in glycolysis and the TCA cycle. Some of these novel interactions were validated by quantitative reverse transcriptase-polymerase chain reaction. More generally, we observe that a large number of mutants directly or indirectly influence acs expression, an effect confirmed for a second promoter, sdhC. The method is applicable to any promoter fused to a luminescent reporter gene in combination with a deletion mutant library. PMID:23892289

  3. Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet Syndrome treatment

    PubMed Central

    Baraban, Scott C.; Dinday, Matthew T.; Hortopan, Gabriela A.

    2013-01-01

    Dravet syndrome (DS) is a catastrophic pediatric epilepsy with severe intellectual disability, impaired social development and persistent drug-resistant seizures. One of its primary monogenic causes are mutations in Nav1.1 (SCN1A), a voltage-gated sodium channel. Here we characterise zebrafish Nav1.1 (scn1Lab) mutants originally identified in a chemical mutagenesis screen. Mutants exhibit spontaneous abnormal electrographic activity, hyperactivity and convulsive behaviors. Although scn1Lab expression is reduced, microarray analysis is remarkable for the small fraction of differentially expressed genes (~3%) and lack of compensatory expression changes in other scn subunits. Ketogenic diet, diazepam, valproate, potassium bromide and stiripentol attenuate mutant seizure activity; seven other antiepileptic drugs have no effect. A phenotype-based screen of 320 compounds identifies a US Food and Drug Administration-approved compound (clemizole) that inhibits convulsive behaviors and electrographic seizures. This approach represents a new direction in modeling pediatric epilepsy and could be used to identify novel therapeutics for any monogenic epilepsy disorder. PMID:24002024

  4. Simplified Screening Approach Identifies Mutated Proteins Expressed in Patient Tumors | Center for Cancer Research

    Cancer.gov

    Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) is a very effective treatment for patients with metastatic melanoma. In phase 2 clinical trials, up to 70 percent of patients with melanoma who received autologous TILs had considerable regressions of metastatic lesions. Recently, in another trial, 40 percent of patients treated had complete regressions of all measurable lesions lasting more than five years after treatment. Identifying antigens associated with TIL-mediated tumor regression has been a difficult task due to the diversity of these large lymphocyte populations and the complexity of current screening approaches.

  5. An in vivo screen identifies ependymoma oncogenes and tumor-suppressor genes.

    PubMed

    Mohankumar, Kumarasamypet M; Currle, David S; White, Elsie; Boulos, Nidal; Dapper, Jason; Eden, Christopher; Nimmervoll, Birgit; Thiruvenkatam, Radhika; Connelly, Michele; Kranenburg, Tanya A; Neale, Geoffrey; Olsen, Scott; Wang, Yong-Dong; Finkelstein, David; Wright, Karen; Gupta, Kirti; Ellison, David W; Thomas, Arzu Onar; Gilbertson, Richard J

    2015-08-01

    Cancers are characterized by non-random chromosome copy number alterations that presumably contain oncogenes and tumor-suppressor genes (TSGs). The affected loci are often large, making it difficult to pinpoint which genes are driving the cancer. Here we report a cross-species in vivo screen of 84 candidate oncogenes and 39 candidate TSGs, located within 28 recurrent chromosomal alterations in ependymoma. Through a series of mouse models, we validate eight new ependymoma oncogenes and ten new ependymoma TSGs that converge on a small number of cell functions, including vesicle trafficking, DNA modification and cholesterol biosynthesis, identifying these as potential new therapeutic targets.

  6. Hits identified in library screening demonstrate selective CYP17A1 lyase inhibition.

    PubMed

    Krug, Sebastian J; Hu, Qingzhong; Hartmann, Rolf W

    2013-03-01

    A screening of structurally different steroid hormone synthesis inhibitors was performed in order to find a starting point for the development of a new inhibitor of the bifunctional steroidogenic enzyme CYP17A1. Emphasis was placed on determination of selectivity between the two catalytic steps, namely 17α-hydroxylase and C(17,20)-lyase. For that purpose a new inhibition assay has been developed. Hits identified within this novel assay demonstrated selective inhibition of CYP17A1 lyase activity, and thus mark the basis for the development of selective C(17,20)-lyase inhibitors for the treatment of prostate cancer.

  7. Visual Genome-Wide RNAi Screening to Identify Human Host Factors Required for Trypanosoma cruzi Infection

    PubMed Central

    de Macedo Dossin, Fernando; Choi, Seo Yeon; Kim, Nam Youl; Kim, Hi Chul; Jung, Sung Yong; Schenkman, Sergio; Almeida, Igor C.; Emans, Neil; Freitas-Junior, Lucio H.

    2011-01-01

    The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy. PMID:21625474

  8. Brief screening items to identify spanish-speaking adults with limited health literacy and numeracy skills.

    PubMed

    Singh, Rashmi; Coyne, Laura Scott; Wallace, Lorraine S

    2015-09-14

    Limited health literacy (HL) and numeracy have been shown to be associated with a wide array of poor health-related outcomes, knowledge, and behaviors. The purpose of this study was to evaluate the clinical utility of brief HL and numeracy screening items in identifying Spanish-speaking adults' HL and numeracy skills. We studied convenience samples of native Spanish-speaking adults in Columbus, Ohio. A trained research assistant administered sociodemographic items, HL and numeracy screening items, Short Assessment of Health Literacy (SAHL), and Newest Vital Sign (NVS) to participants in Spanish. Participants (n = 151) averaged 36.8 ± 11.0 years of age and 54.7% were female. Average SAHL score was 15.7 ± 2.8 (range = 4 to 18), while the average NVS score was 1.7 ± 1.5 (range = 0 to 6). "How confident are you filling out medical forms by yourself?" performed best in detecting limited NVS scores (AUROC = 0.66; 95% confidence interval [CI] = 0.57-0.75), limited/marginal NVS scores (AUROC = 0.75; 95% CI = 0.65-0.84), and inadequate SAHL scores (AUROC = 0.69; 95% CI = 0.58-0.79). A single HL screening item is useful for quickly estimating HL and numeracy skills in native Spanish-speaking adults.

  9. High-throughput screening of mouse gene knockouts identifies established and novel skeletal phenotypes

    PubMed Central

    Brommage, Robert; Liu, Jeff; Hansen, Gwenn M; Kirkpatrick, Laura L; Potter, David G; Sands, Arthur T; Zambrowicz, Brian; Powell, David R; Vogel, Peter

    2014-01-01

    Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening (HTS) data for 3 762 distinct global gene knockout (KO) mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type (WT) cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass: 23 previously reported genes (Calcr, Cebpb, Crtap, Dcstamp, Dkk1, Duoxa2, Enpp1, Fgf23, Kiss1/Kiss1r, Kl (Klotho), Lrp5, Mstn, Neo1, Npr2, Ostm1, Postn, Sfrp4, Slc30a5, Slc39a13, Sost, Sumf1, Src, Wnt10b), five novel genes extensively characterized (Cldn18, Fam20c, Lrrk1, Sgpl1, Wnt16), five novel genes with preliminary characterization (Agpat2, Rassf5, Slc10a7, Slc26a7, Slc30a10) and three novel undisclosed genes coding for potential osteoporosis drug targets. PMID:26273529

  10. Calcification classifications of small nodules identified during CT lung cancer screening

    NASA Astrophysics Data System (ADS)

    Judy, Philip F.; Riva, Roberto; Kadota, Yoshiko; Jacobson, Francine L.

    2004-05-01

    The aim of this study was to determine whether radiologists are more likely to report as calcified the small nodules detected during CT lung-cancer screening, if sharper reconstruction filters are utilized. Images were reconstructed with the 2 filters used at our institution for the lung (B50f) and for the mediastinum (B30f). The 4 lung-cancer screening cases were reconstructed with 1.25-mm section thickness at 0.6-mm section increments. Using a lax criterion, 2 radiologists identified the locations of nodular features and rated the likelihood that the features were calcified. There were 302 nodules reports. More of these (57%) were reported on images reconstructed using the smooth filter. Sixty (60) reports were definitely or possibly calcified. Seventy-three percent (73%) calcification reports were from images reconstructed using B50f. There were 27 calcification reports of one of the radiologist that were classified as non-calcified by the other radiologist. Most of calcification reports (81%) of 27 reports on which radiologists disagree regarding the likelihood of calcification were from images reconstructed using B50f. Radiologists are more likely to report small nodules detected during lung-cancer screening as calcified using the sharper reconstruction filter. Whether these nodules are actually calcified or not remains a question.

  11. Genome-wide Screen of Pseudomonas aeruginosa in Saccharomyces cerevisiae Identifies New Virulence Factors.

    PubMed

    Zrieq, Rafat; Sana, Thibault G; Vergin, Sandra; Garvis, Steve; Volfson, Irina; Bleves, Sophie; Voulhoux, Romé; Hegemann, Johannes H

    2015-01-01

    Pseudomonas aeruginosa is a human opportunistic pathogen that causes mortality in cystic fibrosis and immunocompromised patients. While many virulence factors of this pathogen have already been identified, several remain to be discovered. In this respect we set an unprecedented genome-wide screen of a P. aeruginosa expression library based on a yeast growth phenotype. Fifty-one candidates were selected in athree-round screening process. The robustness of the screen was validated by the selection of three well known secreted proteins including one demonstrated virulence factor, the protease LepA. Further in silico sorting of the 51 candidates highlighted three potential new Pseudomonas effector candidates (Pec). By testing the cytotoxicity of wild type P. aeruginosa vs. pec mutants toward macrophages and the virulence in the Caenorhabditis elegans model, we demonstrated that the three selected Pecs are novel virulence factors of P. aeruginosa. Additional cellular localization experiments in the host revealed specific localization for Pec1 and Pec2 that could inform about their respective functions.

  12. Genome-wide Screen of Pseudomonas aeruginosa in Saccharomyces cerevisiae Identifies New Virulence Factors

    PubMed Central

    Zrieq, Rafat; Sana, Thibault G.; Vergin, Sandra; Garvis, Steve; Volfson, Irina; Bleves, Sophie; Voulhoux, Romé; Hegemann, Johannes H.

    2015-01-01

    Pseudomonas aeruginosa is a human opportunistic pathogen that causes mortality in cystic fibrosis and immunocompromised patients. While many virulence factors of this pathogen have already been identified, several remain to be discovered. In this respect we set an unprecedented genome-wide screen of a P. aeruginosa expression library based on a yeast growth phenotype. Fifty-one candidates were selected in athree-round screening process. The robustness of the screen was validated by the selection of three well known secreted proteins including one demonstrated virulence factor, the protease LepA. Further in silico sorting of the 51 candidates highlighted three potential new Pseudomonas effector candidates (Pec). By testing the cytotoxicity of wild type P. aeruginosa vs. pec mutants toward macrophages and the virulence in the Caenorhabditis elegans model, we demonstrated that the three selected Pecs are novel virulence factors of P. aeruginosa. Additional cellular localization experiments in the host revealed specific localization for Pec1 and Pec2 that could inform about their respective functions. PMID:26636043

  13. Yeast functional screen to identify genes conferring salt stress tolerance in Salicornia europaea

    PubMed Central

    Nakahara, Yoshiki; Sawabe, Shogo; Kainuma, Kenta; Katsuhara, Maki; Shibasaka, Mineo; Suzuki, Masanori; Yamamoto, Kosuke; Oguri, Suguru; Sakamoto, Hikaru

    2015-01-01

    Salinity is a critical environmental factor that adversely affects crop productivity. Halophytes have evolved various mechanisms to adapt to saline environments. Salicornia europaea L. is one of the most salt-tolerant plant species. It does not have special salt-secreting structures like a salt gland or salt bladder, and is therefore a good model for studying the common mechanisms underlying plant salt tolerance. To identify candidate genes encoding key proteins in the mediation of salt tolerance in S. europaea, we performed a functional screen of a cDNA library in yeast. The library was screened for genes that allowed the yeast to grow in the presence of 1.3 M NaCl. We obtained three full-length S. europaea genes that confer salt tolerance. The genes are predicted to encode (1) a novel protein highly homologous to thaumatin-like proteins, (2) a novel coiled-coil protein of unknown function, and (3) a novel short peptide of 32 residues. Exogenous application of a synthetic peptide corresponding to the 32 residues improved salt tolerance of Arabidopsis. The approach described in this report provides a rapid assay system for large-scale screening of S. europaea genes involved in salt stress tolerance and supports the identification of genes responsible for such mechanisms. These genes may be useful candidates for improving crop salt tolerance by genetic transformation. PMID:26579166

  14. A Phenotypic High-Content Screening Assay to Identify Regulators of Membrane Protein Localization.

    PubMed

    Smith, Lorey K; Thomas, Daniel W; Simpson, Kaylene J; Humbert, Patrick O

    2016-10-01

    Correct subcellular localization of proteins is a requirement for appropriate function. This is especially true in epithelial cells, which rely on the precise localization of a diverse array of epithelial polarity and cellular adhesion proteins. Loss of cell polarity and adhesion is a hallmark of cancer, and mislocalization of core polarity proteins, such as Scribble, is observed in a range of human epithelial tumors and is prognostic of poor survival. Despite this, little is known about how Scribble membrane localization is regulated. Here, we describe the development and application of a phenotypic high-content screening assay that is designed to specifically quantify membrane levels of Scribble to identify regulators of its membrane localization. A screening platform that is capable of resolving individual cells and quantifying membrane protein localization in confluent epithelial monolayers was developed by using the cytoplasm-to-cell-membrane bioapplication integrated with the Cellomics ArrayScan high-content imaging platform. Application of this method to a boutique human epithelial polarity and signaling small interfering RNA (siRNA) library resulted in highly robust coefficient-of-variance and Z' factor values. As proof of concept, we present two candidate genes whose depletion specifically reduces Scribble protein levels at the membrane. Data mining revealed that these proteins interact with components of the Scribble polarity complex, providing support for the utility of the screening approach. This method is broadly applicable to genome-wide and large-scale compound screening of membrane-bound proteins, and when coupled with pathway analysis the dataset becomes even more valuable and can provide predictive mechanistic insight.

  15. Identifying the Optimal Age to Perform Newborn Screening for Hearing Loss in Uganda

    PubMed Central

    Walsh, M; Redshaw, E; Crossley, E; Phillips, C

    2015-01-01

    Background: Permanent congenital hearing loss affects up to 6/1000 births in developing countries. Currently, in Uganda there is no newborn screening for hearing loss (NSHL) program and no published work on this topic. Within the existing healthcare system there are two opportunities to deliver screening, at birth or 6 weeks of age when infants receive their immunizations. Aim: This study explored the outcomes of otoacoustic emission (OAE) testing in infants at birth and 6 weeks of age, to identify the optimal age for screening. Subjects and Methods: This cross-sectional pilot study recruited 60 consecutive infants from two health centres in Kampala, Uganda. Thirty infants were newborns recruited from the postnatal ward and 30 were aged 4–8 weeks from the immunization clinic, we performed OAE testing on all infants. Results: The results showed 56.7% (17/30) of newborn infants passed OAE testing compared with 90.0% (27/30) of the immunization infants, P < 0.01. Furthermore, of the 11 newborn infants aged ≥24 h of age 90.9% (10/11) passed, compared with the 19 infants <24 h of age where 37% (7/19) passed, P < 0.01. Conclusions: This study demonstrates a higher pass rate for OAE testing for infants ≥24 h of age compared to those <24 h of age. The overall lower pass rate of the newborn infants could be due to external ear debris and middle ear fluid compromising the OAE testing. These findings would support a NSHL programme in Uganda that offers screening to infants ≥24 h of age, to maximize the cost-effectiveness of the program. PMID:27057378

  16. First Streptococcus pyogenes signature-tagged mutagenesis screen identifies novel virulence determinants.

    PubMed

    Kizy, Anne E; Neely, Melody N

    2009-05-01

    The virulence of bacterial pathogens is a complex process that requires the dynamic expression of many genes for the pathogens to invade and circumvent host defenses, as well as to proliferate in vivo. In this study, we employed a large-scale screen, signature-tagged mutagenesis (STM), to identify Streptococcus pyogenes virulence genes important for pathogenesis within the host. Approximately 1,200 STM mutants were created and screened using the zebrafish infectious disease model. The transposon insertion site was identified for 29 of the 150 mutants that were considered attenuated for virulence. Previously reported streptococcal virulence genes, such as mga, hasA, amrA, smeZ, and two genes in the sil locus, were identified, confirming the utility of the model for revealing genes important for virulence. Multiple genes not previously implicated in virulence were also identified, including genes encoding putative transporters, hypothetical cytosolic proteins, and macrolide efflux pumps. The STM mutant strains display various levels of attenuation, and multiple separate insertions were identified in either the same gene or the same locus, suggesting that these factors are important for this type of acute, invasive infection. We further examined two such genes, silB and silC of a putative quorum-sensing regulon, and determined that they are significant virulence factors in our model of necrotizing fasciitis. sil locus promoter expression was examined under various in vitro conditions, as well as in zebrafish tissues, and was found to be differentially induced. This study was a unique investigation of S. pyogenes factors required for successful invasive infection.

  17. A sensitized mutagenesis screen identifies Gli3 as a modifier of Sox10 neurocristopathy

    PubMed Central

    Matera, Ivana; Watkins-Chow, Dawn E.; Loftus, Stacie K.; Hou, Ling; Incao, Arturo; Silver, Debra L.; Rivas, Cecelia; Elliott, Eugene C.; Baxter, Laura L.; Pavan, William J.

    2008-01-01

    Haploinsufficiency for the transcription factor SOX10 is associated with the pigmentary deficiencies of Waardenburg syndrome (WS) and is modeled in Sox10 haploinsufficient mice (Sox10LacZ/+). As genetic background affects WS severity in both humans and mice, we established an N-ethyl-N-nitrosourea (ENU) mutagenesis screen to identify modifiers that increase the phenotypic severity of Sox10LacZ/+ mice. Analysis of 230 pedigrees identified three modifiers, named modifier of Sox10 neurocristopathies (Mos1, Mos2 and Mos3). Linkage analysis confirmed their locations on mouse chromosomes 13, 4 and 3, respectively, within regions distinct from previously identified WS loci. Positional candidate analysis of Mos1 identified a truncation mutation in a hedgehog(HH)-signaling mediator, GLI-Kruppel family member 3 (Gli3). Complementation tests using a second allele of Gli3 (Gli3Xt-J) confirmed that a null mutation of Gli3 causes the increased hypopigmentation in Sox10LacZ/+;Gli3Mos1/+ double heterozygotes. Early melanoblast markers (Mitf, Sox10, Dct, and Si) are reduced in Gli3Mos1/Mos1 embryos, indicating that loss of GLI3 signaling disrupts melanoblast specification. In contrast, mice expressing only the GLI3 repressor have normal melanoblast specification, indicating that the full-length GLI3 activator is not required for specification of neural crest to the melanocyte lineage. This study demonstrates the feasibility of sensitized screens to identify disease modifier loci and implicates GLI3 and other HH signaling components as modifiers of human neurocristopathies. PMID:18397875

  18. Use of electronic data and existing screening tools to identify clinically significant obstructive sleep apnea

    PubMed Central

    Severson, Carl A; Pendharkar, Sachin R; Ronksley, Paul E; Tsai, Willis H

    2015-01-01

    OBJECTIVES: To assess the ability of electronic health data and existing screening tools to identify clinically significant obstructive sleep apnea (OSA), as defined by symptomatic or severe OSA. METHODS: The present retrospective cohort study of 1041 patients referred for sleep diagnostic testing was undertaken at a tertiary sleep centre in Calgary, Alberta. A diagnosis of clinically significant OSA or an alternative sleep diagnosis was assigned to each patient through blinded independent chart review by two sleep physicians. Predictive variables were identified from online questionnaire data, and diagnostic algorithms were developed. The performance of electronically derived algorithms for identifying patients with clinically significant OSA was determined. Diagnostic performance of these algorithms was compared with versions of the STOP-Bang questionnaire and adjusted neck circumference score (ANC) derived from electronic data. RESULTS: Electronic questionnaire data were highly sensitive (>95%) at identifying clinically significant OSA, but not specific. Sleep diagnostic testing-determined respiratory disturbance index was very specific (specificity ≥95%) for clinically relevant disease, but not sensitive (<35%). Derived algorithms had similar accuracy to the STOP-Bang or ANC, but required fewer questions and calculations. CONCLUSIONS: These data suggest that a two-step process using a small number of clinical variables (maximizing sensitivity) and objective diagnostic testing (maximizing specificity) is required to identify clinically significant OSA. When used in an online setting, simple algorithms can identify clinically relevant OSA with similar performance to existing decision rules such as the STOP-Bang or ANC. PMID:26083542

  19. Yeast-Based High-Throughput Screens to Identify Novel Compounds Active against Brugia malayi

    PubMed Central

    Bilsland, Elizabeth; Bean, Daniel M.; Devaney, Eileen; Oliver, Stephen G.

    2016-01-01

    Background Lymphatic filariasis is caused by the parasitic worms Wuchereria bancrofti, Brugia malayi or B. timori, which are transmitted via the bites from infected mosquitoes. Once in the human body, the parasites develop into adult worms in the lymphatic vessels, causing severe damage and swelling of the affected tissues. According to the World Health Organization, over 1.2 billion people in 58 countries are at risk of contracting lymphatic filariasis. Very few drugs are available to treat patients infected with these parasites, and these have low efficacy against the adult stages of the worms, which can live for 7–15 years in the human body. The requirement for annual treatment increases the risk of drug-resistant worms emerging, making it imperative to develop new drugs against these devastating diseases. Methodology/Principal Findings We have developed a yeast-based, high-throughput screening system whereby essential yeast genes are replaced with their filarial or human counterparts. These strains are labeled with different fluorescent proteins to allow the simultaneous monitoring of strains with parasite or human genes in competition, and hence the identification of compounds that inhibit the parasite target without affecting its human ortholog. We constructed yeast strains expressing eight different Brugia malayi drug targets (as well as seven of their human counterparts), and performed medium-throughput drug screens for compounds that specifically inhibit the parasite enzymes. Using the Malaria Box collection (400 compounds), we identified nine filarial specific inhibitors and confirmed the antifilarial activity of five of these using in vitro assays against Brugia pahangi. Conclusions/Significance We were able to functionally complement yeast deletions with eight different Brugia malayi enzymes that represent potential drug targets. We demonstrated that our yeast-based screening platform is efficient in identifying compounds that can discriminate between

  20. RNA interference (RNAi) screening approach identifies agents that enhance paclitaxel activity in breast cancer cells

    PubMed Central

    2010-01-01

    Introduction Paclitaxel is a widely used drug in the treatment of patients with locally advanced and metastatic breast cancer. However, only a small portion of patients have a complete response to paclitaxel-based chemotherapy, and many patients are resistant. Strategies that increase sensitivity and limit resistance to paclitaxel would be of clinical use, especially for patients with triple-negative breast cancer (TNBC). Methods We generated a gene set from overlay of the druggable genome and a collection of genomically deregulated gene transcripts in breast cancer. We used loss-of-function RNA interference (RNAi) to identify gene products in this set that, when targeted, increase paclitaxel sensitivity. Pharmacological agents that targeted the top scoring hits/genes from our RNAi screens were used in combination with paclitaxel, and the effects on the growth of various breast cancer cell lines were determined. Results RNAi screens performed herein were validated by identification of genes in pathways that, when previously targeted, enhanced paclitaxel sensitivity in the pre-clinical and clinical settings. When chemical inhibitors, CCT007093 and mithramycin, against two top hits in our screen, PPMID and SP1, respectively, were used in combination with paclitaxel, we observed synergistic growth inhibition in both 2D and 3D breast cancer cell cultures. The transforming growth factor beta (TGFβ) receptor inhibitor, LY2109761, that targets the signaling pathway of another top scoring hit, TGFβ1, was synergistic with paclitaxel when used in combination on select breast cancer cell lines grown in 3D culture. We also determined the relative paclitaxel sensitivity of 22 TNBC cell lines and identified 18 drug-sensitive and four drug-resistant cell lines. Of significance, we found that both CCT007093 and mithramycin, when used in combination with paclitaxel, resulted in synergistic inhibition of the four paclitaxel-resistant TNBC cell lines. Conclusions RNAi screening can

  1. An AICD-based functional screen to identify APP metabolism regulators

    PubMed Central

    Zhang, Can; Khandelwal, Preeti J; Chakraborty, Ranjita; Cuellar, Trinna L; Sarangi, Srikant; Patel, Shyam A; Cosentino, Christopher P; O'Connor, Michael; Lee, Jeremy C; Tanzi, Rudolph E; Saunders, Aleister J

    2007-01-01

    Background A central event in Alzheimer's disease (AD) is the regulated intramembraneous proteolysis of the β-amyloid precursor protein (APP), to generate the β-amyloid (Aβ) peptide and the APP intracellular domain (AICD). Aβ is the major component of amyloid plaques and AICD displays transcriptional activation properties. We have taken advantage of AICD transactivation properties to develop a genetic screen to identify regulators of APP metabolism. This screen relies on an APP-Gal4 fusion protein, which upon normal proteolysis, produces AICD-Gal4. Production of AICD-Gal4 induces Gal4-UAS driven luciferase expression. Therefore, when regulators of APP metabolism are modulated, luciferase expression is altered. Results To validate this experimental approach we modulated α-, β-, and γ-secretase levels and activities. Changes in AICD-Gal4 levels as measured by Western blot analysis were strongly and significantly correlated to the observed changes in AICD-Gal4 mediated luciferase activity. To determine if a known regulator of APP trafficking/maturation and Presenilin1 endoproteolysis could be detected using the AICD-Gal4 mediated luciferase assay, we knocked-down Ubiquilin 1 and observed decreased luciferase activity. We confirmed that Ubiquilin 1 modulated AICD-Gal4 levels by Western blot analysis and also observed that Ubiquilin 1 modulated total APP levels, the ratio of mature to immature APP, as well as PS1 endoproteolysis. Conclusion Taken together, we have shown that this screen can identify known APP metabolism regulators that control proteolysis, intracellular trafficking, maturation and levels of APP and its proteolytic products. We demonstrate for the first time that Ubiquilin 1 regulates APP metabolism in the human neuroblastoma cell line, SH-SY5Y. PMID:17718916

  2. Quantitative High-Throughput Screening Identifies 8-Hydroxyquinolines as Cell-Active Histone Demethylase Inhibitors

    PubMed Central

    Kawamura, Akane; Rose, Nathan R.; Ng, Stanley S.; Quinn, Amy M.; Rai, Ganesha; Mott, Bryan T.; Beswick, Paul; Klose, Robert J.; Oppermann, Udo; Jadhav, Ajit; Heightman, Tom D.; Maloney, David J.; Schofield, Christopher J.; Simeonov, Anton

    2010-01-01

    Background Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. Nε-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. Nε-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors. Principal Findings High-throughput screening of a ∼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4) family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II) and to modulate demethylation at the H3K9 locus in a cell-based assay. Conclusions These studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation. PMID:21124847

  3. An ENU mutagenesis screen identifies novel and known genes involved in epigenetic processes in the mouse

    PubMed Central

    2013-01-01

    Background We have used a sensitized ENU mutagenesis screen to produce mouse lines that carry mutations in genes required for epigenetic regulation. We call these lines Modifiers of murine metastable epialleles (Mommes). Results We report a basic molecular and phenotypic characterization for twenty of the Momme mouse lines, and in each case we also identify the causative mutation. Three of the lines carry a mutation in a novel epigenetic modifier, Rearranged L-myc fusion (Rlf), and one gene, Rap-interacting factor 1 (Rif1), has not previously been reported to be involved in transcriptional regulation in mammals. Many of the other lines are novel alleles of known epigenetic regulators. For two genes, Rlf and Widely-interspaced zinc finger (Wiz), we describe the first mouse mutants. All of the Momme mutants show some degree of homozygous embryonic lethality, emphasizing the importance of epigenetic processes. The penetrance of lethality is incomplete in a number of cases. Similarly, abnormalities in phenotype seen in the heterozygous individuals of some lines occur with incomplete penetrance. Conclusions Recent advances in sequencing enhance the power of sensitized mutagenesis screens to identify the function of previously uncharacterized factors and to discover additional functions for previously characterized proteins. The observation of incomplete penetrance of phenotypes in these inbred mutant mice, at various stages of development, is of interest. Overall, the Momme collection of mouse mutants provides a valuable resource for researchers across many disciplines. PMID:24025402

  4. A genome-wide screen identifies genes that affect somatic homolog pairing in Drosophila.

    PubMed

    Bateman, Jack R; Larschan, Erica; D'Souza, Ryan; Marshall, Lauren S; Dempsey, Kyle E; Johnson, Justine E; Mellone, Barbara G; Kuroda, Mitzi I

    2012-07-01

    In Drosophila and other Dipterans, homologous chromosomes are in close contact in virtually all nuclei, a phenomenon known as somatic homolog pairing. Although homolog pairing has been recognized for over a century, relatively little is known about its regulation. We performed a genome-wide RNAi-based screen that monitored the X-specific localization of the male-specific lethal (MSL) complex, and we identified 59 candidate genes whose knockdown via RNAi causes a change in the pattern of MSL staining that is consistent with a disruption of X-chromosomal homolog pairing. Using DNA fluorescent in situ hybridization (FISH), we confirmed that knockdown of 17 of these genes has a dramatic effect on pairing of the 359 bp repeat at the base of the X. Furthermore, dsRNAs targeting Pr-set7, which encodes an H4K20 methyltransferase, cause a modest disruption in somatic homolog pairing. Consistent with our results in cultured cells, a classical mutation in one of the strongest candidate genes, pebble (pbl), causes a decrease in somatic homolog pairing in developing embryos. Interestingly, many of the genes identified by our screen have known roles in diverse cell-cycle events, suggesting an important link between somatic homolog pairing and the choreography of chromosomes during the cell cycle.

  5. An interaction screen identifies headcase as a regulator of large-scale pruning

    PubMed Central

    Loncle, Nicolas; Williams, Darren W

    2012-01-01

    Large-scale pruning, the removal of long neuronal processes, is deployed widely within the developing nervous system and is essential for proper circuit formation. In Drosophila the dendrites of the class IV dendritic arborization sensory neuron ddaC undergo large-scale pruning by local degeneration controlled by the steroid hormone ecdysone. The molecular mechanisms that control such events are largely unknown. To identify new molecules that orchestrate this developmental degeneration we performed a genetic interaction screen. Our approach combines the strength of Drosophila forward genetics with detailed in vivo imaging of ddaC neurons. This screen allowed us to identify headcase (hdc) as a new gene involved in dendrite pruning. hdc is evolutionarily conserved, but the protein’s function is unknown. Here we show that hdc is expressed just prior to metamorphosis in sensory neurons that undergo remodeling. hdc is required in a cell autonomous manner to control dendrite severing, the first phase of pruning. Our epistasis experiments with known regulators of dendrite pruning reveal hdc as a founding member of a new pathway downstream of ecdysone signaling. PMID:23197702

  6. Kinase-interacting substrate screening is a novel method to identify kinase substrates

    PubMed Central

    Amano, Mutsuki; Hamaguchi, Tomonari; Shohag, Md. Hasanuzzaman; Kozawa, Kei; Kato, Katsuhiro; Zhang, Xinjian; Yura, Yoshimitsu; Matsuura, Yoshiharu; Kataoka, Chikako; Nishioka, Tomoki

    2015-01-01

    Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases. PMID:26101221

  7. A chemical screen for medulloblastoma identifies quercetin as a putative radiosensitizer.

    PubMed

    Lagerweij, Tonny; Hiddingh, Lotte; Biesmans, Dennis; Crommentuijn, Matheus H W; Cloos, Jacqueline; Li, Xiao-Nan; Kogiso, Mari; Tannous, Bakhos A; Vandertop, W Peter; Noske, David P; Kaspers, Gertjan J L; Würdinger, Tom; Hulleman, Esther

    2016-06-14

    Treatment of medulloblastoma in children fails in approximately 30% of patients, and is often accompanied by severe late sequelae. Therefore, more effective drugs are needed that spare normal tissue and diminish long-term side effects. Since radiotherapy plays a pivotal role in the treatment of medulloblastoma, we set out to identify novel drugs that could potentiate the effect of ionizing radiation.Thereto, a small molecule library, consisting of 960 chemical compounds, was screened for its ability to sensitize towards irradiation. This small molecule screen identified the flavonoid quercetin as a novel radiosensitizer for the medulloblastoma cell lines DAOY, D283-med, and, to a lesser extent, D458-med at low micromolar concentrations and irradiation doses used in fractionated radiation schemes. Quercetin did not affect the proliferation of neural precursor cells or normal human fibroblasts. Importantly, in vivo experiments confirmed the radiosensitizing properties of quercetin. Administration of this flavonoid at the time of irradiation significantly prolonged survival in orthotopically xenografted mice. Together, these findings indicate that quercetin is a potent radiosensitizer for medulloblastoma cells that may be a promising lead for the treatment of medulloblastoma in patients.

  8. Integrative screening approach identifies regulators of polyploidization and targets for acute megakaryocytic leukemia

    PubMed Central

    Wen, Qiang; Goldenson, Benjamin; Silver, Serena J.; Schenone, Monica; Dancik, Vladimir; Huang, Zan; Wang, Ling-Zhi; Lewis, Timothy; An, W. Frank; Li, Xiaoyu; Bray, Mark-Anthony; Thiollier, Clarisse; Diebold, Lauren; Gilles, Laure; Vokes, Martha S.; Moore, Christopher B.; Bliss-Moreau, Meghan; VerPlank, Lynn; Tolliday, Nicola J.; Mishra, Rama; Vemula, Sasidhar; Shi, Jianjian; Wei, Lei; Kapur, Reuben; Lopez, Cécile K.; Gerby, Bastien; Ballerini, Paola; Pflumio, Francoise; Gilliland, D. Gary; Goldberg, Liat; Birger, Yehudit; Izraeli, Shai; Gamis, Alan S.; Smith, Franklin O.; Woods, William G.; Taub, Jeffrey; Scherer, Christina A.; Bradner, James; Goh, Boon-Cher; Mercher, Thomas; Carpenter, Anne E.; Gould, Robert J.; Clemons, Paul A.; Carr, Steven A.; Root, David E.; Schreiber, Stuart L.; Stern, Andrew M.; Crispino, John D.

    2012-01-01

    Summary The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. We found that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. A broadly applicable, highly integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora A kinase (AURKA), which has not been studied extensively in megakaryocytes. Moreover, we discovered that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in AMKL blasts and displayed potent anti-AMKL activity in vivo. This research provides the rationale to support clinical trials of MLN8237 and other inducers of polyploidization in AMKL. Finally, we have identified five networks of kinases that regulate the switch to polyploidy. PMID:22863010

  9. Unique drug screening approach for prion diseases identifies tacrolimus and astemizole as antiprion agents

    PubMed Central

    Karapetyan, Yervand Eduard; Sferrazza, Gian Franco; Zhou, Minghai; Ottenberg, Gregory; Spicer, Timothy; Chase, Peter; Fallahi, Mohammad; Hodder, Peter; Weissmann, Charles; Lasmézas, Corinne Ida

    2013-01-01

    Prion diseases such as Creutzfeldt–Jakob disease (CJD) are incurable and rapidly fatal neurodegenerative diseases. Because prion protein (PrP) is necessary for prion replication but dispensable for the host, we developed the PrP–FRET-enabled high throughput assay (PrP–FEHTA) to screen for compounds that decrease PrP expression. We screened a collection of drugs approved for human use and identified astemizole and tacrolimus, which reduced cell-surface PrP and inhibited prion replication in neuroblastoma cells. Tacrolimus reduced total cellular PrP levels by a nontranscriptional mechanism. Astemizole stimulated autophagy, a hitherto unreported mode of action for this pharmacophore. Astemizole, but not tacrolimus, prolonged the survival time of prion-infected mice. Astemizole is used in humans to treat seasonal allergic rhinitis in a chronic setting. Given the absence of any treatment option for CJD patients and the favorable drug characteristics of astemizole, including its ability to cross the blood–brain barrier, it may be considered as therapy for CJD patients and for prophylactic use in familial prion diseases. Importantly, our results validate PrP-FEHTA as a method to identify antiprion compounds and, more generally, FEHTA as a unique drug discovery platform. PMID:23576755

  10. A chemical screen for medulloblastoma identifies quercetin as a putative radiosensitizer

    PubMed Central

    Biesmans, Dennis; Crommentuijn, Matheus H.W.; Cloos, Jacqueline; Li, Xiao-Nan; Kogiso, Mari; Tannous, Bakhos A.; Vandertop, W. Peter; Noske, David P.; Kaspers, Gertjan J.L.; Würdinger, Tom; Hulleman, Esther

    2016-01-01

    Treatment of medulloblastoma in children fails in approximately 30% of patients, and is often accompanied by severe late sequelae. Therefore, more effective drugs are needed that spare normal tissue and diminish long-term side effects. Since radiotherapy plays a pivotal role in the treatment of medulloblastoma, we set out to identify novel drugs that could potentiate the effect of ionizing radiation. Thereto, a small molecule library, consisting of 960 chemical compounds, was screened for its ability to sensitize towards irradiation. This small molecule screen identified the flavonoid quercetin as a novel radiosensitizer for the medulloblastoma cell lines DAOY, D283-med, and, to a lesser extent, D458-med at low micromolar concentrations and irradiation doses used in fractionated radiation schemes. Quercetin did not affect the proliferation of neural precursor cells or normal human fibroblasts. Importantly, in vivo experiments confirmed the radiosensitizing properties of quercetin. Administration of this flavonoid at the time of irradiation significantly prolonged survival in orthotopically xenografted mice. Together, these findings indicate that quercetin is a potent radiosensitizer for medulloblastoma cells that may be a promising lead for the treatment of medulloblastoma in patients. PMID:26967057

  11. High-Throughput Chemical Screens Identify Disulfiram as an Inhibitor of Human Glioblastoma Stem Cells

    PubMed Central

    Hothi, Parvinder; Martins, Timothy J.; Chen, LiPing; Deleyrolle, Loic; Yoon, Jae-Geun; Reynolds, Brent; Foltz, Greg

    2012-01-01

    Glioblastoma Multiforme (GBM) continues to have a poor patient prognosis despite optimal standard of care. Glioma stem cells (GSCs) have been implicated as the presumed cause of tumor recurrence and resistance to therapy. With this in mind, we screened a diverse chemical library of 2,000 compounds to identify therapeutic agents that inhibit GSC proliferation and therefore have the potential to extend patient survival. High-throughput screens (HTS) identified 78 compounds that repeatedly inhibited cellular proliferation, of which 47 are clinically approved for other indications and 31 are experimental drugs. Several compounds (such as digitoxin, deguelin, patulin and phenethyl caffeate) exhibited high cytotoxicity, with half maximal inhibitory concentrations (IC50) in the low nanomolar range. In particular, the FDA approved drug for the treatment of alcoholism, disulfiram (DSF), was significantly potent across multiple patient samples (IC50 of 31.1 nM). The activity of DSF was potentiated by copper (Cu), which markedly increased GSC death. DSF–Cu inhibited the chymotrypsin-like proteasomal activity in cultured GSCs, consistent with inactivation of the ubiquitin-proteasome pathway and the subsequent induction of tumor cell death. Given that DSF is a relatively non-toxic drug that can penetrate the blood-brain barrier, we suggest that DSF should be tested (as either a monotherapy or as an adjuvant) in pre-clinical models of human GBM. Data also support targeting of the ubiquitin-proteasome pathway as a therapeutic approach in the treatment of GBM. PMID:23165409

  12. Live cell screening platform identifies PPARδ as a regulator of cardiomyocyte proliferation and cardiac repair

    PubMed Central

    Magadum, Ajit; Ding, Yishu; He, Lan; Kim, Teayoun; Vasudevarao, Mohankrishna Dalvoy; Long, Qinqiang; Yang, Kevin; Wickramasinghe, Nadeera; Renikunta, Harsha V; Dubois, Nicole; Weidinger, Gilbert; Yang, Qinglin; Engel, Felix B

    2017-01-01

    Zebrafish can efficiently regenerate their heart through cardiomyocyte proliferation. In contrast, mammalian cardiomyocytes stop proliferating shortly after birth, limiting the regenerative capacity of the postnatal mammalian heart. Therefore, if the endogenous potential of postnatal cardiomyocyte proliferation could be enhanced, it could offer a promising future therapy for heart failure patients. Here, we set out to systematically identify small molecules triggering postnatal cardiomyocyte proliferation. By screening chemical compound libraries utilizing a Fucci-based system for assessing cell cycle stages, we identified carbacyclin as an inducer of postnatal cardiomyocyte proliferation. In vitro, carbacyclin induced proliferation of neonatal and adult mononuclear rat cardiomyocytes via a peroxisome proliferator-activated receptor δ (PPARδ)/PDK1/p308Akt/GSK3β/β-catenin pathway. Inhibition of PPARδ reduced cardiomyocyte proliferation during zebrafish heart regeneration. Notably, inducible cardiomyocyte-specific overexpression of constitutively active PPARδ as well as treatment with PPARδ agonist after myocardial infarction in mice induced cell cycle progression in cardiomyocytes, reduced scarring, and improved cardiac function. Collectively, we established a cardiomyocyte proliferation screening system and present a new drugable target with promise for the treatment of cardiac pathologies caused by cardiomyocyte loss. PMID:28621328

  13. A functional siRNA screen identifies genes modulating angiotensin II-mediated EGFR transactivation

    PubMed Central

    George, Amee J.; Purdue, Brooke W.; Gould, Cathryn M.; Thomas, Daniel W.; Handoko, Yanny; Qian, Hongwei; Quaife-Ryan, Gregory A.; Morgan, Kylie A.; Simpson, Kaylene J.; Thomas, Walter G.; Hannan, Ross D.

    2013-01-01

    Summary The angiotensin type 1 receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR) to mediate cellular growth, however, the molecular mechanisms involved have not yet been resolved. To address this, we performed a functional siRNA screen of the human kinome in human mammary epithelial cells that demonstrate a robust AT1R–EGFR transactivation. We identified a suite of genes encoding proteins that both positively and negatively regulate AT1R–EGFR transactivation. Many candidates are components of EGFR signalling networks, whereas others, including TRIO, BMX and CHKA, have not been previously linked to EGFR transactivation. Individual knockdown of TRIO, BMX or CHKA attenuated tyrosine phosphorylation of the EGFR by angiotensin II stimulation, but this did not occur following direct stimulation of the EGFR with EGF, indicating that these proteins function between the activated AT1R and the EGFR. Further investigation of TRIO and CHKA revealed that their activity is likely to be required for AT1R–EGFR transactivation. CHKA also mediated EGFR transactivation in response to another G protein-coupled receptor (GPCR) ligand, thrombin, indicating a pervasive role for CHKA in GPCR–EGFR crosstalk. Our study reveals the power of unbiased, functional genomic screens to identify new signalling mediators important for tissue remodelling in cardiovascular disease and cancer. PMID:24046455

  14. An ENU mutagenesis screen identifies novel and known genes involved in epigenetic processes in the mouse.

    PubMed

    Daxinger, Lucia; Harten, Sarah K; Oey, Harald; Epp, Trevor; Isbel, Luke; Huang, Edward; Whitelaw, Nadia; Apedaile, Anwyn; Sorolla, Anabel; Yong, Joan; Bharti, Vandhana; Sutton, Joanne; Ashe, Alyson; Pang, Zhenyi; Wallace, Nathan; Gerhardt, Daniel J; Blewitt, Marnie E; Jeddeloh, Jeffrey A; Whitelaw, Emma

    2013-01-01

    We have used a sensitized ENU mutagenesis screen to produce mouse lines that carry mutations in genes required for epigenetic regulation. We call these lines Modifiers of murine metastable epialleles (Mommes). We report a basic molecular and phenotypic characterization for twenty of the Momme mouse lines, and in each case we also identify the causative mutation. Three of the lines carry a mutation in a novel epigenetic modifier, Rearranged L-myc fusion (Rlf), and one gene, Rap-interacting factor 1 (Rif1), has not previously been reported to be involved in transcriptional regulation in mammals. Many of the other lines are novel alleles of known epigenetic regulators. For two genes, Rlf and Widely-interspaced zinc finger (Wiz), we describe the first mouse mutants. All of the Momme mutants show some degree of homozygous embryonic lethality, emphasizing the importance of epigenetic processes. The penetrance of lethality is incomplete in a number of cases. Similarly ,abnormalities in phenotype seen in the heterozygous individuals of some lines occur with incomplete penetrance. Recent advances in sequencing enhance the power of sensitized mutagenesis screens to identify the function of previously uncharacterized factors and to discover additional functions for previously characterized proteins. The observation of incomplete penetrance of phenotypes in these inbred mutant mice, at various stages of development, is of interest. Overall, the Momme collection of mouse mutants provides a valuable resource for researchers across many disciplines.

  15. Research resource: modulators of glucocorticoid receptor activity identified by a new high-throughput screening assay.

    PubMed

    Blackford, John A; Brimacombe, Kyle R; Dougherty, Edward J; Pradhan, Madhumita; Shen, Min; Li, Zhuyin; Auld, Douglas S; Chow, Carson C; Austin, Christopher P; Simons, S Stoney

    2014-07-01

    Glucocorticoid steroids affect almost every type of tissue and thus are widely used to treat a variety of human pathological conditions. However, the severity of numerous side effects limits the frequency and duration of glucocorticoid treatments. Of the numerous approaches to control off-target responses to glucocorticoids, small molecules and pharmaceuticals offer several advantages. Here we describe a new, extended high-throughput screen in intact cells to identify small molecule modulators of dexamethasone-induced glucocorticoid receptor (GR) transcriptional activity. The novelty of this assay is that it monitors changes in both GR maximal activity (A(max)) and EC(50) (the position of the dexamethasone dose-response curve). Upon screening 1280 chemicals, 10 with the greatest changes in the absolute value of A(max) or EC(50) were selected for further examination. Qualitatively identical behaviors for 60% to 90% of the chemicals were observed in a completely different system, suggesting that other systems will be similarly affected by these chemicals. Additional analysis of the 10 chemicals in a recently described competition assay determined their kinetically defined mechanism and site of action. Some chemicals had similar mechanisms of action despite divergent effects on the level of the GR-induced product. These combined assays offer a straightforward method of identifying numerous new pharmaceuticals that can alter GR transactivation in ways that could be clinically useful.

  16. Genome-wide functional screen identifies a compendium of genes affecting sensitivity to tamoxifen.

    PubMed

    Mendes-Pereira, Ana M; Sims, David; Dexter, Tim; Fenwick, Kerry; Assiotis, Ioannis; Kozarewa, Iwanka; Mitsopoulos, Costas; Hakas, Jarle; Zvelebil, Marketa; Lord, Christopher J; Ashworth, Alan

    2012-02-21

    Therapies that target estrogen signaling have made a very considerable contribution to reducing mortality from breast cancer. However, resistance to tamoxifen remains a major clinical problem. Here we have used a genome-wide functional profiling approach to identify multiple genes that confer resistance or sensitivity to tamoxifen. Combining whole-genome shRNA screening with massively parallel sequencing, we have profiled the impact of more than 56,670 RNA interference reagents targeting 16,487 genes on the cellular response to tamoxifen. This screen, along with subsequent validation experiments, identifies a compendium of genes whose silencing causes tamoxifen resistance (including BAP1, CLPP, GPRC5D, NAE1, NF1, NIPBL, NSD1, RAD21, RARG, SMC3, and UBA3) and also a set of genes whose silencing causes sensitivity to this endocrine agent (C10orf72, C15orf55/NUT, EDF1, ING5, KRAS, NOC3L, PPP1R15B, RRAS2, TMPRSS2, and TPM4). Multiple individual genes, including NF1, a regulator of RAS signaling, also correlate with clinical outcome after tamoxifen treatment.

  17. A Genome-Wide Screen for Dendritically Localized RNAs Identifies Genes Required for Dendrite Morphogenesis

    PubMed Central

    Misra, Mala; Edmund, Hendia; Ennis, Darragh; Schlueter, Marissa A.; Marot, Jessica E.; Tambasco, Janet; Barlow, Ida; Sigurbjornsdottir, Sara; Mathew, Renjith; Vallés, Ana Maria; Wojciech, Waldemar; Roth, Siegfried; Davis, Ilan; Leptin, Maria; Gavis, Elizabeth R.

    2016-01-01

    Localizing messenger RNAs at specific subcellular sites is a conserved mechanism for targeting the synthesis of cytoplasmic proteins to distinct subcellular domains, thereby generating the asymmetric protein distributions necessary for cellular and developmental polarity. However, the full range of transcripts that are asymmetrically distributed in specialized cell types, and the significance of their localization, especially in the nervous system, are not known. We used the EP-MS2 method, which combines EP transposon insertion with the MS2/MCP in vivo fluorescent labeling system, to screen for novel localized transcripts in polarized cells, focusing on the highly branched Drosophila class IV dendritic arborization neurons. Of a total of 541 lines screened, we identified 55 EP-MS2 insertions producing transcripts that were enriched in neuronal processes, particularly in dendrites. The 47 genes identified by these insertions encode molecularly diverse proteins, and are enriched for genes that function in neuronal development and physiology. RNAi-mediated knockdown confirmed roles for many of the candidate genes in dendrite morphogenesis. We propose that the transport of mRNAs encoded by these genes into the dendrites allows their expression to be regulated on a local scale during the dynamic developmental processes of dendrite outgrowth, branching, and/or remodeling. PMID:27260999

  18. A Genome-Wide Screen for Dendritically Localized RNAs Identifies Genes Required for Dendrite Morphogenesis.

    PubMed

    Misra, Mala; Edmund, Hendia; Ennis, Darragh; Schlueter, Marissa A; Marot, Jessica E; Tambasco, Janet; Barlow, Ida; Sigurbjornsdottir, Sara; Mathew, Renjith; Vallés, Ana Maria; Wojciech, Waldemar; Roth, Siegfried; Davis, Ilan; Leptin, Maria; Gavis, Elizabeth R

    2016-08-09

    Localizing messenger RNAs at specific subcellular sites is a conserved mechanism for targeting the synthesis of cytoplasmic proteins to distinct subcellular domains, thereby generating the asymmetric protein distributions necessary for cellular and developmental polarity. However, the full range of transcripts that are asymmetrically distributed in specialized cell types, and the significance of their localization, especially in the nervous system, are not known. We used the EP-MS2 method, which combines EP transposon insertion with the MS2/MCP in vivo fluorescent labeling system, to screen for novel localized transcripts in polarized cells, focusing on the highly branched Drosophila class IV dendritic arborization neurons. Of a total of 541 lines screened, we identified 55 EP-MS2 insertions producing transcripts that were enriched in neuronal processes, particularly in dendrites. The 47 genes identified by these insertions encode molecularly diverse proteins, and are enriched for genes that function in neuronal development and physiology. RNAi-mediated knockdown confirmed roles for many of the candidate genes in dendrite morphogenesis. We propose that the transport of mRNAs encoded by these genes into the dendrites allows their expression to be regulated on a local scale during the dynamic developmental processes of dendrite outgrowth, branching, and/or remodeling.

  19. A functional genomic screen in planarians identifies novel regulators of germ cell development.

    PubMed

    Wang, Yuying; Stary, Joel M; Wilhelm, James E; Newmark, Phillip A

    2010-09-15

    Germ cells serve as intriguing examples of differentiated cells that retain the capacity to generate all cell types of an organism. Here we used functional genomic approaches in planarians to identify genes required for proper germ cell development. We conducted microarray analyses and in situ hybridization to discover and validate germ cell-enriched transcripts, and then used RNAi to screen for genes required for discrete stages of germ cell development. The majority of genes we identified encode conserved RNA-binding proteins, several of which have not been implicated previously in germ cell development. We also show that a germ cell-specific subunit of the conserved transcription factor CCAAT-binding protein/nuclear factor-Y is required for maintaining spermatogonial stem cells. Our results demonstrate that conserved transcriptional and post-transcriptional mechanisms regulate germ cell development in planarians. These findings suggest that studies of planarians will inform our understanding of germ cell biology in higher organisms.

  20. Novel anti- Plasmodial hits identified by virtual screening of the ZINC database

    NASA Astrophysics Data System (ADS)

    Mugumbate, Grace; Newton, Ana S.; Rosenthal, Philip J.; Gut, Jiri; Moreira, Rui; Chibale, Kelly; Guedes, Rita C.

    2013-10-01

    Increased resistance of Plasmodium falciparum to most available drugs challenges the control of malaria. Studies with protease inhibitors have suggested important roles for the falcipain family of cysteine proteases. These enzymes act in concert with other proteases to hydrolyze host erythrocyte hemoglobin in the parasite food vacuole. In order to find potential new antimalarial drugs, we screened in silico the ZINC database using two different protocols involving structure- and ligand-based methodologies. Our search identified 19 novel low micromolar inhibitors of cultured chloroquine resistant P. falciparum. The most active compound presented an IC50 value of 0.5 μM against cultured parasites and it also inhibited the cysteine protease falcipain-2 (IC50 = 25.5 μM). These results identify novel classes of antimalarials that are structurally different from those currently in use and which can be further derivatized to deliver leads suitable for optimisation.

  1. Application of modified in vitro screening procedure for identifying herbals possessing sulfonylurea-like activity.

    PubMed

    Rotshteyn, Y; Zito, S W

    2004-08-01

    We describe here the application of a modified in vitro procedure for identifying herbs potentially possessing sulfonylurea-like activity. The procedure consists of the combination of an SUR1 receptor binding assay and an insulin secretion assay in cultures of HIT-T15 cells. This procedure could be used as an initial step in identifying new safe and efficacious agents for the management of Type II diabetes. The application of this screening procedure to a set of selected herbs produced results that were consistent with the previously reported properties of those herbs. The collected data suggest that the hypoglycemic properties of bitter melon (Momordica charantia, Linn. Family, Cucurbitacea), cerasse (Momordica charantia, Linn. wild variety, Family, Cucurbitacea) and American ginseng (Panax quinquefolius, Linn., Family Araliacea) are at least partially due to their sulfonylurea-like activity.

  2. A functional genomic screen in planarians identifies novel regulators of germ cell development

    PubMed Central

    Wang, Yuying; Stary, Joel M.; Wilhelm, James E.; Newmark, Phillip A.

    2010-01-01

    Germ cells serve as intriguing examples of differentiated cells that retain the capacity to generate all cell types of an organism. Here we used functional genomic approaches in planarians to identify genes required for proper germ cell development. We conducted microarray analyses and in situ hybridization to discover and validate germ cell-enriched transcripts, and then used RNAi to screen for genes required for discrete stages of germ cell development. The majority of genes we identified encode conserved RNA-binding proteins, several of which have not been implicated previously in germ cell development. We also show that a germ cell-specific subunit of the conserved transcription factor CCAAT-binding protein/nuclear factor-Y is required for maintaining spermatogonial stem cells. Our results demonstrate that conserved transcriptional and post-transcriptional mechanisms regulate germ cell development in planarians. These findings suggest that studies of planarians will inform our understanding of germ cell biology in higher organisms. PMID:20844018

  3. Novel Dual-Reporter Preclinical Screen for Anti-Astrocytoma Agents Identifies Cytostatic and Cytotoxic Compounds

    PubMed Central

    Hawes, Jessica J.; Nerva, John D.; Reilly, Karlyne M.

    2009-01-01

    Astrocytoma/glioblastoma is the most common malignant form of brain cancer and is often unresponsive to current pharmacological therapies and surgical interventions. Despite several potential therapeutic agents against astrocytoma and glioblastoma (1), there are currently no effective therapies for astrocytoma, creating a great need for the identification of effective anti-tumor agents. We have developed a novel dual-reporter system in Trp53/Nf1-null astrocytoma cells to simultaneously and rapidly assay cell viability and cell cycle progression as evidenced by activity of the human E2F1 promoter in vitro. The dual-reporter high-throughput assay was used to screen experimental therapeutics for activity in Trp53/Nf1-null astrocytoma. Several compounds were identified demonstrating selectivity for astrocytoma over primary astrocytes. The dual-reporter system described here may be a valuable tool for identifying potential anti-tumor treatments that specifically target astrocytoma. PMID:18664715

  4. Comparative RNAi screening identifies a conserved core metazoan actinome by phenotype

    PubMed Central

    Sims, David; Liu, Tao; Fedorova, Marina; Schöck, Frieder; Dopie, Joseph; Vartiainen, Maria K.; Kiger, Amy A.; Perrimon, Norbert

    2011-01-01

    Although a large number of actin-binding proteins and their regulators have been identified through classical approaches, gaps in our knowledge remain. Here, we used genome-wide RNA interference as a systematic method to define metazoan actin regulators based on visual phenotype. Using comparative screens in cultured Drosophila and human cells, we generated phenotypic profiles for annotated actin regulators together with proteins bearing predicted actin-binding domains. These phenotypic clusters for the known metazoan “actinome” were used to identify putative new core actin regulators, together with a number of genes with conserved but poorly studied roles in the regulation of the actin cytoskeleton, several of which we studied in detail. This work suggests that although our search for new components of the core actin machinery is nearing saturation, regulation at the level of nuclear actin export, RNA splicing, ubiquitination, and other upstream processes remains an important but unexplored frontier of actin biology. PMID:21893601

  5. A high-content morphological screen identifies novel microRNAs that regulate neuroblastoma cell differentiation.

    PubMed

    Zhao, Zhenze; Ma, Xiuye; Hsiao, Tzu-Hung; Lin, Gregory; Kosti, Adam; Yu, Xiaojie; Suresh, Uthra; Chen, Yidong; Tomlinson, Gail E; Pertsemlidis, Alexander; Du, Liqin

    2014-05-15

    Neuroblastoma, the most common extracranial solid tumor of childhood, arises from neural crest cell precursors that fail to differentiate. Inducing cell differentiation is an important therapeutic strategy for neuroblastoma. We developed a direct functional high-content screen to identify differentiation-inducing microRNAs, in order to develop microRNA-based differentiation therapy for neuroblastoma. We discovered novel microRNAs, and more strikingly, three microRNA seed families that induce neuroblastoma cell differentiation. In addition, we showed that microRNA seed families were overrepresented in the identified group of fourteen differentiation-inducing microRNAs, suggesting that microRNA seed families are functionally more important in neuroblastoma differentiation than microRNAs with unique sequences. We further investigated the differentiation-inducing function of the microRNA-506-3p/microRNA-124-3p seed family, which was the most potent inducer of differentiation. We showed that the differentiation-inducing function of microRNA-506-3p/microRNA-124-3p is mediated, at least partially, by down-regulating expression of their targets CDK4 and STAT3. We further showed that expression of miR-506-3p, but not miR-124-3p, is dramatically upregulated in differentiated neuroblastoma cells, suggesting the important role of endogenous miR-506-3p in differentiation and tumorigenesis. Overall, our functional screen on microRNAs provided the first comprehensive analysis on the involvements of microRNA species in neuroblastoma cell differentiation and identified novel differentiation-inducing microRNAs. Further investigations are certainly warranted to fully characterize the function of the identified microRNAs in order to eventually benefit neuroblastoma therapy.

  6. Iterative carotenogenic screens identify combinations of yeast gene deletions that enhance sclareol production.

    PubMed

    Trikka, Fotini A; Nikolaidis, Alexandros; Athanasakoglou, Anastasia; Andreadelli, Aggeliki; Ignea, Codruta; Kotta, Konstantia; Argiriou, Anagnostis; Kampranis, Sotirios C; Makris, Antonios M

    2015-04-24

    Terpenoids (isoprenoids) have numerous applications in flavors, fragrances, drugs and biofuels. The number of microbially produced terpenoids is increasing as new biosynthetic pathways are being elucidated. However, efforts to improve terpenoid production in yeast have mostly taken advantage of existing knowledge of the sterol biosynthetic pathway, while many additional factors may affect the output of the engineered system. Aiming to develop a yeast strain that can support high titers of sclareol, a diterpene of great importance for the perfume industry, we sought to identify gene deletions that improved carotenoid, and thus potentially sclareol, production. Using a carotenogenic screen, the best 100 deletion mutants, out of 4,700 mutant strains, were selected to create a subset for further analysis. To identify combinations of deletions that cooperate to further boost production, iterative carotenogenic screens were applied, and each time the top performing gene deletions were further ranked according to the number of genetic and physical interactions known for each specific gene. The gene selected in each round was deleted and the resulting strain was employed in a new round of selection. This approach led to the development of an EG60 derived haploid strain combining six deletions (rox1, dos2, yer134c, vba5, ynr063w and ygr259c) and exhibiting a 40-fold increase in carotenoid and 12-fold increase in sclareol titers, reaching 750 mg/L sclareol in shake flask cultivation. Using an iterative approach, we identified novel combinations of yeast gene deletions that improve carotenoid and sclareol production titers without compromising strain growth and viability. Most of the identified deletions have not previously been implicated in sterol pathway control. Applying the same approach using a different starting point could yield alternative sets of deletions with similar or improved outcome.

  7. A high-content morphological screen identifies novel microRNAs that regulate neuroblastoma cell differentiation

    PubMed Central

    Zhao, Zhenze; Ma, Xiuye; Hsiao, Tzu-Hung; Lin, Gregory; Kosti, Adam; Yu, Xiaojie; Suresh, Uthra; Chen, Yidong; Tomlinson, Gail E.; Pertsemlidis, Alexander; Du, Liqin

    2014-01-01

    Neuroblastoma, the most common extracranial solid tumor of childhood, arises from neural crest cell precursors that fail to differentiate. Inducing cell differentiation is an important therapeutic strategy for neuroblastoma. We developed a direct functional high-content screen to identify differentiation-inducing microRNAs, in order to develop microRNA-based differentiation therapy for neuroblastoma. We discovered novel microRNAs, and more strikingly, three microRNA seed families that induce neuroblastoma cell differentiation. In addition, we showed that microRNA seed families were overrepresented in the identified group of fourteen differentiation-inducing microRNAs, suggesting that microRNA seed families are functionally more important in neuroblastoma differentiation than microRNAs with unique sequences. We further investigated the differentiation-inducing function of the microRNA-506-3p/microRNA-124-3p seed family, which was the most potent inducer of differentiation. We showed that the differentiation-inducing function of microRNA-506-3p/microRNA-124-3p is mediated, at least partially, by down-regulating expression of their targets CDK4 and STAT3. We further showed that expression of miR-506-3p, but not miR-124-3p, is dramatically upregulated in differentiated neuroblastoma cells, suggesting the important role of endogenous miR-506-3p in differentiation and tumorigenesis. Overall, our functional screen on microRNAs provided the first comprehensive analysis on the involvements of microRNA species in neuroblastoma cell differentiation and identified novel differentiation-inducing microRNAs. Further investigations are certainly warranted to fully characterize the function of the identified microRNAs in order to eventually benefit neuroblastoma therapy. PMID:24811707

  8. A Sleeping Beauty forward genetic screen identifies new genes and pathways driving osteosarcoma development and metastasis

    PubMed Central

    Moriarity, Branden S; Otto, George M; Rahrmann, Eric P; Rathe, Susan K; Wolf, Natalie K; Weg, Madison T; Manlove, Luke A; LaRue, Rebecca S; Temiz, Nuri A; Molyneux, Sam D; Choi, Kwangmin; Holly, Kevin J; Sarver, Aaron L; Scott, Milcah C; Forster, Colleen L; Modiano, Jaime F; Khanna, Chand; Hewitt, Stephen M; Khokha, Rama; Yang, Yi; Gorlick, Richard; Dyer, Michael A; Largaespada, David A

    2016-01-01

    Osteosarcomas are sarcomas of the bone, derived from osteoblasts or their precursors, with a high propensity to metastasize. Osteosarcoma is associated with massive genomic instability, making it problematic to identify driver genes using human tumors or prototypical mouse models, many of which involve loss of Trp53 function. To identify the genes driving osteosarcoma development and metastasis, we performed a Sleeping Beauty (SB) transposon-based forward genetic screen in mice with and without somatic loss of Trp53. Common insertion site (CIS) analysis of 119 primary tumors and 134 metastatic nodules identified 232 sites associated with osteosarcoma development and 43 sites associated with metastasis, respectively. Analysis of CIS-associated genes identified numerous known and new osteosarcoma-associated genes enriched in the ErbB, PI3K-AKT-mTOR and MAPK signaling pathways. Lastly, we identified several oncogenes involved in axon guidance, including Sema4d and Sema6d, which we functionally validated as oncogenes in human osteosarcoma. PMID:25961939

  9. A small molecule screen identifies in vivo modulators of peripheral nerve regeneration in zebrafish

    PubMed Central

    Skinner, Julianne; Granato, Michael

    2017-01-01

    Adult vertebrates have retained the ability to regenerate peripheral nerves after injury, although regeneration is frequently incomplete, often leading to functional impairments. Small molecule screens using whole organisms have high potential to identify biologically relevant targets, yet currently available assays for in vivo peripheral nerve regeneration are either very laborious and/or require complex technology. Here we take advantage of the optical transparency of larval zebrafish to develop a simple and fast pectoral fin removal assay that measures peripheral nerve regeneration in vivo. Twenty-four hours after fin amputation we observe robust and stereotyped nerve regrowth at the fin base. Similar to laser mediated nerve transection, nerve regrowth after fin amputation requires Schwann cells and FGF signaling, confirming that the fin amputation assay identifies pathways relevant for peripheral nerve regeneration. From a library of small molecules with known targets, we identified 21 compounds that impair peripheral nerve regeneration. Several of these compounds target known regulators of nerve regeneration, further validating the fin removal assay. Twelve of the identified compounds affect targets not previously known to control peripheral nerve regeneration. Using a laser-mediated nerve transection assay we tested ten of those compounds and confirmed six of these compounds to impair peripheral nerve regeneration: an EGFR inhibitor, a glucocorticoid, prostaglandin D2, a retinoic acid agonist, an inhibitor of calcium channels and a topoisomerase I inhibitor. Thus, we established a technically simple assay to rapidly identify valuable entry points into pathways critical for vertebrate peripheral nerve regeneration. PMID:28575069

  10. A Sleeping Beauty forward genetic screen identifies new genes and pathways driving osteosarcoma development and metastasis.

    PubMed

    Moriarity, Branden S; Otto, George M; Rahrmann, Eric P; Rathe, Susan K; Wolf, Natalie K; Weg, Madison T; Manlove, Luke A; LaRue, Rebecca S; Temiz, Nuri A; Molyneux, Sam D; Choi, Kwangmin; Holly, Kevin J; Sarver, Aaron L; Scott, Milcah C; Forster, Colleen L; Modiano, Jaime F; Khanna, Chand; Hewitt, Stephen M; Khokha, Rama; Yang, Yi; Gorlick, Richard; Dyer, Michael A; Largaespada, David A

    2015-06-01

    Osteosarcomas are sarcomas of the bone, derived from osteoblasts or their precursors, with a high propensity to metastasize. Osteosarcoma is associated with massive genomic instability, making it problematic to identify driver genes using human tumors or prototypical mouse models, many of which involve loss of Trp53 function. To identify the genes driving osteosarcoma development and metastasis, we performed a Sleeping Beauty (SB) transposon-based forward genetic screen in mice with and without somatic loss of Trp53. Common insertion site (CIS) analysis of 119 primary tumors and 134 metastatic nodules identified 232 sites associated with osteosarcoma development and 43 sites associated with metastasis, respectively. Analysis of CIS-associated genes identified numerous known and new osteosarcoma-associated genes enriched in the ErbB, PI3K-AKT-mTOR and MAPK signaling pathways. Lastly, we identified several oncogenes involved in axon guidance, including Sema4d and Sema6d, which we functionally validated as oncogenes in human osteosarcoma.

  11. A loss-of-function genetic screening identifies novel mediators of thyroid cancer cell viability

    PubMed Central

    Cantisani, Maria Carmela; Parascandolo, Alessia; Perälä, Merja; Allocca, Chiara; Fey, Vidal; Sahlberg, Niko; Merolla, Francesco; Basolo, Fulvio; Laukkanen, Mikko O.; Kallioniemi, Olli Pekka; Santoro, Massimo; Castellone, Maria Domenica

    2016-01-01

    RET, BRAF and other protein kinases have been identified as major molecular players in thyroid cancer. To identify novel kinases required for the viability of thyroid carcinoma cells, we performed a RNA interference screening in the RET/PTC1(CCDC6-RET)-positive papillary thyroid cancer cell line TPC1 using a library of synthetic small interfering RNAs (siRNAs) targeting the human kinome and related proteins. We identified 14 hits whose silencing was able to significantly reduce the viability and the proliferation of TPC1 cells; most of them were active also in BRAF-mutant BCPAP (papillary thyroid cancer) and 8505C (anaplastic thyroid cancer) and in RAS-mutant CAL62 (anaplastic thyroid cancer) cells. These included members of EPH receptor tyrosine kinase family as well as SRC and MAPK (mitogen activated protein kinases) families. Importantly, silencing of the identified hits did not affect significantly the viability of Nthy-ori 3-1 (hereafter referred to as NTHY) cells derived from normal thyroid tissue, suggesting cancer cell specificity. The identified proteins are worth exploring as potential novel druggable thyroid cancer targets. PMID:27058903

  12. BFH-OST, a new predictive screening tool for identifying osteoporosis in postmenopausal Han Chinese women

    PubMed Central

    Ma, Zhao; Yang, Yong; Lin, JiSheng; Zhang, XiaoDong; Meng, Qian; Wang, BingQiang; Fei, Qi

    2016-01-01

    Purpose To develop a simple new clinical screening tool to identify primary osteoporosis by dual-energy X-ray absorptiometry (DXA) in postmenopausal women and to compare its validity with the Osteoporosis Self-Assessment Tool for Asians (OSTA) in a Han Chinese population. Methods A cross-sectional study was conducted, enrolling 1,721 community-dwelling postmenopausal Han Chinese women. All the subjects completed a structured questionnaire and had their bone mineral density measured using DXA. Using logistic regression analysis, we assessed the ability of numerous potential risk factors examined in the questionnaire to identify women with osteoporosis. Based on this analysis, we build a new predictive model, the Beijing Friendship Hospital Osteoporosis Self-Assessment Tool (BFH-OST). Receiver operating characteristic curves were generated to compare the validity of the new model and OSTA in identifying postmenopausal women at increased risk of primary osteoporosis as defined according to the World Health Organization criteria. Results At screening, it was found that of the 1,721 subjects with DXA, 22.66% had osteoporosis and a further 47.36% had osteopenia. Of the items screened in the questionnaire, it was found that age, weight, height, body mass index, personal history of fracture after the age of 45 years, history of fragility fracture in either parent, current smoking, and consumption of three of more alcoholic drinks per day were all predictive of osteoporosis. However, age at menarche and menopause, years since menopause, and number of pregnancies and live births were irrelevant in this study. The logistic regression analysis and item reduction yielded a final tool (BFH-OST) based on age, body weight, height, and history of fracture after the age of 45 years. The BFH-OST index (cutoff =9.1), which performed better than OSTA, had a sensitivity of 73.6% and a specificity of 72.7% for identifying osteoporosis, with an area under the receiver operating

  13. BFH-OST, a new predictive screening tool for identifying osteoporosis in postmenopausal Han Chinese women.

    PubMed

    Ma, Zhao; Yang, Yong; Lin, JiSheng; Zhang, XiaoDong; Meng, Qian; Wang, BingQiang; Fei, Qi

    2016-01-01

    To develop a simple new clinical screening tool to identify primary osteoporosis by dual-energy X-ray absorptiometry (DXA) in postmenopausal women and to compare its validity with the Osteoporosis Self-Assessment Tool for Asians (OSTA) in a Han Chinese population. A cross-sectional study was conducted, enrolling 1,721 community-dwelling postmenopausal Han Chinese women. All the subjects completed a structured questionnaire and had their bone mineral density measured using DXA. Using logistic regression analysis, we assessed the ability of numerous potential risk factors examined in the questionnaire to identify women with osteoporosis. Based on this analysis, we build a new predictive model, the Beijing Friendship Hospital Osteoporosis Self-Assessment Tool (BFH-OST). Receiver operating characteristic curves were generated to compare the validity of the new model and OSTA in identifying postmenopausal women at increased risk of primary osteoporosis as defined according to the World Health Organization criteria. At screening, it was found that of the 1,721 subjects with DXA, 22.66% had osteoporosis and a further 47.36% had osteopenia. Of the items screened in the questionnaire, it was found that age, weight, height, body mass index, personal history of fracture after the age of 45 years, history of fragility fracture in either parent, current smoking, and consumption of three of more alcoholic drinks per day were all predictive of osteoporosis. However, age at menarche and menopause, years since menopause, and number of pregnancies and live births were irrelevant in this study. The logistic regression analysis and item reduction yielded a final tool (BFH-OST) based on age, body weight, height, and history of fracture after the age of 45 years. The BFH-OST index (cutoff =9.1), which performed better than OSTA, had a sensitivity of 73.6% and a specificity of 72.7% for identifying osteoporosis, with an area under the receiver operating characteristic curve of 0.797. BFH

  14. Drug Repurposing Screening Identifies Novel Compounds That Effectively Inhibit Toxoplasma gondii Growth

    PubMed Central

    Dittmar, Ashley J.; Drozda, Allison A.

    2016-01-01

    ABSTRACT The urgent need to develop new antimicrobial therapies has spawned the development of repurposing screens in which well-studied drugs and other types of compounds are tested for potential off-label uses. As a proof-of-principle screen to identify compounds effective against Toxoplasma gondii, we screened a collection of 1,120 compounds for the ability to significantly reduce Toxoplasma replication. A total of 94 compounds blocked parasite replication with 50% inhibitory concentrations of <5 µM. A significant number of these compounds are established inhibitors of dopamine or estrogen signaling. Follow-up experiments with the dopamine receptor inhibitor pimozide revealed that the drug impacted both parasite invasion and replication but did so independently of inhibition of dopamine or other neurotransmitter receptor signaling. Tamoxifen, which is an established inhibitor of the estrogen receptor, also reduced parasite invasion and replication. Even though Toxoplasma can activate the estrogen receptor, tamoxifen inhibits parasite growth independently of this transcription factor. Tamoxifen is also a potent inducer of autophagy, and we find that the drug stimulates recruitment of the autophagy marker light chain 3-green fluorescent protein onto the membrane of the vacuolar compartment in which the parasite resides and replicates. In contrast to other antiparasitic drugs, including pimozide, tamoxifen treatment of infected cells leads to a time-dependent elimination of intracellular parasites. Taken together, these data suggest that tamoxifen restricts Toxoplasma growth by inducing xenophagy or autophagic destruction of this obligate intracellular parasite. IMPORTANCE There is an urgent need to develop new therapies to treat microbial infections, and the repurposing of well-characterized compounds is emerging as one approach to achieving this goal. Using the protozoan parasite Toxoplasma gondii, we screened a library of 1,120 compounds and identified several

  15. A high-throughput phenotypic screen identifies clofazimine as a potential treatment for cryptosporidiosis

    PubMed Central

    Jumani, Rajiv S.; Wright, Timothy M.; Chatterjee, Arnab K.; Huston, Christopher D.; Schultz, Peter G.; McNamara, Case W.

    2017-01-01

    Cryptosporidiosis has emerged as a leading cause of non-viral diarrhea in children under five years of age in the developing world, yet the current standard of care to treat Cryptosporidium infections, nitazoxanide, demonstrates limited and immune-dependent efficacy. Given the lack of treatments with universal efficacy, drug discovery efforts against cryptosporidiosis are necessary to find therapeutics more efficacious than the standard of care. To date, cryptosporidiosis drug discovery efforts have been limited to a few targeted mechanisms in the parasite and whole cell phenotypic screens against small, focused collections of compounds. Using a previous screen as a basis, we initiated the largest known drug discovery effort to identify novel anticryptosporidial agents. A high-content imaging assay for inhibitors of Cryptosporidium parvum proliferation within a human intestinal epithelial cell line was miniaturized and automated to enable high-throughput phenotypic screening against a large, diverse library of small molecules. A screen of 78,942 compounds identified 12 anticryptosporidial hits with sub-micromolar activity, including clofazimine, an FDA-approved drug for the treatment of leprosy, which demonstrated potent and selective in vitro activity (EC50 = 15 nM) against C. parvum. Clofazimine also displayed activity against C. hominis–the other most clinically-relevant species of Cryptosporidium. Importantly, clofazimine is known to accumulate within epithelial cells of the small intestine, the primary site of Cryptosporidium infection. In a mouse model of acute cryptosporidiosis, a once daily dosage regimen for three consecutive days or a single high dose resulted in reduction of oocyst shedding below the limit detectable by flow cytometry. Recently, a target product profile (TPP) for an anticryptosporidial compound was proposed by Huston et al. and highlights the need for a short dosing regimen (< 7 days) and formulations for children < 2 years

  16. A high-throughput phenotypic screen identifies clofazimine as a potential treatment for cryptosporidiosis.

    PubMed

    Love, Melissa S; Beasley, Federico C; Jumani, Rajiv S; Wright, Timothy M; Chatterjee, Arnab K; Huston, Christopher D; Schultz, Peter G; McNamara, Case W

    2017-02-01

    Cryptosporidiosis has emerged as a leading cause of non-viral diarrhea in children under five years of age in the developing world, yet the current standard of care to treat Cryptosporidium infections, nitazoxanide, demonstrates limited and immune-dependent efficacy. Given the lack of treatments with universal efficacy, drug discovery efforts against cryptosporidiosis are necessary to find therapeutics more efficacious than the standard of care. To date, cryptosporidiosis drug discovery efforts have been limited to a few targeted mechanisms in the parasite and whole cell phenotypic screens against small, focused collections of compounds. Using a previous screen as a basis, we initiated the largest known drug discovery effort to identify novel anticryptosporidial agents. A high-content imaging assay for inhibitors of Cryptosporidium parvum proliferation within a human intestinal epithelial cell line was miniaturized and automated to enable high-throughput phenotypic screening against a large, diverse library of small molecules. A screen of 78,942 compounds identified 12 anticryptosporidial hits with sub-micromolar activity, including clofazimine, an FDA-approved drug for the treatment of leprosy, which demonstrated potent and selective in vitro activity (EC50 = 15 nM) against C. parvum. Clofazimine also displayed activity against C. hominis-the other most clinically-relevant species of Cryptosporidium. Importantly, clofazimine is known to accumulate within epithelial cells of the small intestine, the primary site of Cryptosporidium infection. In a mouse model of acute cryptosporidiosis, a once daily dosage regimen for three consecutive days or a single high dose resulted in reduction of oocyst shedding below the limit detectable by flow cytometry. Recently, a target product profile (TPP) for an anticryptosporidial compound was proposed by Huston et al. and highlights the need for a short dosing regimen (< 7 days) and formulations for children < 2 years. Clofazimine

  17. Overexpression screens identify conserved dosage chromosome instability genes in yeast and human cancer

    PubMed Central

    Duffy, Supipi; Fam, Hok Khim; Wang, Yi Kan; Styles, Erin B.; Kim, Jung-Hyun; Ang, J. Sidney; Singh, Tejomayee; Larionov, Vladimir; Shah, Sohrab P.; Andrews, Brenda; Boerkoel, Cornelius F.; Hieter, Philip

    2016-01-01

    Somatic copy number amplification and gene overexpression are common features of many cancers. To determine the role of gene overexpression on chromosome instability (CIN), we performed genome-wide screens in the budding yeast for yeast genes that cause CIN when overexpressed, a phenotype we refer to as dosage CIN (dCIN), and identified 245 dCIN genes. This catalog of genes reveals human orthologs known to be recurrently overexpressed and/or amplified in tumors. We show that two genes, TDP1, a tyrosyl-DNA-phosphdiesterase, and TAF12, an RNA polymerase II TATA-box binding factor, cause CIN when overexpressed in human cells. Rhabdomyosarcoma lines with elevated human Tdp1 levels also exhibit CIN that can be partially rescued by siRNA-mediated knockdown of TDP1. Overexpression of dCIN genes represents a genetic vulnerability that could be leveraged for selective killing of cancer cells through targeting of an unlinked synthetic dosage lethal (SDL) partner. Using SDL screens in yeast, we identified a set of genes that when deleted specifically kill cells with high levels of Tdp1. One gene was the histone deacetylase RPD3, for which there are known inhibitors. Both HT1080 cells overexpressing hTDP1 and rhabdomyosarcoma cells with elevated levels of hTdp1 were more sensitive to histone deacetylase inhibitors valproic acid (VPA) and trichostatin A (TSA), recapitulating the SDL interaction in human cells and suggesting VPA and TSA as potential therapeutic agents for tumors with elevated levels of hTdp1. The catalog of dCIN genes presented here provides a candidate list to identify genes that cause CIN when overexpressed in cancer, which can then be leveraged through SDL to selectively target tumors. PMID:27551064

  18. Potent Plasmodium falciparum Gametocytocidal Activity of Diaminonaphthoquinones, Lead Antimalarial Chemotypes Identified in an Antimalarial Compound Screen

    PubMed Central

    Tanaka, Takeshi Q; Guiguemde, W. Armand; Barnett, David S.; Maron, Maxim I.; Min, Jaeki; Connelly, Michele C.; Suryadevara, Praveen Kumar; Guy, R. Kiplin

    2014-01-01

    Forty percent of the world's population is threatened by malaria, which is caused by Plasmodium parasites and results in an estimated 200 million clinical cases and 650,000 deaths each year. Drug resistance has been reported for all commonly used antimalarials and has prompted screens to identify new drug candidates. However, many of these new candidates have not been evaluated against the parasite stage responsible for transmission, gametocytes. If Plasmodium falciparum gametocytes are not eliminated, patients continue to spread malaria for weeks after asexual parasite clearance. Asymptomatic individuals can also harbor gametocyte burdens sufficient for transmission, and a safe, effective gametocytocidal agent could also be used in community-wide malaria control programs. Here, we identify 15 small molecules with nanomolar activity against late-stage gametocytes. Fourteen are diaminonaphthoquinones (DANQs), and one is a 2-imino-benzo[d]imidazole (IBI). One of the DANQs identified, SJ000030570, is a lead antimalarial candidate. In contrast, 94% of the 650 compounds tested are inactive against late-stage gametocytes. Consistent with the ineffectiveness of most approved antimalarials against gametocytes, of the 19 novel compounds with activity against known anti-asexual-stage targets, only 3 had any strong effect on gametocyte viability. These data demonstrate the distinct biology of the transmission stages and emphasize the importance of screening for gametocytocidal activity. The potent gametocytocidal activity of DANQ and IBI coupled with their efficacy against asexual parasites provides leads for the development of antimalarials with the potential to prevent both the symptoms and the spread of malaria. PMID:25512421

  19. Potent Plasmodium falciparum gametocytocidal activity of diaminonaphthoquinones, lead antimalarial chemotypes identified in an antimalarial compound screen.

    PubMed

    Tanaka, Takeshi Q; Guiguemde, W Armand; Barnett, David S; Maron, Maxim I; Min, Jaeki; Connelly, Michele C; Suryadevara, Praveen Kumar; Guy, R Kiplin; Williamson, Kim C

    2015-03-01

    Forty percent of the world's population is threatened by malaria, which is caused by Plasmodium parasites and results in an estimated 200 million clinical cases and 650,000 deaths each year. Drug resistance has been reported for all commonly used antimalarials and has prompted screens to identify new drug candidates. However, many of these new candidates have not been evaluated against the parasite stage responsible for transmission, gametocytes. If Plasmodium falciparum gametocytes are not eliminated, patients continue to spread malaria for weeks after asexual parasite clearance. Asymptomatic individuals can also harbor gametocyte burdens sufficient for transmission, and a safe, effective gametocytocidal agent could also be used in community-wide malaria control programs. Here, we identify 15 small molecules with nanomolar activity against late-stage gametocytes. Fourteen are diaminonaphthoquinones (DANQs), and one is a 2-imino-benzo[d]imidazole (IBI). One of the DANQs identified, SJ000030570, is a lead antimalarial candidate. In contrast, 94% of the 650 compounds tested are inactive against late-stage gametocytes. Consistent with the ineffectiveness of most approved antimalarials against gametocytes, of the 19 novel compounds with activity against known anti-asexual-stage targets, only 3 had any strong effect on gametocyte viability. These data demonstrate the distinct biology of the transmission stages and emphasize the importance of screening for gametocytocidal activity. The potent gametocytocidal activity of DANQ and IBI coupled with their efficacy against asexual parasites provides leads for the development of antimalarials with the potential to prevent both the symptoms and the spread of malaria. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  20. Food Insecurity Screening in Pediatric Primary Care: Can Offering Referrals Help Identify Families in Need?

    PubMed

    Bottino, Clement J; Rhodes, Erinn T; Kreatsoulas, Catherine; Cox, Joanne E; Fleegler, Eric W

    2017-07-01

    To describe a clinical approach for food insecurity screening incorporating a menu offering food-assistance referrals, and to examine relationships between food insecurity and referral selection. Caregivers of 3- to 10-year-old children presenting for well-child care completed a self-administered questionnaire on a laptop computer. Items included the US Household Food Security Survey Module: 6-Item Short Form (food insecurity screen) and a referral menu offering assistance with: 1) finding a food pantry, 2) getting hot meals, 3) applying for Supplemental Nutrition Assistance Program (SNAP), and 4) applying for Special Supplemental Nutrition Program for Women, Infants, and Children (WIC). Referrals were offered independent of food insecurity status or eligibility. We examined associations between food insecurity and referral selection using multiple logistic regression while adjusting for covariates. A total of 340 caregivers participated; 106 (31.2%) reported food insecurity, and 107 (31.5%) selected one or more referrals. Forty-nine caregivers (14.4%) reported food insecurity but selected no referrals; 50 caregivers (14.7%) selected one or more referrals but did not report food insecurity; and 57 caregivers (16.8%) both reported food insecurity and selected one or more referrals. After adjustment, caregivers who selected one or more referrals had greater odds of food insecurity compared to caregivers who selected no referrals (adjusted odds ratio 4.0; 95% confidence interval 2.4-7.0). In this sample, there was incomplete overlap between food insecurity and referral selection. Offering referrals may be a helpful adjunct to standard screening for eliciting family preferences and identifying unmet social needs. Copyright © 2016 Academic Pediatric Association. Published by Elsevier Inc. All rights reserved.

  1. Gametocytocidal Screen Identifies Novel Chemical Classes with Plasmodium falciparum Transmission Blocking Activity

    PubMed Central

    Sanders, Natalie G.; Sullivan, David J.; Mlambo, Godfree; Dimopoulos, George; Tripathi, Abhai K.

    2014-01-01

    Discovery of transmission blocking compounds is an important intervention strategy necessary to eliminate and eradicate malaria. To date only a small number of drugs that inhibit gametocyte development and thereby transmission from the mosquito to the human host exist. This limitation is largely due to a lack of screening assays easily adaptable to high throughput because of multiple incubation steps or the requirement for high gametocytemia. Here we report the discovery of new compounds with gametocytocidal activity using a simple and robust SYBR Green I- based DNA assay. Our assay utilizes the exflagellation step in male gametocytes and a background suppressor, which masks the staining of dead cells to achieve healthy signal to noise ratio by increasing signal of viable parasites and subtracting signal from dead parasites. By determining the contribution of exflagellation to fluorescent signal and using appropriate cutoff values, we were able to screen for gametocytocidal compounds. After assay validation and optimization, we screened an FDA approved drug library of approximately 1500 compounds, as well as the 400 compound MMV malaria box and identified 44 gametocytocidal compounds with sub to low micromolar IC50s. Major classes of compounds with gametocytocidal activity included quaternary ammonium compounds with structural similarity to choline, acridine-like compounds similar to quinacrine and pyronaridine, as well as antidepressant, antineoplastic, and anthelminthic compounds. Top drug candidates showed near complete transmission blocking in membrane feeding assays. This assay is simple, reproducible and demonstrated robust Z-factor values at low gametocytemia levels, making it amenable to HTS for identification of novel and potent gametocytocidal compounds. PMID:25157792

  2. Chemical and metabolomic screens identify novel biomarkers and antidotes for cyanide exposure

    PubMed Central

    Nath, Anjali K.; Roberts, Lee D.; Liu, Yan; Mahon, Sari B.; Kim, Sonia; Ryu, Justine H.; Werdich, Andreas; Januzzi, James L.; Boss, Gerry R.; Rockwood, Gary A.; MacRae, Calum A.; Brenner, Matthew; Gerszten, Robert E.; Peterson, Randall T.

    2013-01-01

    Exposure to cyanide causes a spectrum of cardiac, neurological, and metabolic dysfunctions that can be fatal. Improved cyanide antidotes are needed, but the ideal biological pathways to target are not known. To understand better the metabolic effects of cyanide and to discover novel cyanide antidotes, we developed a zebrafish model of cyanide exposure and scaled it for high-throughput chemical screening. In a screen of 3120 small molecules, we discovered 4 novel antidotes that block cyanide toxicity. The most potent antidote was riboflavin. Metabolomic profiling of cyanide-treated zebrafish revealed changes in bile acid and purine metabolism, most notably by an increase in inosine levels. Riboflavin normalizes many of the cyanide-induced neurological and metabolic perturbations in zebrafish. The metabolic effects of cyanide observed in zebrafish were conserved in a rabbit model of cyanide toxicity. Further, humans treated with nitroprusside, a drug that releases nitric oxide and cyanide ions, display increased circulating bile acids and inosine. In summary, riboflavin may be a novel treatment for cyanide toxicity and prophylactic measure during nitroprusside treatment, inosine may serve as a biomarker of cyanide exposure, and metabolites in the bile acid and purine metabolism pathways may shed light on the pathways critical to reversing cyanide toxicity.—Nath, A. K., Roberts, L. D., Liu, Y., Mahon, S. B., Kim, S., Ryu, J. H., Werdich, A., Januzzi, J. L., Boss, G. R., Rockwood, G. A., MacRae, C. A., Brenner, M., Gerszten, R. E., Peterson, R. T. Chemical and metabolomic screens identify novel biomarkers and antidotes for cyanide exposure. PMID:23345455

  3. A high throughput genetic screen identifies new early meiotic recombination functions in Arabidopsis thaliana.

    PubMed

    De Muyt, Arnaud; Pereira, Lucie; Vezon, Daniel; Chelysheva, Liudmila; Gendrot, Ghislaine; Chambon, Aurélie; Lainé-Choinard, Sandrine; Pelletier, Georges; Mercier, Raphaël; Nogué, Fabien; Grelon, Mathilde

    2009-09-01

    Meiotic recombination is initiated by the formation of numerous DNA double-strand breaks (DSBs) catalysed by the widely conserved Spo11 protein. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation; however, unlike Spo11, few of these are conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we took advantage of a high-throughput meiotic mutant screen carried out in the model plant Arabidopsis thaliana. A collection of 55,000 mutant lines was screened, and spo11-like mutations, characterised by a drastic decrease in chiasma formation at metaphase I associated with an absence of synapsis at prophase, were selected. This screen led to the identification of two populations of mutants classified according to their recombination defects: mutants that repair meiotic DSBs using the sister chromatid such as Atdmc1 or mutants that are unable to make DSBs like Atspo11-1. We found that in Arabidopsis thaliana at least four proteins are necessary for driving meiotic DSB repair via the homologous chromosomes. These include the previously characterised DMC1 and the Hop1-related ASY1 proteins, but also the meiotic specific cyclin SDS as well as the Hop2 Arabidopsis homologue AHP2. Analysing the mutants defective in DSB formation, we identified the previously characterised AtSPO11-1, AtSPO11-2, and AtPRD1 as well as two new genes, AtPRD2 and AtPRD3. Our data thus increase the number of proteins necessary for DSB formation in Arabidopsis thaliana to five. Unlike SPO11 and (to a minor extent) PRD1, these two new proteins are poorly conserved among species, suggesting that the DSB formation mechanism, but not its regulation, is conserved among eukaryotes.

  4. Accuracy of Depression Screening Tools for Identifying Postpartum Depression Among Urban Mothers

    PubMed Central

    Chaudron, Linda H.; Szilagyi, Peter G.; Tang, Wan; Anson, Elizabeth; Talbot, Nancy L.; Wadkins, Holly I.M.; Tu, Xin; Wisner, Katherine L.

    2011-01-01

    Objective The goal was to describe the accuracy of the Edinburgh Postnatal Depression Scale (EPDS), Beck Depression Inventory II (BDI-II), and Postpartum Depression Screening Scale (PDSS) in identifying major depressive disorder (MDD) or minor depressive disorder (MnDD) in low-income, urban mothers attending well childcare (WCC) visits during the postpartum year. Design/Methods Mothers (N=198) attending WCC visits with their infants 0 to 14 months of age completed a psychiatric diagnostic interview (standard method) and 3 screening tools. The sensitivity and specificity of each screening tool were calculated in comparison with diagnoses of MDD or MDD/MnDD. Receiver operating characteristic curves were calculated and the areas under the curves for each tool were compared to assess accuracy for the entire sample (representing the postpartum year) and sub-samples (representing early, middle and late postpartum time frames). Optimal cut-points were calculated. Results At some point between 2 weeks and 14 months postpartum, 56% of mothers met criteria for either MDD (37%) or MnDD (19%). When used as a continuous measures, all scales performed equally well (areas under the curves of ≥ 0.8). With traditional cut-points, the measures did not perform at the expected levels of sensitivity and specificity. Optimal cut-points for the BDI-II (≥14 for MDD, ≥11 for MDD/MnDD) and EPDS (≥9 for MDD, ≥7 for MDD/MnDD) were lower than currently recommended. For the PDSS, the optimal cut-point was consistent with current guidelines for MDD (≥80) but higher than recommended for MDD/MnDD (≥ 77). Conclusions Large proportions of low-income, urban mothers attending WCC visits experience MDD or MnDD during the postpartum year. The EPDS, BDI-II and PDSS have high accuracy in identifying depression but cutoff points may need to be altered to more accurately identify depression in urban, low-income mothers. PMID:20156899

  5. Spiperone, identified through compound screening, activates calcium-dependent chloride secretion in the airway

    PubMed Central

    Liang, Lihua; MacDonald, Kelvin; Schwiebert, Erik M.; Zeitlin, Pamela L.; Guggino, William B.

    2009-01-01

    Cystic fibrosis (CF) is caused by mutations in the gene producing the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR functions as a Cl− channel. Its dysfunction limits Cl− secretion and enhances Na+ absorption, leading to viscous mucus in the airway. Ca2+-activated Cl− channels (CaCCs) are coexpressed with CFTR in the airway surface epithelia. Increases in cytosolic Ca2+ activate the epithelial CaCCs, which provides an alternative Cl− secretory pathway in CF. We developed a screening assay and screened a library for compounds that could enhance cytoplasmic Ca2+, activate the CaCC, and increase Cl− secretion. We found that spiperone, a known antipsychotic drug, is a potent intracellular Ca2+ enhancer and demonstrated that it stimulates intracellular Ca2+, not by acting in its well-known role as an antagonist of serotonin 5-HT2 or dopamine D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Spiperone activates CaCCs, which stimulates Cl− secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro and in CFTR-knockout mice in vivo. In conclusion, we have identified spiperone as a new therapeutic platform for correction of defective Cl− secretion in CF via a pathway independent of CFTR. PMID:18987251

  6. High efficacy vasopermeability drug candidates identified by screening in an ex ovo chorioallantoic membrane model

    PubMed Central

    Pink, Desmond; Luhrs, Keith A.; Zhou, Longen; Schulte, Wendy; Chase, Jennifer; Frosch, Christian; Haberl, Udo; Nguyen, Van; Roy, Aparna I.; Lewis, John D.; Zijlstra, Andries; Parseghian, Missag H.

    2015-01-01

    The use of rodent models to evaluate efficacy during testing is accompanied by significant economic and regulatory hurdles which compound the costs of screening for promising drug candidates. Vasopermeation Enhancement Agents (VEAs) are a new class of biologics that are designed to increase the uptake of cancer therapeutics at the tumor site by modifying vascular permeability in the tumor to increase the therapeutic index of co-administered drugs. To evaluate the efficacy of a panel of VEA clinical candidates, we compared the rodent Miles assay to an equivalent assay in the ex ovo chicken embryo model. Both model systems identified the same candidate (PVL 10) as the most active promoter of vasopermeation in non-tumor tissues. An ex ovo chicken embryo system was utilized to test each candidate VEA in two human tumor models at a range of concentrations. Vasopermeation activity due to VEA was dependent on tumor type, with HEp3 tumors displaying higher levels of vasopermeation than MDA-MB-435. One candidate (PVL 10) proved optimal for HEp3 tumors and another (PVL 2) for MDA-MB-435. The use of the ex ovo chicken embryo model provides a rapid and less costly alternative to the use of rodent models for preclinical screening of drug candidates. PMID:26510887

  7. Functional screen identifies kinases driving prostate cancer visceral and bone metastasis.

    PubMed

    Faltermeier, Claire M; Drake, Justin M; Clark, Peter M; Smith, Bryan A; Zong, Yang; Volpe, Carmen; Mathis, Colleen; Morrissey, Colm; Castor, Brandon; Huang, Jiaoti; Witte, Owen N

    2016-01-12

    Mutationally activated kinases play an important role in the progression and metastasis of many cancers. Despite numerous oncogenic alterations implicated in metastatic prostate cancer, mutations of kinases are rare. Several lines of evidence suggest that nonmutated kinases and their pathways are involved in prostate cancer progression, but few kinases have been mechanistically linked to metastasis. Using a mass spectrometry-based phosphoproteomics dataset in concert with gene expression analysis, we selected over 100 kinases potentially implicated in human metastatic prostate cancer for functional evaluation. A primary in vivo screen based on overexpression of candidate kinases in murine prostate cells identified 20 wild-type kinases that promote metastasis. We queried these 20 kinases in a secondary in vivo screen using human prostate cells. Strikingly, all three RAF family members, MERTK, and NTRK2 drove the formation of bone and visceral metastasis confirmed by positron-emission tomography combined with computed tomography imaging and histology. Immunohistochemistry of tissue microarrays indicated that these kinases are highly expressed in human metastatic castration-resistant prostate cancer tissues. Our functional studies reveal the strong capability of select wild-type protein kinases to drive critical steps of the metastatic cascade, and implicate these kinases in possible therapeutic intervention.

  8. A Drosophila screen identifies neurofibromatosis-1 genetic modifiers involved in systemic and synaptic growth

    PubMed Central

    Walker, James A; Bernards, André

    2014-01-01

    Neurofibromatosis type 1 (NF1) is caused by loss of a negative regulator of Ras oncoproteins. Unknown genetic modifiers have been implicated in NF1’s characteristic variability. Drosophila melanogaster dNf1 phenotypes include cognitive deficits and reduced growth, both of which resemble human symptoms. We recently reported results of a screen for dominant modifiers of dNf1 growth. Suppressors include the dAlk tyrosine kinase and its activating ligand, two other genes involved in Ras/ERK signal transduction, the synaptic scaffold Dap160 and the CCKLR-17D1 drosulfakinin receptor. Additional modifiers include several genes involved in cAMP/PKA signaling. Providing mechanistic insights, dAlk, jeb, and CCKLR-17D1 also suppress a dNf1 synaptic overgrowth defect, and increasing cAMP/PKA signaling in the neuroendocrine ring gland rescued the dNf1 growth deficiency. Finally, among the several suppressors identified in our screen, we specifically implicate ALK as a potential therapeutic target by showing that NF1-regulated ALK/RAS/ERK signaling is conserved in human cells. PMID:25054093

  9. A Screening Approach for Identifying Gliadin Neutralizing Antibodies on Epithelial Intestinal Caco-2 Cells.

    PubMed

    Hundsberger, Harald; Koppensteiner, Anita; Hofmann, Elisabeth; Ripper, Doris; Pflüger, Maren; Stadlmann, Valerie; Klein, Christian Theodor; Kreiseder, Birgit; Katzlinger, Michael; Eger, Andreas; Forster, Florian; Missbichler, Albert; Wiesner, Christoph

    2017-09-01

    Celiac disease (CD) is a chronic inflammatory condition caused by the ingestion of gliadin-containing food in genetically susceptible individuals. Undigested peptides of gliadin exert various effects, including increased intestinal permeability and inflammation in the small intestine. Although many therapeutic approaches are in development, a gluten-free diet is the only effective treatment for CD. Affecting at least 1% of the population in industrialized countries, it is important to generate therapeutic options against CD. Here, we describe the establishment of a high-throughput screening (HTS) platform based on AlphaLISA and electrical cell-substrate impedance sensing (ECIS) technology for the identification of anti-inflammatory and barrier-protective compounds in human enterocytes after pepsin-trypsin-digested gliadin (PT-gliadin) treatment. Our results show that the combination of these HTS technologies enables fast, reliable, simple, and label-free screening of IgY antibodies against PT-gliadin. Using this platform, we have identified a new chicken anti-PT-gliadin IgY antibody as a potential anti-CD agent.

  10. Mutational screening of PARKIN identified a 3' UTR variant (rs62637702) associated with Parkinson's disease.

    PubMed

    de Mena, Lorena; Samaranch, L Luís; Coto, Eliecer; Cardo, Lucía F; Ribacoba, René; Lorenzo-Betancor, Oswaldo; Pastor, Pau; Wang, Li; Irigoyen, Jaione; Mata, Ignacio F; Díaz, Marta; Moris, Germán; Menéndez, Manuel; Corao, Ana I; Lorenzo, Elena; Alvarez, Victoria

    2013-06-01

    PRKN mutations have been linked to Parkinson's disease (PD). Most of the mutational screenings have focused on the coding exons. The 3' untranslated region (UTR) could also harbor functionally relevant nucleotide changes. We performed a mutational screening of PRKN in a cohort of early-onset PD patients (n = 235) from Spain. We found 16 mutations (five new): 16 patients (7 %) carried two mutations and only one mutation was found in 28 (12 %). Patients with two mutations had significantly lower mean age (30 ± 9 years) compared to patients with one (40 ± 7) or no mutation (42 ± 7). We found a total of 15 nucleotide variants (three new) in the 3' UTR region. The frequency of carriers of the rare rs62637702 G allele (*94A/G) was significantly lower among the patients compared to healthy controls (n = 418) (0.03 vs. 0.004; p < 0.001), suggesting a protective role for this allele. In order to investigate the basal effect of this variant, we performed luciferase assays. No different basal activity was observed between the two alleles. In conclusion, the rs62637702 polymorphism was associated with PD. This could be a surrogate marker for disease risk, in linkage disequilibrium with other non-identified functional variant.

  11. Nickel-resistance determinants in Acidiphilium sp. PM identified by genome-wide functional screening.

    PubMed

    San Martin-Uriz, Patxi; Mirete, Salvador; Alcolea, Pedro J; Gomez, Manuel J; Amils, Ricardo; Gonzalez-Pastor, Jose E

    2014-01-01

    Acidiphilium spp. are conspicuous dwellers of acidic, metal-rich environments. Indeed, they are among the most metal-resistant organisms; yet little is known about the mechanisms behind the metal tolerance in this genus. Acidiphilium sp. PM is an environmental isolate from Rio Tinto, an acidic, metal-laden river located in southwestern Spain. The characterization of its metal resistance revealed a remarkable ability to tolerate high Ni concentrations. Here we report the screening of a genomic library of Acidiphilium sp. PM to identify genes involved in Ni resistance. This approach revealed seven different genes conferring Ni resistance to E. coli, two of which form an operon encoding the ATP-dependent protease HslVU (ClpQY). This protease was found to enhance resistance to both Ni and Co in E. coli, a function not previously reported. Other Ni-resistance determinants include genes involved in lipopolysaccharide biosynthesis and the synthesis of branched amino acids. The diversity of molecular functions of the genes recovered in the screening suggests that Ni resistance in Acidiphilium sp. PM probably relies on different molecular mechanisms.

  12. Screening of human SNP database identifies recoding sites of A-to-I RNA editing

    PubMed Central

    Gommans, Willemijn M.; Tatalias, Nicholas E.; Sie, Christina P.; Dupuis, Dylan; Vendetti, Nicholas; Smith, Lauren; Kaushal, Rikhi; Maas, Stefan

    2008-01-01

    Single nucleotide polymorphisms (SNPs) are DNA sequence variations that can affect the expression or function of genes. As a result, they may lead to phenotypic differences between individuals, such as susceptibility to disease, response to medications, and disease progression. Millions of SNPs have been mapped within the human genome providing a rich resource for genetic variation studies. Adenosine-to-inosine RNA editing also leads to the production of RNA and protein sequence variants, but it acts on the level of primary gene transcripts. Sequence variations due to RNA editing may be misannotated as SNPs when relying solely on expressed sequence data instead of genomic material. In this study, we screened the human SNP database for potential cases of A-to-I RNA editing that cause amino acid changes in the encoded protein. Our search strategy applies five molecular features to score candidate sites. It identifies all previously known cases of editing present in the SNP database and successfully uncovers novel, bona fide targets of adenosine deamination editing. Our approach sets the stage for effective and comprehensive genome-wide screens for A-to-I editing targets. PMID:18772245

  13. A Genome-Wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes

    PubMed Central

    Sidik, Saima M.; Huet, Diego; Ganesan, Suresh M.; Huynh, My-Hang; Wang, Tim; Nasamu, Armiyaw S.; Thiru, Prathapan; Saeij, Jeroen P.J.; Carruthers, Vern B.; Niles, Jacquin C.; Lourido, Sebastian

    2016-01-01

    SUMMARY Apicomplexan parasites are leading causes of human and livestock diseases—like malaria and toxoplasmosis—yet most of their genes remain uncharacterized. Here, we present the first genome-wide genetic screen of an apicomplexan. We adapted CRISPR/Cas9 to assess the contribution of each gene from the human parasite Toxoplasma gondii during infection of fibroblasts. Our analysis defines ~200 previously uncharacterized, fitness-conferring genes unique to the phylum, from which 16 were investigated, revealing essential functions during infection of human cells. Secondary screens identify as an invasion factor the claudin-like apicomplexan microneme protein (CLAMP), which resembles mammalian tight-junction proteins and localizes to secretory organelles, making it critical to the initiation of infection. CLAMP is present throughout sequenced apicomplexan genomes, and is essential during the asexual stages of the malaria parasite Plasmodium falciparum. These results provide broad-based functional information on T. gondii genes and will facilitate future approaches to expand the horizon of antiparasitic interventions. PMID:27594426

  14. Discovery of dual binding site acetylcholinesterase inhibitors identified by pharmacophore modeling and sequential virtual screening techniques.

    PubMed

    Gupta, Shikhar; Fallarero, Adyary; Järvinen, Päivi; Karlsson, Daniela; Johnson, Mark S; Vuorela, Pia M; Mohan, C Gopi

    2011-02-15

    Dual binding site acetylcholinesterase (AChE) inhibitors are promising for the treatment of Alzheimer's disease (AD). They alleviate the cognitive deficits and AD-modifying agents, by inhibiting the β-amyloid (Aβ) peptide aggregation, through binding to both the catalytic and peripheral anionic sites, the so called dual binding site of the AChE enzyme. In this Letter, chemical features based 3D-pharmacophore models were developed based on the eight potent and structurally diverse AChE inhibitors (I-VIII) obtained from high-throughput in vitro screening technique. The best 3D-pharmacophore model, Hypo1, consists of two hydrogen-bond acceptor lipid, one hydrophobe, and two hydrophobic aliphatic features obtained by Catalyst/HIPHOP algorithm adopted in Discovery studio program. Hypo1 was used as a 3D query in sequential virtual screening study to filter three small compound databases. Further, a total of nine compounds were selected and followed on in vitro analysis. Finally, we identified two leads--Specs1 (IC(50)=3.279 μM) and Spec2 (IC(50)=5.986 μM) dual binding site compounds from Specs database, having good AChE enzyme inhibitory activity.

  15. RNAi screening identifies mediators of NOD2 signaling: Implications for spatial specificity of MDP recognition

    PubMed Central

    Lipinski, Simone; Grabe, Nils; Jacobs, Gunnar; Billmann-Born, Susanne; Till, Andreas; Häsler, Robert; Aden, Konrad; Paulsen, Maren; Arlt, Alexander; Kraemer, Lars; Hagemann, Nina; Erdmann, Kai Sven; Schreiber, Stefan; Rosenstiel, Philip

    2012-01-01

    The intracellular nucleotide-binding oligomerization domain-2 (NOD2) receptor detects bacteria-derived muramyl dipeptide (MDP) and activates the transcription factor NF-κB. Here we describe the regulatome of NOD2 signaling using a systematic RNAi screen. Using three consecutive screens, we identified a set of 20 positive NF-κB regulators including the known pathway members RIPK2, RELA, and BIRC4 (XIAP) as well as FRMPD2 (FERM and PDZ domain-containing 2). FRMPD2 interacts with NOD2 via leucine-rich repeats and forms a complex with the membrane-associated protein ERBB2IP. We demonstrate that FRMPD2 spatially assembles the NOD2-signaling complex, hereby restricting NOD2-mediated immune responses to the basolateral compartment of polarized intestinal epithelial cells. We show that genetic truncation of the NOD2 leucine-rich repeat domain, which is associated with Crohn disease, impairs the interaction with FRMPD2, and that intestinal inflammation leads to down-regulation of FRMPD2. These results suggest a structural mechanism for how polarity of epithelial cells acts on intestinal NOD-like receptor signaling to mediate spatial specificity of bacterial recognition and control of immune responses. PMID:23213202

  16. RNAi screening identifies mediators of NOD2 signaling: implications for spatial specificity of MDP recognition.

    PubMed

    Lipinski, Simone; Grabe, Nils; Jacobs, Gunnar; Billmann-Born, Susanne; Till, Andreas; Häsler, Robert; Aden, Konrad; Paulsen, Maren; Arlt, Alexander; Kraemer, Lars; Hagemann, Nina; Erdmann, Kai Sven; Schreiber, Stefan; Rosenstiel, Philip

    2012-12-26

    The intracellular nucleotide-binding oligomerization domain-2 (NOD2) receptor detects bacteria-derived muramyl dipeptide (MDP) and activates the transcription factor NF-κB. Here we describe the regulatome of NOD2 signaling using a systematic RNAi screen. Using three consecutive screens, we identified a set of 20 positive NF-κB regulators including the known pathway members RIPK2, RELA, and BIRC4 (XIAP) as well as FRMPD2 (FERM and PDZ domain-containing 2). FRMPD2 interacts with NOD2 via leucine-rich repeats and forms a complex with the membrane-associated protein ERBB2IP. We demonstrate that FRMPD2 spatially assembles the NOD2-signaling complex, hereby restricting NOD2-mediated immune responses to the basolateral compartment of polarized intestinal epithelial cells. We show that genetic truncation of the NOD2 leucine-rich repeat domain, which is associated with Crohn disease, impairs the interaction with FRMPD2, and that intestinal inflammation leads to down-regulation of FRMPD2. These results suggest a structural mechanism for how polarity of epithelial cells acts on intestinal NOD-like receptor signaling to mediate spatial specificity of bacterial recognition and control of immune responses.

  17. A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death.

    PubMed

    Ma, Hongming; Dang, Ying; Wu, Yonggan; Jia, Gengxiang; Anaya, Edgar; Zhang, Junli; Abraham, Sojan; Choi, Jang-Gi; Shi, Guojun; Qi, Ling; Manjunath, N; Wu, Haoquan

    2015-07-28

    West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the ER-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  18. A synthetic lethal siRNA screen identifying genes mediating sensitivity to a PARP inhibitor.

    PubMed

    Turner, Nicholas C; Lord, Christopher J; Iorns, Elizabeth; Brough, Rachel; Swift, Sally; Elliott, Richard; Rayter, Sydonia; Tutt, Andrew N; Ashworth, Alan

    2008-05-07

    Inhibitors of poly (ADP-ribose)-polymerase-1 (PARP) are highly lethal to cells with deficiencies in BRCA1, BRCA2 or other components of the homologous recombination pathway. This has led to PARP inhibitors entering clinical trials as a potential therapy for cancer in carriers of BRCA1 and BRCA2 mutations. To discover new determinants of sensitivity to these drugs, we performed a PARP-inhibitor synthetic lethal short interfering RNA (siRNA) screen. We identified a number of kinases whose silencing strongly sensitised to PARP inhibitor, including cyclin-dependent kinase 5 (CDK5), MAPK12, PLK3, PNKP, STK22c and STK36. How CDK5 silencing mediates sensitivity was investigated. Previously, CDK5 has been suggested to be active only in a neuronal context, but here we show that CDK5 is required in non-neuronal cells for the DNA-damage response and, in particular, intra-S and G(2)/M cell-cycle checkpoints. These results highlight the potential of synthetic lethal siRNA screens with chemical inhibitors to define new determinants of sensitivity and potential therapeutic targets.

  19. A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes.

    PubMed

    Sidik, Saima M; Huet, Diego; Ganesan, Suresh M; Huynh, My-Hang; Wang, Tim; Nasamu, Armiyaw S; Thiru, Prathapan; Saeij, Jeroen P J; Carruthers, Vern B; Niles, Jacquin C; Lourido, Sebastian

    2016-09-08

    Apicomplexan parasites are leading causes of human and livestock diseases such as malaria and toxoplasmosis, yet most of their genes remain uncharacterized. Here, we present the first genome-wide genetic screen of an apicomplexan. We adapted CRISPR/Cas9 to assess the contribution of each gene from the parasite Toxoplasma gondii during infection of human fibroblasts. Our analysis defines ∼200 previously uncharacterized, fitness-conferring genes unique to the phylum, from which 16 were investigated, revealing essential functions during infection of human cells. Secondary screens identify as an invasion factor the claudin-like apicomplexan microneme protein (CLAMP), which resembles mammalian tight-junction proteins and localizes to secretory organelles, making it critical to the initiation of infection. CLAMP is present throughout sequenced apicomplexan genomes and is essential during the asexual stages of the malaria parasite Plasmodium falciparum. These results provide broad-based functional information on T. gondii genes and will facilitate future approaches to expand the horizon of antiparasitic interventions.

  20. Nickel-Resistance Determinants in Acidiphilium sp. PM Identified by Genome-Wide Functional Screening

    PubMed Central

    San Martin-Uriz, Patxi; Mirete, Salvador; Alcolea, Pedro J.; Gomez, Manuel J.; Amils, Ricardo; Gonzalez-Pastor, Jose E.

    2014-01-01

    Acidiphilium spp. are conspicuous dwellers of acidic, metal-rich environments. Indeed, they are among the most metal-resistant organisms; yet little is known about the mechanisms behind the metal tolerance in this genus. Acidiphilium sp. PM is an environmental isolate from Rio Tinto, an acidic, metal-laden river located in southwestern Spain. The characterization of its metal resistance revealed a remarkable ability to tolerate high Ni concentrations. Here we report the screening of a genomic library of Acidiphilium sp. PM to identify genes involved in Ni resistance. This approach revealed seven different genes conferring Ni resistance to E. coli, two of which form an operon encoding the ATP-dependent protease HslVU (ClpQY). This protease was found to enhance resistance to both Ni and Co in E. coli, a function not previously reported. Other Ni-resistance determinants include genes involved in lipopolysaccharide biosynthesis and the synthesis of branched amino acids. The diversity of molecular functions of the genes recovered in the screening suggests that Ni resistance in Acidiphilium sp. PM probably relies on different molecular mechanisms. PMID:24740277

  1. Novel protein kinase signaling systems regulating lifespan identified by small molecule library screening using Drosophila.

    PubMed

    Spindler, Stephen R; Li, Rui; Dhahbi, Joseph M; Yamakawa, Amy; Sauer, Frank

    2012-01-01

    Protein kinase signaling cascades control most aspects of cellular function. The ATP binding domains of signaling protein kinases are the targets of most available inhibitors. These domains are highly conserved from mammals to flies. Herein we describe screening of a library of small molecule inhibitors of protein kinases for their ability to increase Drosophila lifespan. We developed an assay system which allowed screening using the small amounts of materials normally present in commercial chemical libraries. The studies identified 17 inhibitors, the majority of which targeted tyrosine kinases associated with the epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) receptors, G-protein coupled receptor (GPCR), Janus kinase (JAK)/signal transducer and activator of transcription (STAT), the insulin and insulin-like growth factor (IGFI) receptors. Comparison of the protein kinase signaling effects of the inhibitors in vitro defined a consensus intracellular signaling profile which included decreased signaling by p38MAPK (p38), c-Jun N-terminal kinase (JNK) and protein kinase C (PKC). If confirmed, many of these kinases will be novel additions to the signaling cascades known to regulate metazoan longevity.

  2. A new screening method to identify inhibitors of the Lol (localization of lipoproteins) system, a novel antibacterial target.

    PubMed

    Ito, Hideaki; Ura, Atsushi; Oyamada, Yoshihiro; Yoshida, Hiroaki; Yamagishi, Jun-Ichi; Narita, Shin-Ichiro; Matsuyama, Shin-Ichi; Tokuda, Hajime

    2007-01-01

    As the Lol system, which is involved in localization of lipoproteins, is essential for Escherichia coli growth and widely conserved among gram-negative bacteria, it is considered to be a promising target for the development of anti-gram-negative bacterial agents. However, no high-throughput screening method has so far been developed to screen for Lol system inhibitors. By combining three assay systems (anucleate cell blue assay, Lpp assay, and LolA-dependent release inhibition assay) and a drug susceptibility test, we have successfully developed a new screening method for identification of compounds that inhibit the Lol system. Using this new screening method, we screened 23,600 in-house chemical compounds and found 2 Lol system inhibitors. We therefore conclude that our new screening method can efficiently identify new antibacterial agents that target the Lol system.

  3. Identifying preschool children at risk of later reading difficulties: evaluation of two emergent literacy screening tools.

    PubMed

    Wilson, Shauna B; Lonigan, Christopher J

    2010-01-01

    Emergent literacy skills are predictive of children's early reading success, and literacy achievement in early schooling declines more rapidly for children who are below-average readers. It is therefore important for teachers to identify accurately children at risk for later reading difficulty so children can be exposed to effective emergent literacy interventions. In this study, 176 preschoolers were administered two screening tools, the Revised Get Ready to Read! (GRTR-R) and the Individual Growth and Development Indicators (IGDIs), and a diagnostic measure at two time points. Receiver operating characteristic curve analyses revealed that, at optimal cut scores, GRTR-R provided more accurate classification of children's overall emergent literacy skills than did IGDIs. However, neither measure was particularly good at classifying specific emergent literacy skills.

  4. An image-based screen identifies a small molecule regulator of megakaryopoiesis

    PubMed Central

    Boitano, Anthony E.; de Lichtervelde, Lorenzo; Snead, Jennifer L.; Cooke, Michael P.; Schultz, Peter G.

    2012-01-01

    Molecules that control the lineage commitment of hematopoietic stem cells (HSCs) may allow the expansion of enriched progenitor populations for both research and therapeutic uses. In an effort to better understand and control the differentiation of HSCs to megakaryocytes, we carried out an image-based screen of a library of 50,000 heterocycles using primary human CD34+ cells. A class of naphthyridinone derivatives was identified that induces the differentiation of common myeloid progenitors (CMP) to megakaryocytes. Kinase profiling and subsequent functional assays revealed that these compounds act through inhibition of platelet-derived growth factor receptor (PDGFR) signaling in CMPs. Such molecules may ultimately have clinical utility in the treatment of thrombocytopenia. PMID:22891346

  5. Axon regeneration pathways identified by systematic genetic screening in C. elegans.

    PubMed

    Chen, Lizhen; Wang, Zhiping; Ghosh-Roy, Anindya; Hubert, Thomas; Yan, Dong; O'Rourke, Sean; Bowerman, Bruce; Wu, Zilu; Jin, Yishi; Chisholm, Andrew D

    2011-09-22

    The mechanisms underlying the ability of axons to regrow after injury remain poorly explored at the molecular genetic level. We used a laser injury model in Caenorhabditis elegans mechanosensory neurons to screen 654 conserved genes for regulators of axonal regrowth. We uncover several functional clusters of genes that promote or repress regrowth, including genes classically known to affect axon guidance, membrane excitability, neurotransmission, and synaptic vesicle endocytosis. The conserved Arf Guanine nucleotide Exchange Factor (GEF), EFA-6, acts as an intrinsic inhibitor of regrowth. By combining genetics and in vivo imaging, we show that EFA-6 inhibits regrowth via microtubule dynamics, independent of its Arf GEF activity. Among newly identified regrowth inhibitors, only loss of function in EFA-6 partially bypasses the requirement for DLK-1 kinase. Identification of these pathways significantly expands our understanding of the genetic basis of axonal injury responses and repair.

  6. A mouse forward genetics screen identifies LISTERIN as an E3 ubiquitin ligase involved in neurodegeneration.

    PubMed

    Chu, Jessie; Hong, Nancy A; Masuda, Claudio A; Jenkins, Brian V; Nelms, Keats A; Goodnow, Christopher C; Glynne, Richard J; Wu, Hua; Masliah, Eliezer; Joazeiro, Claudio A P; Kay, Steve A

    2009-02-17

    A mouse neurological mutant, lister, was identified through a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Homozygous lister mice exhibit profound early-onset and progressive neurological and motor dysfunction. lister encodes a RING finger protein, LISTERIN, which functions as an E3 ubiquitin ligase in vitro. Although lister is widely expressed in all tissues, motor and sensory neurons and neuronal processes in the brainstem and spinal cord are primarily affected in the mutant. Pathological signs include gliosis, dystrophic neurites, vacuolated mitochondria, and accumulation of soluble hyperphosphorylated tau. Analysis with a different lister allele generated through targeted gene trap insertion reveals LISTERIN is required for embryonic development and confirms that direct perturbation of a LISTERIN-regulated process causes neurodegeneration. The lister mouse uncovers a pathway involved in neurodegeneration and may serves as a model for understanding the molecular mechanisms underlying human neurodegenerative disorders.

  7. Virtual screening studies to identify novel inhibitors for Sigma F protein of Mycobacterium tuberculosis.

    PubMed

    Mustyala, Kiran Kumar; Malkhed, Vasavi; Chittireddy, Venkataramana Reddy; Vuruputuri, Uma

    2015-12-01

    Tuberculosis (TB) is one of the oldest threats to public health. TB is caused by the pathogen Mycobacterium tuberculosis (MTB). The Sigma factors are essential for the survival of MTB. The Sigma factor Sigma F (SigF) regulates genes expression under stress conditions. The SigF binds to RNA polymerase and forms a holoenzyme, which initiates the transcription of various genes. The Usfx, an anti-SigF protein, binds to SigF and alters the transcription initiation and gene expression. In the present work, virtual screening studies are taken up to identify the interactions between SigF and small molecular inhibitors which can inhibit the formation of holoenzyme. The studies reveal that ARG 104 and ARG 224 amino acid residues of SigF protein are forming important binding interactions with the ligands. The in silico ADME properties for the ligand data set are calculated to check the druggability of the molecules.

  8. A mouse forward genetics screen identifies LISTERIN as an E3 ubiquitin ligase involved in neurodegeneration

    PubMed Central

    Chu, Jessie; Hong, Nancy A.; Masuda, Claudio A.; Jenkins, Brian V.; Nelms, Keats A.; Goodnow, Christopher C.; Glynne, Richard J.; Wu, Hua; Masliah, Eliezer; Joazeiro, Claudio A. P.; Kay, Steve A.

    2009-01-01

    A mouse neurological mutant, lister, was identified through a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Homozygous lister mice exhibit profound early-onset and progressive neurological and motor dysfunction. lister encodes a RING finger protein, LISTERIN, which functions as an E3 ubiquitin ligase in vitro. Although lister is widely expressed in all tissues, motor and sensory neurons and neuronal processes in the brainstem and spinal cord are primarily affected in the mutant. Pathological signs include gliosis, dystrophic neurites, vacuolated mitochondria, and accumulation of soluble hyperphosphorylated tau. Analysis with a different lister allele generated through targeted gene trap insertion reveals LISTERIN is required for embryonic development and confirms that direct perturbation of a LISTERIN-regulated process causes neurodegeneration. The lister mouse uncovers a pathway involved in neurodegeneration and may serves as a model for understanding the molecular mechanisms underlying human neurodegenerative disorders. PMID:19196968

  9. Challenges in identifying target skills for math disability screening and intervention.

    PubMed

    Mazzocco, Michèle M M

    2005-01-01

    Gersten, Jordan, and Flojo (in this series) review their research on math difficulties, with an emphasis on applying current knowledge to inform practices of early identification and intervention. On a practical level, educators are in dire need of empirically based screening and intervention tools. From a scientific perspective, it is important to recognize the need to clearly define what we seek to identify and remediate, and to acknowledge that we are currently far from achieving this goal despite recent advances in the field. Among the studies reviewed by Gersten et al., as well as other studies by several other researchers, there is much variability in how mathematics difficulties are defined and measured, and even in the terms used to refer to them. I address the degree of consensus and controversy currently characterizing the state of math learning disabilities research, with an emphasis on the usefulness of a developmental perspective in appraising this young field.

  10. A chemical screen to identify inducers of the mitochondrial unfolded protein response in C. elegans

    PubMed Central

    Rauthan, Manish; Pilon, Marc

    2015-01-01

    We previously showed that inhibition of the mevalonate pathway in C. elegans causes inhibition of protein prenylation, developmental arrest and lethality. We also showed that constitutive activation of the mitochondrial unfolded protein response, UPRmt, is an effective way for C. elegans to become resistant to the negative effects of mevalonate pathway inhibition. This was an important finding since statins, a drug class prescribed to lower cholesterol levels in patients, act by inhibiting the mevalonate pathway, and it is therefore possible that some of their undesirable side effects could be alleviated by activating the UPRmt. Here, we screened a chemical library and identified 4 compounds that specifically activated the UPRmt. One of these compounds, methacycline hydrochloride (a tetracycline antibiotic) also protected C. elegans and mammalian cells from statin toxicity. Methacycline hydrochloride and ethidium bromide, a known UPRmt activator, were also tested in mice: only ethidium bromide significantly activate the UPRmt in skeletal muscles. PMID:27123370

  11. Whole-genome screening identifies proteins localized to distinct nuclear bodies.

    PubMed

    Fong, Ka-Wing; Li, Yujing; Wang, Wenqi; Ma, Wenbin; Li, Kunpeng; Qi, Robert Z; Liu, Dan; Songyang, Zhou; Chen, Junjie

    2013-10-14

    The nucleus is a unique organelle that contains essential genetic materials in chromosome territories. The interchromatin space is composed of nuclear subcompartments, which are defined by several distinctive nuclear bodies believed to be factories of DNA or RNA processing and sites of transcriptional and/or posttranscriptional regulation. In this paper, we performed a genome-wide microscopy-based screening for proteins that form nuclear foci and characterized their localizations using markers of known nuclear bodies. In total, we identified 325 proteins localized to distinct nuclear bodies, including nucleoli (148), promyelocytic leukemia nuclear bodies (38), nuclear speckles (27), paraspeckles (24), Cajal bodies (17), Sam68 nuclear bodies (5), Polycomb bodies (2), and uncharacterized nuclear bodies (64). Functional validation revealed several proteins potentially involved in the assembly of Cajal bodies and paraspeckles. Together, these data establish the first atlas of human proteins in different nuclear bodies and provide key information for research on nuclear bodies.

  12. An In Vivo shRNA-Drug Screen to Identify Novel Targeted Therapy Combinations for KRAS Mutant Cancers

    DTIC Science & Technology

    2013-09-01

    identify novel targeted therapy combinations for KRAS mutant cancers ” PRINCIPAL INVESTIGATOR: Ryan B. Corcoran, MD PhD CONTRACTING...screen to identify novel targeted 5a. CONTRACT NUMBER therapy combinations for KRAS mutant cancers 5b. GRANT NUMBER W81XWH-12-1-0420 5c. PROGRAM...underway. Upon completion, this shRNA mini-pool will be utilized to perform the secondary shRNA-drug screen in an orthotopic mouse pancreatic cancer

  13. An in Vivo shRNA-Drug Screen to Identify Novel Targeted Therapy Combinations for KRAS Mutant Cancers

    DTIC Science & Technology

    2014-12-01

    AWARD NUMBER: W81XWH-12-1-0420 TITLE: An in Vivo shRNA-Drug Screen to Identify Novel Targeted Therapy Combinations for KRAS Mutant Cancers...2012 - 31 Aug 2014 4. TITLE AND SUBTITLE An in Vivo shRNA-Drug Screen to Identify Novel Targeted Therapy Combinations for KRAS Mutant Cancers...through the MAPK pathway, a key KRAS effector) to kill KRAS mutant pancreatic cancer cells in order to develop novel therapeutic combinations for these

  14. Phenotypic Screening Identifies Protein Synthesis Inhibitors as H-Ras-Nanocluster-Increasing Tumor Growth Inducers.

    PubMed

    Najumudeen, Arafath K; Posada, Itziar M D; Lectez, Benoit; Zhou, Yong; Landor, Sebastian K-J; Fallarero, Adyary; Vuorela, Pia; Hancock, John; Abankwa, Daniel

    2015-12-15

    Ras isoforms H-, N-, and K-ras are each mutated in specific cancer types at varying frequencies and have different activities in cell fate control. On the plasma membrane, Ras proteins are laterally segregated into isoform-specific nanoscale signaling hubs, termed nanoclusters. As Ras nanoclusters are required for Ras signaling, chemical modulators of nanoclusters represent ideal candidates for the specific modulation of Ras activity in cancer drug development. We therefore conducted a chemical screen with commercial and in-house natural product libraries using a cell-based H-ras-nanoclustering FRET assay. Next to established Ras inhibitors, such as a statin and farnesyl-transferase inhibitor, we surprisingly identified five protein synthesis inhibitors as positive regulators. Using commonly employed cycloheximide as a representative compound, we show that protein synthesis inhibition increased nanoclustering and effector recruitment specifically of active H-ras but not of K-ras. Consistent with these data, cycloheximide treatment activated both Erk and Akt kinases and specifically promoted H-rasG12V-induced, but not K-rasG12V-induced, PC12 cell differentiation. Intriguingly, cycloheximide increased the number of mammospheres, which are enriched for cancer stem cells. Depletion of H-ras in combination with cycloheximide significantly reduced mammosphere formation, suggesting an exquisite synthetic lethality. The potential of cycloheximide to promote tumor cell growth was also reflected in its ability to increase breast cancer cell tumors grown in ovo. These results illustrate the possibility of identifying Ras-isoform-specific modulators using nanocluster-directed screening. They also suggest an unexpected feedback from protein synthesis inhibition to Ras signaling, which might present a vulnerability in certain tumor cell types.

  15. Screening to Identify Multidrug Resistance-Associated Protein Inhibitors with Neuroblastoma-Selective Cytotoxicity.

    PubMed

    Nakamichi, Noritaka; Ishimoto, Takahiro; Yamauchi, Yoshihide; Masuo, Yusuke; Kato, Yukio

    2016-01-01

    The aim of the present study is to discover multidrug resistance-associated protein (MRP) inhibitors with neuroblastoma-selective cytotoxicity by means of fluorescence assay with a membrane-permeable fluorescent dye, Fluo-8 AM, based on our observation that gene expression of Mrp3 in neuroblastoma Neuro2a cells was remarkably higher than that in primary cultured cortical neurons, as determined by real-time PCR. Neuro2a cells showed minimal fluorescence upon incubation with Fluo-8 AM. However, blocking of Mrp3 efflux function by small interfering RNA (siRNA) transfection or inhibition with probenecid resulted in significant dye accumulation, observed as an increase of fluorescence. Interestingly, Mrp3 siRNA or probenecid treatment also resulted in increased cytotoxicity, as evidenced by decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-reducing activity of Neuro2a, with a concomitant increase in release of lactate dehydrogenase. On the other hand, primary cultured neurons exhibited higher fluorescence intensity after incubation with Fluo-8 AM regardless of addition of probenecid. Also, probenecid only minimally affected MTT-reducing activity. Thus, probenecid showed selective cytotoxicity towards Neuro2a cells. Based on these findings, we screened a series of established therapeutic agents for ability to induce Fluo-8 accumulation in Neuro2a cells. Several uricosuric and nonsteroidal anti-inflammatory drugs were identified, and these drugs were confirmed to decrease MTT-reducing activity selectively in Neuro2a. There was a negative linear correlation between Fluo-8 accumulation and cytotoxicity of these agents. Although the compounds identified here are insufficiently potent for practical application, further screening to discover higher-affinity MRP3 inhibitors using larger chemical libraries may uncover drug candidates with potent neuroblastoma-selective cytotoxicity.

  16. From The Cover: Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation

    NASA Astrophysics Data System (ADS)

    Nollen, Ellen A. A.; Garcia, Susana M.; van Haaften, Gijs; Kim, Soojin; Chavez, Alejandro; Morimoto, Richard I.; Plasterk, Ronald H. A.

    2004-04-01

    Protein misfolding and the formation of aggregates are increasingly recognized components of the pathology of human genetic disease and hallmarks of many neurodegenerative disorders. As exemplified by polyglutamine diseases, the propensity for protein misfolding is associated with the length of polyglutamine expansions and age-dependent changes in protein-folding homeostasis, suggesting a critical role for a protein homeostatic buffer. To identify the complement of protein factors that protects cells against the formation of protein aggregates, we tested transgenic Caenorhabditis elegans strains expressing polyglutamine expansion yellow fluorescent protein fusion proteins at the threshold length associated with the age-dependent appearance of protein aggregation. We used genome-wide RNA interference to identify genes that, when suppressed, resulted in the premature appearance of protein aggregates. Our screen identified 186 genes corresponding to five principal classes of polyglutamine regulators: genes involved in RNA metabolism, protein synthesis, protein folding, and protein degradation; and those involved in protein trafficking. We propose that each of these classes represents a molecular machine collectively comprising the protein homeostatic buffer that responds to the expression of damaged proteins to prevent their misfolding and aggregation. protein misfolding | neurodegenerative diseases

  17. NAFL screening score: A basic score identifying ultrasound-diagnosed non-alcoholic fatty liver.

    PubMed

    Zhou, Yu-Jie; Zhou, Yi-Fan; Zheng, Ji-Na; Liu, Wen-Yue; Van Poucke, Sven; Zou, Tian-Tian; Zhang, Dong-Chu; Shen, Shengrong; Shi, Ke-Qing; Wang, Xiao-Dong; Zheng, Ming-Hua

    2017-09-27

    Several non-invasive diagnostic scores for non-alcoholic fatty liver (NAFL) have been developed, but the clinical application is limited because of their complexity. To develop and validate an easy-to-calculate scoring system to identify ultrasound-diagnosed NAFL. 48,489 patients from 2 centers were included in this study. Multivariable logistic regression models were employed for model development. Ultrasonography was applied to diagnose NAFL. The selected variables were assigned an integer score proportional to the estimated coefficient from the logistic regression analysis, namely NAFL Screening Score (NSS). The ability of the NSS to identify NAFL was assessed by analyzing the area under the receiver operating characteristic curve (AUROC) and was tested in an independent validation cohort. Additionally, the performance of NSS was compared with existing models. NSS was developed as a basic score comprising of age, body mass index (BMI), triglyceride (TG), ALT/AST, fasting plasma glucose (FPG) and uric acid (UA) in both sexes. NSS showed a relatively good discriminative power (AUROC=0.825 for males, 0.861 for females in the validation cohort) in comparison with other models. The optimal cut-off point was 32 for males and 29 for females. We developed and validated NSS, an easy-to-use score sheet identify ultrasound-diagnosed NAFL. NSS may be clinically useful for initial diagnosing NAFL. Copyright © 2017. Published by Elsevier B.V.

  18. Network-wide Screen Identifies Variation of Novel Precise On-module Targets Using Conformational Modudaoism.

    PubMed

    Li, Bing; Liu, Jun; Yu, Yanan; Wang, Pengqian; Zhang, Yingying; Ni, Xumin; Liu, Qiong; Zhang, Xiaoxu; Wang, Zhong; Wang, Yongyan

    2017-09-19

    Modular targeting is promising in drug research at network level, but it's challenging to quantificationally identify the precise On-modules. Based on a proposed Modudaoism (MD), we defined conserved MD (MDc) and varied MD (MDv) to quantitatively evaluate the conformational and energy variations of modules, and thereby identify the conserved and discrepant allosteric modules. Compared with the Zsummary , MDc/MDv got an optimized result of module preserved ratio and modular structure. In mice anti-ischemic networks, 3, 5, and 1 conserved allosteric modules as well as 4, 1, and 3 On-modules of baicalin (BA), jasminoidin (JA), and ursodeoxycholic acid (UA) were identified by MDc and MDv, 5 unique allosteric modules and their characteristic actions were revealed. Besides, Co-IP experiments validated the representative modular structure. MDc/MDv method can quantitatively define the conformational variations of modules and screen the precise On-modules at network-wide, which may provide a promising strategy for drug discovery. This article is protected by copyright. All rights reserved. © 2017 ASCPT.

  19. Screening of cell cycle fusion proteins to identify kinase signaling networks.

    PubMed

    Trojanowsky, Michelle; Vidovic, Dusica; Simanski, Scott; Penas, Clara; Schurer, Stephan; Ayad, Nagi G

    2015-01-01

    Kinase signaling networks are well-established mediators of cell cycle transitions. However, how kinases interact with the ubiquitin proteasome system (UPS) to elicit protein turnover is not fully understood. We sought a means of identifying kinase-substrate interactions to better understand signaling pathways controlling protein degradation. Our prior studies used a luciferase fusion protein to uncover kinase networks controlling protein turnover. In this study, we utilized a similar approach to identify pathways controlling the cell cycle protein p27(Kip1). We generated a p27(Kip1)-luciferase fusion and expressed it in cells incubated with compounds from a library of pharmacologically active compounds. We then compared the relative effects of the compounds on p27(Kip1)-luciferase fusion stabilization. This was combined with in silico kinome profiling to identify potential kinases inhibited by each compound. This approach effectively uncovered known kinases regulating p27(Kip1) turnover. Collectively, our studies suggest that this parallel screening approach is robust and can be applied to fully understand kinase-ubiquitin pathway interactions.

  20. Process Evaluation of Serial Screening Criteria to Identify Injured Patients That Benefit From Brief Intervention: Practical Implications

    PubMed Central

    Field, Craig; Caetano, Raul; Pezzia, Carla

    2010-01-01

    Background The aim of the current study is to evaluate the effectiveness of serial screening methods for the identification of injured patients at risk for alcohol problems and are most likely to benefit from brief interventions. We hypothesize that blood alcohol concentration (BAC) alone is not sufficient to effectively identify at-risk drinkers in the trauma care setting. Methods During a 2-year period, patients admitted to an urban Level I trauma center for treatment of an injury were screened for alcohol problems. Screening consisted of four serial screening criteria: (1) clinical indication of acute intoxication including positive BAC; (2) self-reported drinking 6 hours before injury; (3) at-risk drinking as defined by National Institutes on Alcohol Abuse and Alcoholism or (4) by responding yes to one or more items on the CAGE within the last year. Results In all, 11,028 patients were seen. Fifty-eight percent were eligible for screening and 90% of eligible patients were screened. Of screened patients, 41% screened positive for an alcohol-related injury. Of patients that did not have a BAC drawn, 39% (n = 935) went on to screen positive using serial screening procedures. Additionally, 36% (n = 339) of patients with a negative BAC went on to screen positive using serial screening procedures. Conclusions This evaluation clearly suggests that BAC alone is not sufficient to identify patients who are most likely to benefit from brief alcohol interventions. Self-reported drinking in conjunction with BAC facilitates identification and intervention of injured patients with alcohol problems. PMID:18797416

  1. Clinical manifestations of hemochromatosis in HFE C282Y homozygotes identified by screening

    PubMed Central

    McLaren, Gordon D; McLaren, Christine E; Adams, Paul C; Barton, James C; Reboussin, David M; Gordeuk, Victor R; Acton, Ronald T; Harris, Emily L; Speechley, Mark R; Sholinsky, Phyliss; Dawkins, Fitzroy W; Snively, Beverly M; Vogt, Thomas M; Eckfeldt, John H

    2008-01-01

    BACKGROUND: Patients with hemochromatosis may suffer organ damage from iron overload, often with serious clinical consequences. OBJECTIVE: To assess prevalences of self-reported symptoms and clinical signs and conditions in persons homozygous for the hemochromatosis gene (HFE) mutation (C282Y) identified by screening. METHODS: Participants were adults 25 years of age or older enrolled in the Hemochromatosis and Iron Overload Screening (HEIRS) Study. C282Y homozygotes (n=282) were compared with control participants without the HFE C282Y or H63D alleles (ie, wild type/wild type; n=364). RESULTS: Previously diagnosed C282Y homozygotes and newly diagnosed homozygotes with elevated serum ferritin levels had higher prevalences of certain symptoms such as chronic fatigue (OR 2.8; 95% CI 1.34 to 5.95, and OR 2.0; 95% CI 1.07 to 3.75, respectively), and had more hyperpigmentation on physical examination (OR 4.7; 95% CI 1.50 to 15.06, and OR 3.7; 95% CI 1.10 to 12.16, respectively) and swelling or tenderness of the second and third metacarpophalangeal joints (OR 4.2; 95% CI 1.37 to 13.03, and OR 3.3; 95% CI 1.17 to 9.49, respectively) than control subjects. Joint stiffness was also more common among newly diagnosed C282Y homozygotes with elevated serum ferritin than among control subjects (OR 2.7; 95% CI 1.38 to 5.30). However, the sex- and age-adjusted prevalences of self-reported symptoms and signs of liver disease, heart disease, diabetes and most other major clinical manifestations of hemochromatosis were similar in C282Y homozygotes and control subjects. CONCLUSIONS: Some symptoms and conditions associated with hemochromatosis were more prevalent among C282Y homozygotes identified by screening than among control subjects, but prevalences of most outcomes were similar in C282Y homozygotes and controls in this primary care-based study. PMID:19018338

  2. Clinical manifestations of hemochromatosis in HFE C282Y homozygotes identified by screening.

    PubMed

    McLaren, Gordon D; McLaren, Christine E; Adams, Paul C; Barton, James C; Reboussin, David M; Gordeuk, Victor R; Acton, Ronald T; Harris, Emily L; Speechley, Mark R; Sholinsky, Phyliss; Dawkins, Fitzroy W; Snively, Beverly M; Vogt, Thomas M; Eckfeldt, John H

    2008-11-01

    Patients with hemochromatosis may suffer organ damage from iron overload, often with serious clinical consequences. To assess prevalences of self-reported symptoms and clinical signs and conditions in persons homozygous for the hemochromatosis gene (HFE) mutation (C282Y) identified by screening. Participants were adults 25 years of age or older enrolled in the Hemochromatosis and Iron Overload Screening (HEIRS) Study. C282Y homozygotes (n=282) were compared with control participants without the HFE C282Y or H63D alleles (ie, wild type/wild type; n=364). Previously diagnosed C282Y homozygotes and newly diagnosed homozygotes with elevated serum ferritin levels had higher prevalences of certain symptoms such as chronic fatigue (OR 2.8; 95% CI 1.34 to 5.95, and OR 2.0; 95% CI 1.07 to 3.75, respectively), and had more hyperpigmentation on physical examination (OR 4.7; 95% CI 1.50 to 15.06, and OR 3.7; 95% CI 1.10 to 12.16, respectively) and swelling or tenderness of the second and third metacarpophalangeal joints (OR 4.2; 95% CI 1.37 to 13.03, and OR 3.3; 95% CI 1.17 to 9.49, respectively) than control subjects. Joint stiffness was also more common among newly diagnosed C282Y homozygotes with elevated serum ferritin than among control subjects (OR 2.7; 95% CI 1.38 to 5.30). However, the sex- and age-adjusted prevalences of self-reported symptoms and signs of liver disease, heart disease, diabetes and most other major clinical manifestations of hemochromatosis were similar in C282Y homozygotes and control subjects. Some symptoms and conditions associated with hemochromatosis were more prevalent among C282Y homozygotes identified by screening than among control subjects, but prevalences of most outcomes were similar in C282Y homozygotes and controls in this primary care-based study.

  3. A Screening Instrument for Identifying Elderly at Risk of Abuse and Neglect.

    ERIC Educational Resources Information Center

    Hwalek, Melanie A.; Sengstock, Mary C.

    Recently more attention has been focused on elder abuse, with laws enacted requiring reporting of this crime. Since service providers often do not recognize elder abuse, a validated screening tool for elder abuse is needed. A screening tool called the Hwalek-Sengstock Elder Abuse Screening Protocol has been developed and is currently being…

  4. Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci.

    PubMed

    Westrick, Randal J; Tomberg, Kärt; Siebert, Amy E; Zhu, Guojing; Winn, Mary E; Dobies, Sarah L; Manning, Sara L; Brake, Marisa A; Cleuren, Audrey C; Hobbs, Linzi M; Mishack, Lena M; Johnston, Alexander J; Kotnik, Emilee; Siemieniak, David R; Xu, Jishu; Li, Jun Z; Saunders, Thomas L; Ginsburg, David

    2017-09-05

    Factor V Leiden (F5(L) ) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized N-ethyl-N-nitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for F5(L) (F5(L/L) ) and haploinsufficient for tissue factor pathway inhibitor (Tfpi(+/-) ). F8 deficiency enhanced the survival of F5(L/L)Tfpi(+/-) mice, demonstrating that F5(L/L)Tfpi(+/-) lethality is genetically suppressible. ENU-mutagenized F5(L/L) males and F5(L/+)Tfpi(+/-) females were crossed to generate 6,729 progeny, with 98 F5(L/L)Tfpi(+/-) offspring surviving until weaning. Sixteen lines, referred to as "modifier of Factor 5 Leiden (MF5L1-16)," exhibited transmission of a putative thrombosuppressor to subsequent generations. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene (F3). Although no ENU-induced F3 mutation was identified, haploinsufficiency for F3 (F3(+/-) ) suppressed F5(L/L)Tfpi(+/-) lethality. Whole-exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of F5(L/L)Tfpi(+/-) lethality (P = 1.7 × 10(-6)), suggesting that Actr2(p.R258G) is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent Actr2 knockin/knockout demonstrated that Actr2 haploinsufficiency is lethal, supporting a hypomorphic or gain-of-function mechanism of action for Actr2(p.R258G) Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5(L) and also suggest a role for Actr2 in this process.

  5. Identifying novel mycobacterial stress associated genes using a random mutagenesis screen in Mycobacterium smegmatis.

    PubMed

    Viswanathan, Gopinath; Joshi, Shrilaxmi V; Sridhar, Aditi; Dutta, Sayantanee; Raghunand, Tirumalai R

    2015-12-10

    Cell envelope associated components of Mycobacterium tuberculosis (M.tb) have been implicated in stress response, immune modulation and in vivo survival of the pathogen. Although many such factors have been identified, there is a large disparity between the number of genes predicted to be involved in functions linked to the envelope and those described in the literature. To identify and characterise novel stress related factors associated with the mycobacterial cell envelope, we isolated colony morphotype mutants of Mycobacterium smegmatis (M. smegmatis), based on the hypothesis that mutants with unusual colony morphology may have defects in the biosynthesis of cell envelope components. On testing their susceptibility to stress conditions relevant to M.tb physiology, multiple mutants were found to be sensitive to Isoniazid, Diamide and H2O2, indicative of altered permeability due to changes in cell envelope composition. Two mutants showed defects in biofilm formation implying possible roles for the target genes in antibiotic tolerance and/or virulence. These assays identified novel stress associated roles for several mycobacterial genes including sahH, tatB and aceE. Complementation analysis of selected mutants with the M. smegmatis genes and their M.tb homologues showed phenotypic restoration, validating their link to the observed phenotypes. A mutant carrying an insertion in fhaA encoding a forkhead associated domain containing protein, showed reduced survival in THP-1 macrophages, providing in vivo validation to this screen. Taken together, these results suggest that the M.tb homologues of a majority of the identified genes may play significant roles in the pathogenesis of tuberculosis.

  6. An update on the use of C. elegans for preclinical drug discovery: screening and identifying anti-infective drugs.

    PubMed

    Kim, Wooseong; Hendricks, Gabriel Lambert; Lee, Kiho; Mylonakis, Eleftherios

    2017-06-01

    The emergence of antibiotic-resistant and -tolerant bacteria is a major threat to human health. Although efforts for drug discovery are ongoing, conventional bacteria-centered screening strategies have thus far failed to yield new classes of effective antibiotics. Therefore, new paradigms for discovering novel antibiotics are of critical importance. Caenorhabditis elegans, a model organism used for in vivo, offers a promising solution for identification of anti-infective compounds. Areas covered: This review examines the advantages of C. elegans-based high-throughput screening over conventional, bacteria-centered in vitro screens. It discusses major anti-infective compounds identified from large-scale C. elegans-based screens and presents the first clinically-approved drugs, then known bioactive compounds, and finally novel small molecules. Expert opinion: There are clear advantages of using a C. elegans-infection based screening method. A C. elegans-based screen produces an enriched pool of non-toxic, efficacious, potential anti-infectives, covering: conventional antimicrobial agents, immunomodulators, and anti-virulence agents. Although C. elegans-based screens do not denote the mode of action of hit compounds, this can be elucidated in secondary studies by comparing the results to target-based screens, or conducting subsequent target-based screens, including the genetic knock-down of host or bacterial genes.

  7. A Transposon Screen Identifies Genetic Determinants of Vibrio cholerae Resistance to High-Molecular-Weight Antibiotics.

    PubMed

    Dörr, Tobias; Delgado, Fernanda; Umans, Benjamin D; Gerding, Matthew A; Davis, Brigid M; Waldor, Matthew K

    2016-08-01

    Gram-negative bacteria are notoriously resistant to a variety of high-molecular-weight antibiotics due to the limited permeability of their outer membrane (OM). The basis of OM barrier function and the genetic factors required for its maintenance remain incompletely understood. Here, we employed transposon insertion sequencing to identify genes required for Vibrio cholerae resistance to vancomycin and bacitracin, antibiotics that are thought to be too large to efficiently penetrate the OM. The screen yielded several genes whose protein products are predicted to participate in processes important for OM barrier functions and for biofilm formation. In addition, we identified a novel factor, designated vigA (for vancomycin inhibits growth), that has not previously been characterized or linked to outer membrane function. The vigA open reading frame (ORF) codes for an inner membrane protein, and in its absence, cells became highly sensitive to glycopeptide antibiotics (vancomycin and ramoplanin) and bacitracin but not to other large antibiotics or detergents. In contrast to wild-type (WT) cells, the vigA mutant was stained with fluorescent vancomycin. These observations suggest that VigA specifically prevents the periplasmic accumulation of certain large antibiotics without exerting a general role in the maintenance of OM integrity. We also observed marked interspecies variability in the susceptibilities of Gram-negative pathogens to glycopeptides and bacitracin. Collectively, our findings suggest that the OM barrier is not absolute but rather depends on specific OM-antibiotic interactions. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  8. Presenilin-Based Genetic Screens in Drosophila melanogaster Identify Novel Notch Pathway Modifiers

    PubMed Central

    Mahoney, Matt B.; Parks, Annette L.; Ruddy, David A.; Tiong, Stanley Y. K.; Esengil, Hanife; Phan, Alexander C.; Philandrinos, Panos; Winter, Christopher G.; Chatterjee, Runa; Huppert, Kari; Fisher, William W.; L'Archeveque, Lynn; Mapa, Felipa A.; Woo, Wendy; Ellis, Michael C.; Curtis, Daniel

    2006-01-01

    Presenilin is the enzymatic component of γ-secretase, a multisubunit intramembrane protease that processes several transmembrane receptors, such as the amyloid precursor protein (APP). Mutations in human Presenilins lead to altered APP cleavage and early-onset Alzheimer's disease. Presenilins also play an essential role in Notch receptor cleavage and signaling. The Notch pathway is a highly conserved signaling pathway that functions during the development of multicellular organisms, including vertebrates, Drosophila, and C. elegans. Recent studies have shown that Notch signaling is sensitive to perturbations in subcellular trafficking, although the specific mechanisms are largely unknown. To identify genes that regulate Notch pathway function, we have performed two genetic screens in Drosophila for modifiers of Presenilin-dependent Notch phenotypes. We describe here the cloning and identification of 19 modifiers, including nicastrin and several genes with previously undescribed involvement in Notch biology. The predicted functions of these newly identified genes are consistent with extracellular matrix and vesicular trafficking mechanisms in Presenilin and Notch pathway regulation and suggest a novel role for γ-tubulin in the pathway. PMID:16415372

  9. Presenilin-based genetic screens in Drosophila melanogaster identify novel notch pathway modifiers.

    PubMed

    Mahoney, Matt B; Parks, Annette L; Ruddy, David A; Tiong, Stanley Y K; Esengil, Hanife; Phan, Alexander C; Philandrinos, Panos; Winter, Christopher G; Chatterjee, Runa; Huppert, Kari; Fisher, William W; L'Archeveque, Lynn; Mapa, Felipa A; Woo, Wendy; Ellis, Michael C; Curtis, Daniel

    2006-04-01

    Presenilin is the enzymatic component of gamma-secretase, a multisubunit intramembrane protease that processes several transmembrane receptors, such as the amyloid precursor protein (APP). Mutations in human Presenilins lead to altered APP cleavage and early-onset Alzheimer's disease. Presenilins also play an essential role in Notch receptor cleavage and signaling. The Notch pathway is a highly conserved signaling pathway that functions during the development of multicellular organisms, including vertebrates, Drosophila, and C. elegans. Recent studies have shown that Notch signaling is sensitive to perturbations in subcellular trafficking, although the specific mechanisms are largely unknown. To identify genes that regulate Notch pathway function, we have performed two genetic screens in Drosophila for modifiers of Presenilin-dependent Notch phenotypes. We describe here the cloning and identification of 19 modifiers, including nicastrin and several genes with previously undescribed involvement in Notch biology. The predicted functions of these newly identified genes are consistent with extracellular matrix and vesicular trafficking mechanisms in Presenilin and Notch pathway regulation and suggest a novel role for gamma-tubulin in the pathway.

  10. Two children with thalassemia identified during screening for anemia in junior high school.

    PubMed

    Maeda, Miho; Fukunaga, Yoshitaka; Kaizu, Kiyohiko; Harano, Keiko; Harano, Teruo

    2004-08-01

    We present two Japanese students with thalassemia identified during screening for anemia in their junior high school. Blood test results revealed marked hypochromic and microcytic erythrocytosis in one patient and microcytic anemia in the other. Both cases showed a mean corpuscular volume/red blood cell (MCV/RBC) ratio less than 13. Their beta/alpha synthesis ratio was elevated. Deletion of psialpha2, psialpha1, alpha2, alpha1 and theta1 genes in the alpha-globin gene clusters were noted in the first case. This pattern of gene deletion was consistent with heterozygous alpha-thalassemia 1 of the Southeast Asian type. On the other hand, an increased hemoglobin A2 level and reduced beta/alpha synthesis ratio were found in the second case. Direct cloning and DNA sequencing identified a point mutation (guanine to adenine) at position 1 of intervening sequence II in the beta-globin gene (IVS II-1 G-->A). These results suggest that this patient had heterozygous beta0-thalassemia. Diagnosis of thalassemia should be confirmed by molecular analysis in cases with microcytic anemia or hypochromic microcytosis with a MCV/RBC ratio of 13 or less.

  11. A Phenotypic Screening Approach to Identify Anticancer Compounds Derived from Marine Fungi

    PubMed Central

    Silber, Johanna; Prashar, Anjali; Landskron, Johannes; Weber, Jonas; Rehermann, Sarah; Müller, Franz-Josef; Smith, Stephen; Wrigley, Stephen; Taskén, Kjetil; Gribbon, Philip; Labes, Antje; Imhoff, Johannes F.

    2014-01-01

    Abstract This study covers the isolation, testing, and identification of natural products with anticancer properties. Secondary metabolites were isolated from fungal strains originating from a variety of marine habitats. Strain culture protocols were optimized with respect to growth media composition and fermentation conditions. From these producers, isolated compounds were screened for their effect on the viability and proliferation of a subset of the NCI60 panel of cancer cell lines. Active compounds of interest were identified and selected for detailed assessments and structural elucidation using nuclear magnetic resonance. This revealed the majority of fungal-derived compounds represented known anticancer chemotypes, confirming the integrity of the process and the ability to identify suitable compounds. Examination of effects of selected compounds on cancer-associated cell signaling pathways used phospho flow cytometry in combination with 3D fluorescent cell barcoding. In parallel, the study addressed the logistical aspects of maintaining multiple cancer cell lines in culture simultaneously. A potential solution involving microbead-based cell culture was investigated (BioLevitator, Hamilton). Selected cell lines were cultured in microbead and 2D methods and cell viability tests showed comparable compound inhibition in both methods (R2=0.95). In a further technology assessment, an image-based assay system was investigated for its utility as a possible complement to ATP-based detection for quantifying cell growth and viability in a label-free manner. PMID:24735443

  12. Genome-wide RNAi screen identifies networks involved in intestinal stem cell regulation in Drosophila.

    PubMed

    Zeng, Xiankun; Han, Lili; Singh, Shree Ram; Liu, Hanhan; Neumüller, Ralph A; Yan, Dong; Hu, Yanhui; Liu, Ying; Liu, Wei; Lin, Xinhua; Hou, Steven X

    2015-02-24

    The intestinal epithelium is the most rapidly self-renewing tissue in adult animals and maintained by intestinal stem cells (ISCs) in both Drosophila and mammals. To comprehensively identify genes and pathways that regulate ISC fates, we performed a genome-wide transgenic RNAi screen in adult Drosophila intestine and identified 405 genes that regulate ISC maintenance and lineage-specific differentiation. By integrating these genes into publicly available interaction databases, we further developed functional networks that regulate ISC self-renewal, ISC proliferation, ISC maintenance of diploid status, ISC survival, ISC-to-enterocyte (EC) lineage differentiation, and ISC-to-enteroendocrine (EE) lineage differentiation. By comparing regulators among ISCs, female germline stem cells, and neural stem cells, we found that factors related to basic stem cell cellular processes are commonly required in all stem cells, and stem-cell-specific, niche-related signals are required only in the unique stem cell type. Our findings provide valuable insights into stem cell maintenance and lineage-specific differentiation.

  13. Whole-Animal Chemical Screen Identifies Colistin as a New Immunomodulator That Targets Conserved Pathways

    PubMed Central

    Cai, Yun; Cao, Xiou

    2014-01-01

    ABSTRACT The purpose of this study was to take advantage of the nematode Caenorhabditis elegans to perform a whole-animal chemical screen to identify potential immune activators that may confer protection against bacterial infections. We identified 45 marketed drugs, out of 1,120 studied compounds, that are capable of activating a conserved p38/PMK-1 mitogen-activated protein kinase pathway required for innate immunity. One of these drugs, the last-resort antibiotic colistin, protected against infections by the Gram-negative pathogens Yersinia pestis and Pseudomonas aeruginosa but not by the Gram-positive pathogens Enterococcus faecalis and Staphylococcus aureus. Protection was independent of the antibacterial activity of colistin, since the drug was administered prophylactically prior to the infections and it was also effective against antibiotic-resistant bacteria. Immune activation by colistin is mediated not only by the p38/PMK-1 pathway but also by the conserved FOXO transcription factor DAF-16 and the transcription factor SKN-1. Furthermore, p38/PMK-1 was found to be required in the intestine for immune activation by colistin. Enhanced p38/PMK-1-mediated immune responses by colistin did not reduce the bacterial burden, indicating that the pathway plays a role in the development of host tolerance to infections by Gram-negative bacteria. PMID:25118236

  14. High-content screening identifies kinase inhibitors that overcome venetoclax resistance in activated CLL cells.

    PubMed

    Oppermann, Sina; Ylanko, Jarkko; Shi, Yonghong; Hariharan, Santosh; Oakes, Christopher C; Brauer, Patrick M; Zúñiga-Pflücker, Juan C; Leber, Brian; Spaner, David E; Andrews, David W

    2016-08-18

    Novel agents such as the Bcl-2 inhibitor venetoclax (ABT-199) are changing treatment paradigms for chronic lymphocytic leukemia (CLL) but important problems remain. Although some patients exhibit deep and durable responses to venetoclax as a single agent, other patients harbor subpopulations of resistant leukemia cells that mediate disease recurrence. One hypothesis for the origin of resistance to venetoclax is by kinase-mediated survival signals encountered in proliferation centers that may be unique for individual patients. An in vitro microenvironment model was developed with primary CLL cells that could be incorporated into an automated high-content microscopy-based screen of kinase inhibitors (KIs) to identify agents that may improve venetoclax therapy in a personalized manner. Marked interpatient variability was noted for which KIs were effective; nevertheless, sunitinib was identified as the most common clinically available KI effective in overcoming venetoclax resistance. Examination of the underlying mechanisms indicated that venetoclax resistance may be induced by microenvironmental signals that upregulate antiapoptotic Bcl-xl, Mcl-1, and A1, which can be counteracted more efficiently by sunitinib than by ibrutinib or idelalisib. Although patient-specific drug responses are common, for many patients, combination therapy with sunitinib may significantly improve the therapeutic efficacy of venetoclax.

  15. A FACS-Optimized Screen Identifies Regulators of Genome Stability in Candida albicans

    PubMed Central

    Loll-Krippleber, Raphaël; Feri, Adeline; Nguyen, Marie; Maufrais, Corinne; Yansouni, Jennifer; d'Enfert, Christophe

    2015-01-01

    Loss of heterozygosity (LOH) plays important roles in genome dynamics, notably, during tumorigenesis. In the fungal pathogen Candida albicans, LOH contributes to the acquisition of antifungal resistance. In order to investigate the mechanisms that regulate LOH in C. albicans, we have established a novel method combining an artificial heterozygous locus harboring the blue fluorescent protein and green fluorescent protein markers and flow cytometry to detect LOH events at the single-cell level. Using this fluorescence-based method, we have confirmed that elevated temperature, treatment with methyl methanesulfonate, and inactivation of the Mec1 DNA damage checkpoint kinase triggered an increase in the frequency of LOH. Taking advantage of this system, we have searched for C. albicans genes whose overexpression triggered an increase in LOH and identified four candidates, some of which are known regulators of genome dynamics with human homologues contributing to cancer progression. Hence, the approach presented here will allow the implementation of new screens to identify genes that are important for genome stability in C. albicans and more generally in eukaryotic cells. PMID:25595446

  16. A FACS-optimized screen identifies regulators of genome stability in Candida albicans.

    PubMed

    Loll-Krippleber, Raphaël; Feri, Adeline; Nguyen, Marie; Maufrais, Corinne; Yansouni, Jennifer; d'Enfert, Christophe; Legrand, Mélanie

    2015-03-01

    Loss of heterozygosity (LOH) plays important roles in genome dynamics, notably, during tumorigenesis. In the fungal pathogen Candida albicans, LOH contributes to the acquisition of antifungal resistance. In order to investigate the mechanisms that regulate LOH in C. albicans, we have established a novel method combining an artificial heterozygous locus harboring the blue fluorescent protein and green fluorescent protein markers and flow cytometry to detect LOH events at the single-cell level. Using this fluorescence-based method, we have confirmed that elevated temperature, treatment with methyl methanesulfonate, and inactivation of the Mec1 DNA damage checkpoint kinase triggered an increase in the frequency of LOH. Taking advantage of this system, we have searched for C. albicans genes whose overexpression triggered an increase in LOH and identified four candidates, some of which are known regulators of genome dynamics with human homologues contributing to cancer progression. Hence, the approach presented here will allow the implementation of new screens to identify genes that are important for genome stability in C. albicans and more generally in eukaryotic cells.

  17. Genome-wide in vivo screen identifies novel host regulators of metastatic colonization.

    PubMed

    van der Weyden, Louise; Arends, Mark J; Campbell, Andrew D; Bald, Tobias; Wardle-Jones, Hannah; Griggs, Nicola; Velasco-Herrera, Martin Del Castillo; Tüting, Thomas; Sansom, Owen J; Karp, Natasha A; Clare, Simon; Gleeson, Diane; Ryder, Edward; Galli, Antonella; Tuck, Elizabeth; Cambridge, Emma L; Voet, Thierry; Macaulay, Iain C; Wong, Kim; Spiegel, Sarah; Speak, Anneliese O; Adams, David J

    2017-01-12

    Metastasis is the leading cause of death for cancer patients. This multi-stage process requires tumour cells to survive in the circulation, extravasate at distant sites, then proliferate; it involves contributions from both the tumour cell and tumour microenvironment ('host', which includes stromal cells and the immune system). Studies suggest the early steps of the metastatic process are relatively efficient, with the post-extravasation regulation of tumour growth ('colonization') being critical in determining metastatic outcome. Here we show the results of screening 810 mutant mouse lines using an in vivo assay to identify microenvironmental regulators of metastatic colonization. We identify 23 genes that, when disrupted in mouse, modify the ability of tumour cells to establish metastatic foci, with 19 of these genes not previously demonstrated to play a role in host control of metastasis. The largest reduction in pulmonary metastasis was observed in sphingosine-1-phosphate (S1P) transporter spinster homologue 2 (Spns2)-deficient mice. We demonstrate a novel outcome of S1P-mediated regulation of lymphocyte trafficking, whereby deletion of Spns2, either globally or in a lymphatic endothelial-specific manner, creates a circulating lymphopenia and a higher percentage of effector T cells and natural killer (NK) cells present in the lung. This allows for potent tumour cell killing, and an overall decreased metastatic burden.

  18. Screening and prioritisation of chemical risks from metal mining operations, identifying exposure media of concern.

    PubMed

    Pan, Jilang; Oates, Christopher J; Ihlenfeld, Christian; Plant, Jane A; Voulvoulis, Nikolaos

    2010-04-01

    Metals have been central to the development of human civilisation from the Bronze Age to modern times, although in the past, metal mining and smelting have been the cause of serious environmental pollution with the potential to harm human health. Despite problems from artisanal mining in some developing countries, modern mining to Western standards now uses the best available mining technology combined with environmental monitoring, mitigation and remediation measures to limit emissions to the environment. This paper develops risk screening and prioritisation methods previously used for contaminated land on military and civilian sites and engineering systems for the analysis and prioritisation of chemical risks from modern metal mining operations. It uses hierarchical holographic modelling and multi-criteria decision making to analyse and prioritise the risks from potentially hazardous inorganic chemical substances released by mining operations. A case study of an active platinum group metals mine in South Africa is used to demonstrate the potential of the method. This risk-based methodology for identifying, filtering and ranking mining-related environmental and human health risks can be used to identify exposure media of greatest concern to inform risk management. It also provides a practical decision-making tool for mine acquisition and helps to communicate risk to all members of mining operation teams.

  19. High-content screening identifies kinase inhibitors that overcome venetoclax resistance in activated CLL cells

    PubMed Central

    Oppermann, Sina; Ylanko, Jarkko; Shi, Yonghong; Hariharan, Santosh; Oakes, Christopher C.; Brauer, Patrick M.; Zúñiga-Pflücker, Juan C.; Leber, Brian; Spaner, David E.

    2016-01-01

    Novel agents such as the Bcl-2 inhibitor venetoclax (ABT-199) are changing treatment paradigms for chronic lymphocytic leukemia (CLL) but important problems remain. Although some patients exhibit deep and durable responses to venetoclax as a single agent, other patients harbor subpopulations of resistant leukemia cells that mediate disease recurrence. One hypothesis for the origin of resistance to venetoclax is by kinase-mediated survival signals encountered in proliferation centers that may be unique for individual patients. An in vitro microenvironment model was developed with primary CLL cells that could be incorporated into an automated high-content microscopy-based screen of kinase inhibitors (KIs) to identify agents that may improve venetoclax therapy in a personalized manner. Marked interpatient variability was noted for which KIs were effective; nevertheless, sunitinib was identified as the most common clinically available KI effective in overcoming venetoclax resistance. Examination of the underlying mechanisms indicated that venetoclax resistance may be induced by microenvironmental signals that upregulate antiapoptotic Bcl-xl, Mcl-1, and A1, which can be counteracted more efficiently by sunitinib than by ibrutinib or idelalisib. Although patient-specific drug responses are common, for many patients, combination therapy with sunitinib may significantly improve the therapeutic efficacy of venetoclax. PMID:27297795

  20. Genome-Wide Overexpression Screen Identifies Genes Able to Bypass p16-Mediated Senescence in Melanoma.

    PubMed

    Lee, Won Jae; Škalamera, Dubravka; Dahmer-Heath, Mareike; Shakhbazov, Konstanin; Ranall, Max V; Fox, Carly; Lambie, Duncan; Stevenson, Alexander J; Yaswen, Paul; Gonda, Thomas J; Gabrielli, Brian

    2017-03-01

    Malignant melanomas often arise from nevi, which result from initial oncogene-induced hyperproliferation of melanocytes that are maintained in a CDKN2A/p16-mediated senescent state. Thus, genes that can bypass this senescence barrier are likely to contribute to melanoma development. We have performed a gain-of-function screen of 17,030 lentivirally expressed human open reading frames (ORFs) in a melanoma cell line containing an inducible p16 construct to identify such genes. Genes known to bypass p16-induced senescence arrest, including the human papilloma virus 18 E7 gene ( HPV18E7), and genes such as the p16-binding CDK6 with expected functions, as well as panel of novel genes, were identified, including high-mobility group box (HMGB) proteins. A number of these were further validated in two other models of p16-induced senescence. Tissue immunohistochemistry demonstrated higher levels of CDK6 in primary melanomas compared with normal skin and nevi. Reduction of CDK6 levels drove melanoma cells expressing functional p16 into senescence, demonstrating its contribution to bypass senescence.

  1. High-throughput screening to identify selective inhibitors of microbial sulfate reduction (and beyond)

    NASA Astrophysics Data System (ADS)

    Carlson, H. K.; Coates, J. D.; Deutschbauer, A. M.

    2015-12-01

    The selective perturbation of complex microbial ecosystems to predictably influence outcomes in engineered and industrial environments remains a grand challenge for geomicrobiology. In some industrial ecosystems, such as oil reservoirs, sulfate reducing microorganisms (SRM) produce hydrogen sulfide which is toxic, explosive and corrosive. Current strategies to selectively inhibit sulfidogenesis are based on non-specific biocide treatments, bio-competitive exclusion by alternative electron acceptors or sulfate-analogs which are competitive inhibitors or futile/alternative substrates of the sulfate reduction pathway. Despite the economic cost of sulfidogenesis, there has been minimal exploration of the chemical space of possible inhibitory compounds, and very little work has quantitatively assessed the selectivity of putative souring treatments. We have developed a high-throughput screening strategy to target SRM, quantitatively ranked the selectivity and potency of hundreds of compounds and identified previously unrecognized SRM selective inhibitors and synergistic interactions between inhibitors. Once inhibitor selectivity is defined, high-throughput characterization of microbial community structure across compound gradients and identification of fitness determinants using isolate bar-coded transposon mutant libraries can give insights into the genetic mechanisms whereby compounds structure microbial communities. The high-throughput (HT) approach we present can be readily applied to target SRM in diverse environments and more broadly, could be used to identify and quantify the potency and selectivity of inhibitors of a variety of microbial metabolisms. Our findings and approach are relevant for engineering environmental ecosystems and also to understand the role of natural gradients in shaping microbial niche space.

  2. A CRISPR screen identifies genes controlling Etv2 threshold expression in murine hemangiogenic fate commitment.

    PubMed

    Zhao, Haiyong; Choi, Kyunghee

    2017-09-14

    The ETS transcription factor Etv2 is necessary and sufficient for the generation of hematopoietic and endothelial cells. However, upstream regulators of Etv2 in hemangiogenesis, generation of hematopoietic and endothelial cells, have not been clearly addressed. Here we track the developmental route of hemangiogenic progenitors from mouse embryonic stem cells, perform genome-wide CRISPR screening, and transcriptome analysis of en route cell populations by utilizing Brachyury, Etv2, or Scl reporter embryonic stem cell lines to further understand the mechanisms that control hemangiogenesis. We identify the forkhead transcription factor Foxh1, in part through Eomes, to be critical for the formation of FLK1(+) mesoderm, from which the hemangiogenic fate is specified. Importantly, hemangiogenic fate is specified not simply by the onset of Etv2 expression, but by a threshold-dependent mechanism, in which VEGF-FLK1 signaling plays an instructive role by promoting Etv2 threshold expression. These studies reveal comprehensive cellular and molecular pathways governing the hemangiogenic cell lineage development.How haematopoietic and endothelial cell lineages are specified is unclear. Here, the authors identify the forkhead transcription factor Foxh1 as regulating FLK1+ mesoderm formation in mouse embryonic stem cells, which in turn specifies hemangiogenic fate via Etv2.

  3. Cell-Based Screening Identifies Paroxetine as an Inhibitor of Diabetic Endothelial Dysfunction

    PubMed Central

    Gerö, Domokos; Szoleczky, Petra; Suzuki, Kunihiro; Módis, Katalin; Oláh, Gabor; Coletta, Ciro; Szabo, Csaba

    2013-01-01

    We have conducted a phenotypic screening in endothelial cells exposed to elevated extracellular glucose (an in vitro model of hyperglycemia) to identify compounds that prevent hyperglycemia-induced reactive oxygen species (ROS) formation without adversely affecting cell viability. From a focused library of >6,000 clinically used drug-like and pharmacologically active compounds, several classes of active compounds emerged, with a confirmed hit rate of <0.5%. Follow-up studies focused on paroxetine, a clinically used antidepressant compound that has not been previously implicated in the context of hyperglycemia or diabetes. Paroxetine reduced hyperglycemia-induced mitochondrial ROS formation, mitochondrial protein oxidation, and mitochondrial and nuclear DNA damage, without interfering with mitochondrial electron transport or cellular bioenergetics. The ability of paroxetine to improve hyperglycemic endothelial cell injury was unique among serotonin reuptake blockers and can be attributed to its antioxidant effect, which primarily resides within its sesamol moiety. Paroxetine maintained the ability of vascular rings to respond to the endothelium-dependent relaxant acetylcholine, both during in vitro hyperglycemia and ex vivo, in a rat model of streptozotocin-induced diabetes. Thus, the current work identifies a novel pharmacological action of paroxetine as a protector of endothelial cells against hyperglycemic injury and raises the potential of repurposing of this drug for the experimental therapy of diabetic cardiovascular complications. PMID:23223176

  4. A Genome-Wide RNAi Screen Identifies Regulators of Cholesterol-Modified Hedgehog Secretion in Drosophila

    PubMed Central

    Ruel, Laurent; Lacas-Gervais, Sandra; Schaub, Sébastien; Thérond, Pascal

    2012-01-01

    Hedgehog (Hh) proteins are secreted molecules that function as organizers in animal development. In addition to being palmitoylated, Hh is the only metazoan protein known to possess a covalently-linked cholesterol moiety. The absence of either modification severely disrupts the organization of numerous tissues during development. It is currently not known how lipid-modified Hh is secreted and released from producing cells. We have performed a genome-wide RNAi screen in Drosophila melanogaster cells to identify regulators of Hh secretion. We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI) but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway. We provide evidence that both proteolysis and cholesterol modification are necessary for the efficient trafficking of Hh through the ER and Golgi. Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg) morphogen secretion in vivo. These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion. PMID:22432040

  5. A Large-Scale Functional Screen to Identify Epigenetic Repressors of Retrotransposon Expression.

    PubMed

    Ecco, Gabriela; Rowe, Helen M; Trono, Didier

    2016-01-01

    Deposition of epigenetic marks is an important layer of the transcriptional control of retrotransposons, especially during early embryogenesis. Krüppel-associated box domain zinc finger proteins (KRAB-ZFPs) are one of the largest families of transcription factors, and collectively partake in this process by tethering to thousands of retroelement-containing genomic loci their cofactor KAP1, which acts as a scaffold for a heterochromatin-inducing machinery. However, while the sequence-specific DNA binding potential of the poly-zinc finger-containing KRAB-ZFPs is recognized, very few members of the family have been assigned specific targets. In this chapter, we describe a large-scale functional screen to identify the retroelements bound by individual murine KRAB-ZFPs. Our method is based on the automated transfection of a library of mouse KRAB-ZFP-containing vectors into 293T cells modified to express GFP from a PGK promoter harboring in its immediate vicinity a KAP1-recruiting retroelement-derived sequence. Analysis is then performed by plate reader and flow cytometry fluorescence readout. Such large-scale DNA-centered functional approach can not only help to identify the trans-acting factors responsible for silencing retrotransposons, but also serve as a model for dissecting the transcriptional networks influenced by retroelement-derived cis-acting sequences.

  6. Drug Repurposing Screen Identifies Foxo1-Dependent Angiopoietin-2 Regulation in Sepsis

    PubMed Central

    Ghosh, Chandra C.; Thamm, Kristina; Berghelli, Anthony V.; Schrimpf, Claudia; Maski, Manish R.; Abid, Tanaz; Milam, Katelyn E.; Rajakumar, Augustine; Santel, Ansgar; Kielstein, Jan T.; Ahmed, Asif; Thickett, David; Wang, Keqin; Chase, Maureen; Donnino, Michael W.; Aird, William C.; Haller, Hermann; David, Sascha; Parikh, Samir M.

    2016-01-01

    Objective The recent withdrawal of a targeted sepsis therapy has diminished pharmaceutical enthusiasm for developing novel drugs for the treatment of sepsis. Angiopoietin-2 is an endothelial-derived protein that potentiates vascular inflammation and leak-age and may be involved in sepsis pathogenesis. We screened approved compounds for putative inhibitors of angiopoietin-2 production and investigated underlying molecular mechanisms. Design Laboratory and animal research plus prospective placebo- controlled randomized controlled trial (NCT00529139) and retrospective analysis (NCT00676897). Setting Research laboratories of Hannover Medical School and Harvard Medical School. Patients Septic patients/C57Bl/6 mice and human endothelial cells. Interventions Food and Drug Administration–approved library screening. Measurements and Main Results In a cell-based screen of more than 650 Food and Drug Administration–approved compounds, we identified multiple members of the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor drug class (referred to as statins) that suppressed angiopoietin-2. Simvastatin inhibited 3-hydroxy-3-methyl-glutaryl-CoA reductase, which in turn activated PI3K-kinase. Downstream of this signaling, PI3K-dependent phosphorylation of the transcription factor Foxo1 at key amino acids inhibited its ability to shuttle to the nucleus and bind cis-elements in the angiopoietin-2 promoter. In septic mice, transient inhibition of angiopoietin-2 expression by liposomal siRNA in vivo improved absolute survival by 50%. Simvastatin had a similar effect, but the combination of angiopoietin-2 siRNA and simvastatin showed no additive benefit. To verify the link between statins and angiopoietin-2 in humans, we performed a pilot matched case-control study and a small randomized placebo-controlled trial demonstrating beneficial effects on angiopoietin-2. Conclusions 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors may operate through a novel Foxo1-angiopoietin-2

  7. Cardiac surgery-specific screening tool identifies preoperative undernutrition in cardiac surgery.

    PubMed

    van Venrooij, Lenny M W; Visser, Marlieke; de Vos, Rien; van Leeuwen, Paul A M; Peters, Ron J G; de Mol, Bas A J M

    2013-02-01

    Loss of body tissue resulting in undernutrition can be caused by reduced food intake, altered metabolism, ageing, and physical inactivity. The predominant cause of undernutrition before cardiac operations is unknown. First, we explored the association of reduced food intake and inactivity with undernutrition in patients before elective cardiac operations. Second, we assessed if adding these reversible, cause-based items to the nutritional screening process improved diagnostic accuracy. A prospective observational study was performed. Undernutrition was defined by low fat-free mass index (LFFMI) measured by bioelectrical impedance spectroscopy and/or unintended weight loss (UWL). Reduced food intake was defined as the patient having a decreased appetite over the previous month. Patients admitted to hospital preoperatively were assumed to be less physically active than patients awaiting cardiac operations at home. Using these data, we developed a new tool and compared this with an existing cardiac surgery-specific tool (Cardiac Surgery-Specific Malnutrition Universal Screening Tool [CSSM]). A total of 325 patients who underwent open cardiac operations were included. Reduced food intake and inactivity were associated with undernutrition (odds ratio [OR], 4.2; 95% confidence interval [CI], 2.1-8.5 and OR, 2.0; 95% CI, 1.0-4.0). Reduced food intake and inactivity were integrated with body mass index (BMI) and UWL into a new scoring system: the Cardiac Surgery-Specific Undernutrition Screening Tool (CSSUST). Sensitivity in identification of undernourished patients was considerably higher with the CSSUST (90%) than with the CSSM (71%) (receiver operating characteristic [ROC] curve-based area under the curve [AUC], 0.79; 95% CI, 0.73-0.86 and ROC AUC, 0.71; 95% CI, 0.63-0.80). Results suggest that reduced food intake and inactivity partly explain undernutrition before cardiac operations. Our new cause-based CSSUST, which includes reduced food intake and inactivity, is

  8. Criteria for Identifying Radiologists with Acceptable Screening Mammography Interpretive Performance based on Multiple Performance Measures

    PubMed Central

    Miglioretti, Diana L.; Ichikawa, Laura; Smith, Robert A.; Bassett, Lawrence W.; Feig, Stephen A.; Monsees, Barbara; Parikh, Jay R.; Rosenberg, Robert D.; Sickles, Edward A.; Carney, Patricia A.

    2014-01-01

    Objective Using a combination of performance measures, we updated previously proposed criteria for identifying physicians whose performance interpreting screening mammograms may indicate suboptimal interpretation skills. Materials and Methods In this Institutional Review Board-approved, HIPAA-compliant study, six expert breast imagers used a method based on the Angoff approach to update criteria for acceptable mammography performance on the basis of combined performance measures: (Group 1) sensitivity and specificity, for facilities with complete capture of false-negative cancers; and (Group 2) cancer detection rate (CDR), recall rate, and positive predictive value of a recall (PPV1), for facilities that cannot capture false negatives, but have reliable cancer follow-up information for positive mammograms. Decisions were informed by normative data from the Breast Cancer Surveillance Consortium (BCSC). Results Updated, combined ranges for acceptable sensitivity and specificity of screening mammography are: (1) sensitivity ≥80% and specificity ≥85% or (2) sensitivity 75–79% and specificity 88–97%. Updated ranges for CDR, recall rate, and PPV1 are: (1) CDR ≥6/1000, recall rate 3–20%, and any PPV1; (2) CDR 4–6/1000, recall rate 3–15%, and PPV1 ≥3%; or (3) CDR 2.5–4/1000, recall rate 5–12%, and PPV1 3–8%. Using the original criteria, 51% of BCSC radiologists had acceptable sensitivity and specificity; 40% had acceptable CDR, recall rate, and PPV1. Using the combined criteria, 69% had acceptable sensitivity and specificity and 62% had acceptable CDR, recall rate, and PPV1. Conclusion The combined criteria improve previous criteria by considering the inter-relationships of multiple performance measures and broaden the acceptable performance ranges compared to previous criteria based on individual measures. PMID:25794100

  9. Chemical Screens Identify Drugs that Enhance or Mitigate Cellular Responses to Antibody-Toxin Fusion Proteins

    PubMed Central

    Guha, Rajarshi; Simon, Nathan; Pasetto, Matteo; Keller, Jonathan; Huang, Manjie; Angelus, Evan; Pastan, Ira; Ferrer, Marc; FitzGerald, David J.; Thomas, Craig J.

    2016-01-01

    The intersection of small molecular weight drugs and antibody-based therapeutics is rarely studied in large scale. Both types of agents are currently part of the cancer armamentarium. However, very little is known about how to combine them in optimal ways. Immunotoxins are antibody-toxin gene fusion proteins engineered to target cancer cells via antibody binding to surface antigens. For fusion proteins derived from Pseudomonas exotoxin (PE), potency relies on the enzymatic domain of the toxin which catalyzes the ADP-ribosylation of EF2 causing inhibition of protein synthesis leading to cell death. Candidate immunotoxins have demonstrated clear value in clinical trials but generally have not been curative as single agents. Therefore we undertook three screens to discover effective combinations that could act synergistically. From the MIPE-3 library of compounds we identified various enhancers of immunotoxin action and at least one major class of inhibitor. Follow-up experiments confirmed the screening data and suggested that immunotoxins when administered with everolimus or nilotinib exhibit favorable combinatory activity and would be candidates for preclinical development. Mechanistic studies revealed that everolimus-immunotoxin combinations acted synergistically on elements of the protein synthetic machinery, including S61 kinase and 4E-BP1 of the mTORC1 pathway. Conversely, PARP inhibitors antagonized immunotoxins and also blocked the toxicity due to native ADP-ribosylating toxins. Thus, our goal of investigating a chemical library was justified based on the identification of several approved compounds that could be developed preclinically as ‘enhancers’ and at least one class of mitigator to be avoided. PMID:27556570

  10. RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia.

    PubMed

    Zuber, Johannes; Shi, Junwei; Wang, Eric; Rappaport, Amy R; Herrmann, Harald; Sison, Edward A; Magoon, Daniel; Qi, Jun; Blatt, Katharina; Wunderlich, Mark; Taylor, Meredith J; Johns, Christopher; Chicas, Agustin; Mulloy, James C; Kogan, Scott C; Brown, Patrick; Valent, Peter; Bradner, James E; Lowe, Scott W; Vakoc, Christopher R

    2011-08-03

    Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.

  11. RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia

    PubMed Central

    Zuber, Johannes; Shi, Junwei; Wang, Eric; Rappaport, Amy R.; Herrmann, Harald; Sison, Edward A.; Magoon, Daniel; Qi, Jun; Blatt, Katharina; Wunderlich, Mark; Taylor, Meredith J.; Johns, Christopher; Chicas, Agustin; Mulloy, James C.; Kogan, Scott C.; Brown, Patrick; Valent, Peter; Bradner, James E.; Lowe, Scott W.; Vakoc, Christopher R.

    2012-01-01

    Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs1. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states2. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention. PMID:21814200

  12. Analysis of high-throughput RNAi screening data in identifying genes mediating sensitivity to chemotherapeutic drugs: statistical approaches and perspectives.

    PubMed

    Ye, Fei; Bauer, Joshua A; Pietenpol, Jennifer A; Shyr, Yu

    2012-01-01

    High-throughput RNA interference (RNAi) screens have been used to find genes that, when silenced, result in sensitivity to certain chemotherapy drugs. Researchers therefore can further identify drug-sensitive targets and novel drug combinations that sensitize cancer cells to chemotherapeutic drugs. Considerable uncertainty exists about the efficiency and accuracy of statistical approaches used for RNAi hit selection in drug sensitivity studies. Researchers require statistical methods suitable for analyzing high-throughput RNAi screening data that will reduce false-positive and false-negative rates. In this study, we carried out a simulation study to evaluate four types of statistical approaches (fold-change/ratio, parametric tests/statistics, sensitivity index, and linear models) with different scenarios of RNAi screenings for drug sensitivity studies. With the simulated datasets, the linear model resulted in significantly lower false-negative and false-positive rates. Based on the results of the simulation study, we then make recommendations of statistical analysis methods for high-throughput RNAi screening data in different scenarios. We assessed promising methods using real data from a loss-of-function RNAi screen to identify hits that modulate paclitaxel sensitivity in breast cancer cells. High-confidence hits with specific inhibitors were further analyzed for their ability to inhibit breast cancer cell growth. Our analysis identified a number of gene targets with inhibitors known to enhance paclitaxel sensitivity, suggesting other genes identified may merit further investigation. RNAi screening can identify druggable targets and novel drug combinations that can sensitize cancer cells to chemotherapeutic drugs. However, applying an inappropriate statistical method or model to the RNAi screening data will result in decreased power to detect the true hits and increase false positive and false negative rates, leading researchers to draw incorrect conclusions. In

  13. RNA Interference Screen to Identify Kinases That Suppress Rescue of ΔF508-CFTR*

    PubMed Central

    Trzcińska-Daneluti, Agata M.; Chen, Anthony; Nguyen, Leo; Murchie, Ryan; Jiang, Chong; Moffat, Jason; Pelletier, Lawrence; Rotin, Daniela

    2015-01-01

    Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding the Cystic fibrosis transmembrane conductance regulator (CFTR). ΔF508-CFTR, the most common disease-causing CF mutant, exhibits folding and trafficking defects and is retained in the endoplasmic reticulum, where it is targeted for proteasomal degradation. To identify signaling pathways involved in ΔF508-CFTR rescue, we screened a library of endoribonuclease-prepared short interfering RNAs (esiRNAs) that target ∼750 different kinases and associated signaling proteins. We identified 20 novel suppressors of ΔF508-CFTR maturation, including the FGFR1. These were subsequently validated by measuring channel activity by the YFP halide-sensitive assay following shRNA-mediated knockdown, immunoblotting for the mature (band C) ΔF508-CFTR and measuring the amount of surface ΔF508-CFTR by ELISA. The role of FGFR signaling on ΔF508-CFTR trafficking was further elucidated by knocking down FGFRs and their downstream signaling proteins: Erk1/2, Akt, PLCγ-1, and FRS2. Interestingly, inhibition of FGFR1 with SU5402 administered to intestinal organoids (mini-guts) generated from the ileum of ΔF508-CFTR homozygous mice resulted in a robust ΔF508-CFTR rescue. Moreover, combination of SU5402 and VX-809 treatments in cells led to an additive enhancement of ΔF508-CFTR rescue, suggesting these compounds operate by different mechanisms. Chaperone array analysis on human bronchial epithelial cells harvested from ΔF508/ΔF508-CFTR transplant patients treated with SU5402 identified altered expression of several chaperones, an effect validated by their overexpression or knockdown experiments. We propose that FGFR signaling regulates specific chaperones that control ΔF508-CFTR maturation, and suggest that FGFRs may serve as important targets for therapeutic intervention for the treatment of CF. PMID:25825526

  14. RNAi Screen Identifies Novel Regulators of RNP Granules in the Caenorhabditis elegans Germ Line

    PubMed Central

    Wood, Megan P.; Hollis, Angela; Severance, Ashley L.; Karrick, Megan L.; Schisa, Jennifer A.

    2016-01-01

    Complexes of RNA and RNA binding proteins form large-scale supramolecular structures under many cellular contexts. In Caenorhabditis elegans, small germ granules are present in the germ line that share characteristics with liquid droplets that undergo phase transitions. In meiotically-arrested oocytes of middle-aged hermaphrodites, the germ granules appear to aggregate or condense into large assemblies of RNA-binding proteins and maternal mRNAs. Prior characterization of the assembly of large-scale RNP structures via candidate approaches has identified a small number of regulators of phase transitions in the C. elegans germ line; however, the assembly, function, and regulation of these large RNP assemblies remain incompletely understood. To identify genes that promote remodeling and assembly of large RNP granules in meiotically-arrested oocytes, we performed a targeted, functional RNAi screen and identified over 300 genes that regulate the assembly of the RNA-binding protein MEX-3 into large granules. Among the most common GO classes are several categories related to RNA biology, as well as novel categories such as cell cortex, ER, and chromosome segregation. We found that arrested oocytes that fail to localize MEX-3 into cortical granules display reduced oocyte quality, consistent with the idea that the larger RNP assemblies promote oocyte quality when fertilization is delayed. Interestingly, a relatively small number of genes overlap with the regulators of germ granule assembly during normal development, or with the regulators of solid RNP granules in cgh-1 oocytes, suggesting fundamental differences in the regulation of RNP granule phase transitions during meiotic arrest. PMID:27317775

  15. RNAi Screen Identifies Novel Regulators of RNP Granules in the Caenorhabditis elegans Germ Line.

    PubMed

    Wood, Megan P; Hollis, Angela; Severance, Ashley L; Karrick, Megan L; Schisa, Jennifer A

    2016-08-09

    Complexes of RNA and RNA binding proteins form large-scale supramolecular structures under many cellular contexts. In Caenorhabditis elegans, small germ granules are present in the germ line that share characteristics with liquid droplets that undergo phase transitions. In meiotically-arrested oocytes of middle-aged hermaphrodites, the germ granules appear to aggregate or condense into large assemblies of RNA-binding proteins and maternal mRNAs. Prior characterization of the assembly of large-scale RNP structures via candidate approaches has identified a small number of regulators of phase transitions in the C. elegans germ line; however, the assembly, function, and regulation of these large RNP assemblies remain incompletely understood. To identify genes that promote remodeling and assembly of large RNP granules in meiotically-arrested oocytes, we performed a targeted, functional RNAi screen and identified over 300 genes that regulate the assembly of the RNA-binding protein MEX-3 into large granules. Among the most common GO classes are several categories related to RNA biology, as well as novel categories such as cell cortex, ER, and chromosome segregation. We found that arrested oocytes that fail to localize MEX-3 into cortical granules display reduced oocyte quality, consistent with the idea that the larger RNP assemblies promote oocyte quality when fertilization is delayed. Interestingly, a relatively small number of genes overlap with the regulators of germ granule assembly during normal development, or with the regulators of solid RNP granules in cgh-1 oocytes, suggesting fundamental differences in the regulation of RNP granule phase transitions during meiotic arrest.

  16. High-throughput screening and whole genome sequencing identifies an antimicrobially active inhibitor of Vibrio cholerae

    PubMed Central

    2014-01-01

    Background Pathogenic serotypes of Vibrio cholerae cause the life-threatening diarrheal disease cholera. The increasing development of bacterial resistances against the known antibiotics necessitates the search for new antimicrobial compounds and targets for this pathogen. Results A high-throughput screening assay with a Vibrio cholerae reporter strain constitutively expressing green fluorescent protein (GFP) was developed and applied in the investigation of the growth inhibitory effect of approximately 28,300 structurally diverse natural compounds and synthetic small molecules. Several compounds with activities in the low micromolar concentration range were identified. The most active structure, designated vz0825, displayed a minimal inhibitory concentration (MIC) of 1.6 μM and a minimal bactericidal concentration (MBC) of 3.2 μM against several strains of V. cholerae and was specific for this pathogen. Mutants with reduced sensitivity against vz0825 were generated and whole genome sequencing of 15 pooled mutants was carried out. Comparison with the genome of the wild type strain identified the gene VC_A0531 (GenBank: AE003853.1) as the major site of single nucleotide polymorphisms in the resistant mutants. VC_A0531 is located on the small chromosome of V. cholerae and encodes the osmosensitive K+-channel sensor histidine kinase (KdpD). Nucleotide exchange of the major mutation site in the wild type strain confirmed the sensitive phenotype. Conclusion The reporter strain MO10 pG13 was successfully used for the identification of new antibacterial compounds against V. cholerae. Generation of resistant mutants and whole genome sequencing was carried out to identify the histidine kinase KdpD as a novel antimicrobial target. PMID:24568688

  17. High-throughput screening and whole genome sequencing identifies an antimicrobially active inhibitor of Vibrio cholerae.

    PubMed

    Sergeev, Galina; Roy, Sambit; Jarek, Michael; Zapolskii, Viktor; Kaufmann, Dieter E; Nandy, Ranjan K; Tegge, Werner

    2014-02-26

    Pathogenic serotypes of Vibrio cholerae cause the life-threatening diarrheal disease cholera. The increasing development of bacterial resistances against the known antibiotics necessitates the search for new antimicrobial compounds and targets for this pathogen. A high-throughput screening assay with a Vibrio cholerae reporter strain constitutively expressing green fluorescent protein (GFP) was developed and applied in the investigation of the growth inhibitory effect of approximately 28,300 structurally diverse natural compounds and synthetic small molecules. Several compounds with activities in the low micromolar concentration range were identified. The most active structure, designated vz0825, displayed a minimal inhibitory concentration (MIC) of 1.6 μM and a minimal bactericidal concentration (MBC) of 3.2 μM against several strains of V. cholerae and was specific for this pathogen. Mutants with reduced sensitivity against vz0825 were generated and whole genome sequencing of 15 pooled mutants was carried out. Comparison with the genome of the wild type strain identified the gene VC_A0531 (GenBank: AE003853.1) as the major site of single nucleotide polymorphisms in the resistant mutants. VC_A0531 is located on the small chromosome of V. cholerae and encodes the osmosensitive K+-channel sensor histidine kinase (KdpD). Nucleotide exchange of the major mutation site in the wild type strain confirmed the sensitive phenotype. The reporter strain MO10 pG13 was successfully used for the identification of new antibacterial compounds against V. cholerae. Generation of resistant mutants and whole genome sequencing was carried out to identify the histidine kinase KdpD as a novel antimicrobial target.

  18. Cell-Based Screen Identifies Human Interferon-Stimulated Regulators of Listeria monocytogenes Infection

    PubMed Central

    Eitson, Jennifer L.; Chen, Didi; Jimenez, Alyssa; Mettlen, Marcel; Schoggins, John W.; Alto, Neal M.

    2016-01-01

    The type I interferon (IFN) activated transcriptional response is a critical antiviral defense mechanism, yet its role in bacterial pathogenesis remains less well characterized. Using an intracellular pathogen Listeria monocytogenes (Lm) as a model bacterial pathogen, we sought to identify the roles of individual interferon-stimulated genes (ISGs) in context of bacterial infection. Previously, IFN has been implicated in both restricting and promoting Lm growth and immune stimulatory functions in vivo. Here we adapted a gain-of-function flow cytometry based approach to screen a library of more than 350 human ISGs for inhibitors and enhancers of Lm infection. We identify 6 genes, including UNC93B1, MYD88, AQP9, and TRIM14 that potently inhibit Lm infection. These inhibitors act through both transcription-mediated (MYD88) and non-transcriptional mechanisms (TRIM14). Further, we identify and characterize the human high affinity immunoglobulin receptor FcγRIa as an enhancer of Lm internalization. Our results reveal that FcγRIa promotes Lm uptake in the absence of known host Lm internalization receptors (E-cadherin and c-Met) as well as bacterial surface internalins (InlA and InlB). Additionally, FcγRIa-mediated uptake occurs independently of Lm opsonization or canonical FcγRIa signaling. Finally, we established the contribution of FcγRIa to Lm infection in phagocytic cells, thus potentially linking the IFN response to a novel bacterial uptake pathway. Together, these studies provide an experimental and conceptual basis for deciphering the role of IFN in bacterial defense and virulence at single-gene resolution. PMID:28002492

  19. Novel inhibitors of Mycobacterium tuberculosis GuaB2 identified by a target based high-throughput phenotypic screen

    PubMed Central

    Cox, Jonathan A. G.; Mugumbate, Grace; Del Peral, Laura Vela-Glez; Jankute, Monika; Abrahams, Katherine A.; Jervis, Peter; Jackenkroll, Stefan; Perez, Arancha; Alemparte, Carlos; Esquivias, Jorge; Lelièvre, Joël; Ramon, Fernando; Barros, David; Ballell, Lluis; Besra, Gurdyal S.

    2016-01-01

    High-throughput phenotypic screens have re-emerged as screening tools in antibiotic discovery. The advent of such technologies has rapidly accelerated the identification of ‘hit’ compounds. A pre-requisite to medicinal chemistry optimisation programmes required to improve the drug-like properties of a ‘hit’ molecule is identification of its mode of action. Herein, we have combined phenotypic screening with a biased target-specific screen. The inosine monophosphate dehydrogenase (IMPDH) protein GuaB2 has been identified as a drugable target in Mycobacterium tuberculosis, however previously identified compounds lack the desired characteristics necessary for further development into lead-like molecules. This study has identified 7 new chemical series from a high-throughput resistance-based phenotypic screen using Mycobacterium bovis BCG over-expressing GuaB2. Hit compounds were identified in a single shot high-throughput screen, validated by dose response and subjected to further biochemical analysis. The compounds were also assessed using molecular docking experiments, providing a platform for their further optimisation using medicinal chemistry. This work demonstrates the versatility and potential of GuaB2 as an anti-tubercular drug target. PMID:27982051

  20. Novel inhibitors of Mycobacterium tuberculosis GuaB2 identified by a target based high-throughput phenotypic screen.

    PubMed

    Cox, Jonathan A G; Mugumbate, Grace; Del Peral, Laura Vela-Glez; Jankute, Monika; Abrahams, Katherine A; Jervis, Peter; Jackenkroll, Stefan; Perez, Arancha; Alemparte, Carlos; Esquivias, Jorge; Lelièvre, Joël; Ramon, Fernando; Barros, David; Ballell, Lluis; Besra, Gurdyal S

    2016-12-16

    High-throughput phenotypic screens have re-emerged as screening tools in antibiotic discovery. The advent of such technologies has rapidly accelerated the identification of 'hit' compounds. A pre-requisite to medicinal chemistry optimisation programmes required to improve the drug-like properties of a 'hit' molecule is identification of its mode of action. Herein, we have combined phenotypic screening with a biased target-specific screen. The inosine monophosphate dehydrogenase (IMPDH) protein GuaB2 has been identified as a drugable target in Mycobacterium tuberculosis, however previously identified compounds lack the desired characteristics necessary for further development into lead-like molecules. This study has identified 7 new chemical series from a high-throughput resistance-based phenotypic screen using Mycobacterium bovis BCG over-expressing GuaB2. Hit compounds were identified in a single shot high-throughput screen, validated by dose response and subjected to further biochemical analysis. The compounds were also assessed using molecular docking experiments, providing a platform for their further optimisation using medicinal chemistry. This work demonstrates the versatility and potential of GuaB2 as an anti-tubercular drug target.

  1. Current and emerging screening methods to identify post-head-emergence frost adaptation in wheat and barley.

    PubMed

    Frederiks, T M; Christopher, J T; Harvey, G L; Sutherland, M W; Borrell, A K

    2012-09-01

    Cereal crops can suffer substantial damage if frosts occur at heading. Identification of post-head-emergence frost (PHEF) resistance in cereals poses a number of unique and difficult challenges. Many decades of research have failed to identify genotypes with PHEF resistance that could offer economically significant benefit to growers. Research and breeding gains have been limited by the available screening systems. Using traditional frost screening systems, genotypes that escape frost injury in trials due to spatial temperature differences and/or small differences in phenology can be misidentified as resistant. We believe that by improving techniques to minimize frost escapes, such 'false-positive' results can be confidently identified and eliminated. Artificial freezing chambers or manipulated natural frost treatments offer many potential advantages but are not yet at the stage where they can be reliably used for frost screening in breeding programmes. Here we describe the development of a novel photoperiod gradient method (PGM) that facilitates screening of genotypes of different phenology under natural field frosts at matched developmental stages. By identifying frost escapes and increasing the efficiency of field screening, the PGM ensures that research effort can be focused on finding genotypes with improved PHEF resistance. To maximize the likelihood of identifying PHEF resistance, we propose that the PGM form part of an integrated strategy to (i) source germplasm;(ii) facilitate high throughput screening; and (iii) permit detailed validation. PGM may also be useful in other studies where either a range of developmental stages and/or synchronized development are desired.

  2. Electronic Screening of Dictated Reports to Identify Patients with Do-Not-Resuscitate Status

    PubMed Central

    Aronsky, Dominik; Kasworm, Evelyn; Jacobson, Jay A.; Haug, Peter J.; Dean, Nathan C.

    2004-01-01

    Objective: Do-not-resuscitate (DNR) orders and advance directives are increasingly prevalent and may affect medical interventions and outcomes. Simple, automated techniques to identify patients with DNR orders do not currently exist but could help avoid costly and time-consuming chart review. This study hypothesized that a decision to withhold cardiopulmonary resuscitation would be included in a patient's dictated reports. The authors developed and validated a simple computerized search method, which screens dictated reports to detect patients with DNR status. Methods A list of concepts related to DNR order documentation was developed using emergency department, hospital admission, consult, and hospital discharge reports of 665 consecutive, hospitalized pneumonia patients during a four-year period (1995–1999). The list was validated in an independent group of 190 consecutive inpatients with pneumonia during a five-month period (1999–2000). The reference standard for the presence of DNR orders was manual chart review of all study patients. Sensitivity, specificity, predictive values, and nonerror rates were calculated for individual and combined concepts. Results The list of concepts included: DNR, Do Not Attempt to Resuscitate (DNAR), DNI, NCR, advanced directive, living will, power of attorney, Cardiopulmonary Resuscitation (CPR), defibrillation, arrest, resuscitate, code, and comfort care. As determined by manual chart review, a DNR order was written for 32.6% of patients in the derivation and for 31.6% in the validation group. Dictated reports included DNR order–related information for 74.5% of patients in the derivation and 73% in the validation group. If mentioned in the dictated report, the combined keyword search had a sensitivity of 74.2% in the derivation group (70.0% in the validation group), a specificity of 91.5% (81.5%), a positive predictive value of 80.9% (63.6%), a negative predictive value of 88.0% (85.5%), and a nonerror rate of 85.9% (77

  3. A Haploid Genetic Screen Identifies Heparan Sulfate Proteoglycans Supporting Rift Valley Fever Virus Infection

    PubMed Central

    Riblett, Amber M.; Blomen, Vincent A.; Jae, Lucas T.; Altamura, Louis A.; Doms, Robert W.; Brummelkamp, Thijn R.

    2015-01-01

    ABSTRACT Rift Valley fever virus (RVFV) causes recurrent insect-borne epizootics throughout the African continent, and infection of humans can lead to a lethal hemorrhagic fever syndrome. Deep mutagenesis of haploid human cells was used to identify host factors required for RVFV infection. This screen identified a suite of enzymes involved in glycosaminoglycan (GAG) biogenesis and transport, including several components of the cis-oligomeric Golgi (COG) complex, one of the central components of Golgi complex trafficking. In addition, disruption of PTAR1 led to RVFV resistance as well as reduced heparan sulfate surface levels, consistent with recent observations that PTAR1-deficient cells exhibit altered Golgi complex morphology and glycosylation defects. A variety of biochemical and genetic approaches were utilized to show that both pathogenic and attenuated RVFV strains require GAGs for efficient infection on some, but not all, cell types, with the block to infection being at the level of virion attachment. Examination of other members of the Bunyaviridae family for GAG-dependent infection suggested that the interaction with GAGs is not universal among bunyaviruses, indicating that these viruses, as well as RVFV on certain cell types, employ additional unidentified virion attachment factors and/or receptors. IMPORTANCE Rift Valley fever virus (RVFV) is an emerging pathogen that can cause severe disease in humans and animals. Epizootics among livestock populations lead to high mortality rates and can be economically devastating. Human epidemics of Rift Valley fever, often initiated by contact with infected animals, are characterized by a febrile disease that sometimes leads to encephalitis or hemorrhagic fever. The global burden of the pathogen is increasing because it has recently disseminated beyond Africa, which is of particular concern because the virus can be transmitted by widely distributed mosquito species. There are no FDA-licensed vaccines or antiviral

  4. The accuracy of the functional movement screen to identify individuals with an elevated risk of musculoskeletal injury.

    PubMed

    Krumrei, Kirk; Flanagan, Molly; Bruner, Josh; Durall, Chris

    2014-11-01

    Injuries are somewhat commonplace in highly active populations. One strategy for reducing injuries is to identify individuals with an elevated injury risk before participation so that remediative interventions can be provided. Preparticipation screenings have traditionally entailed strength and flexibility measures thought to be indicative of inflated injury risk. Some researchers, however, have suggested that functional movements/tasks should be assessed to help identify individuals with a high risk of future injury. One assessment tool used for this purpose is the Functional Movement Screen (FMS). The FMS generates a numeric score based on performance attributes during 7 dynamic tasks; this score is purported to reflect future injury risk. Expanding interest in the FMS has led researchers to investigate how accurately it can identify individuals with an increased risk of injury. Can the Functional Movement Screen accurately identify highly active individuals with an elevated risk of injury?

  5. A Comparison of Teacher Nomination and Screening to Identify Behavioral and Emotional Risk within a Sample of Underrepresented Students

    ERIC Educational Resources Information Center

    Dowdy, Erin; Doane, Kymberly; Eklund, Katie; Dever, Bridget V.

    2013-01-01

    Early identification of behavioral and emotional risk has been identified as one strategy to help decrease rates of childhood behavioral and emotional problems. This study compares two methods for early identification (teacher nomination and universal screening) to determine how each strategy may differentially identify at-risk students. A sample…

  6. A Comparison of Teacher Nomination and Screening to Identify Behavioral and Emotional Risk within a Sample of Underrepresented Students

    ERIC Educational Resources Information Center

    Dowdy, Erin; Doane, Kymberly; Eklund, Katie; Dever, Bridget V.

    2013-01-01

    Early identification of behavioral and emotional risk has been identified as one strategy to help decrease rates of childhood behavioral and emotional problems. This study compares two methods for early identification (teacher nomination and universal screening) to determine how each strategy may differentially identify at-risk students. A sample…

  7. Comparative Genome-Wide Screening Identifies a Conserved Doxorubicin Repair Network That Is Diploid Specific in Saccharomyces cerevisiae

    PubMed Central

    Westmoreland, Tammy J.; Wickramasekara, Sajith M.; Guo, Andrew Y.; Selim, Alice L.; Winsor, Tiffany S.; Greenleaf, Arno L.; Blackwell, Kimberly L.; Olson, John A.; Marks, Jeffrey R.; Bennett, Craig B.

    2009-01-01

    The chemotherapeutic doxorubicin (DOX) induces DNA double-strand break (DSB) damage. In order to identify conserved genes that mediate DOX resistance, we screened the Saccharomyces cerevisiae diploid deletion collection and identified 376 deletion strains in which exposure to DOX was lethal or severely reduced growth fitness. This diploid screen identified 5-fold more DOX resistance genes than a comparable screen using the isogenic haploid derivative. Since DSB damage is repaired primarily by homologous recombination in yeast, and haploid cells lack an available DNA homolog in G1 and early S phase, this suggests that our diploid screen may have detected the loss of repair functions in G1 or early S phase prior to complete DNA replication. To test this, we compared the relative DOX sensitivity of 30 diploid deletion mutants identified under our screening conditions to their isogenic haploid counterpart, most of which (n = 26) were not detected in the haploid screen. For six mutants (bem1Δ, ctf4Δ, ctk1Δ, hfi1Δ,nup133Δ, tho2Δ) DOX-induced lethality was absent or greatly reduced in the haploid as compared to the isogenic diploid derivative. Moreover, unlike WT, all six diploid mutants displayed severe G1/S phase cell cycle progression defects when exposed to DOX and some were significantly enhanced (ctk1Δ and hfi1Δ) or deficient (tho2Δ) for recombination. Using these and other “THO2-like” hypo-recombinogenic, diploid-specific DOX sensitive mutants (mft1Δ, thp1Δ, thp2Δ) we utilized known genetic/proteomic interactions to construct an interactive functional genomic network which predicted additional DOX resistance genes not detected in the primary screen. Most (76%) of the DOX resistance genes detected in this diploid yeast screen are evolutionarily conserved suggesting the human orthologs are candidates for mediating DOX resistance by impacting on checkpoint and recombination functions in G1 and/or early S phases. PMID:19503795

  8. Focused Screening Identifies Evoxine as a Small Molecule That Counteracts CO2-Induced Immune Suppression

    PubMed Central

    Helenius, Iiro Taneli; Nair, Aisha; Trejo Bittar, Humberto E.; Sznajder, Jacob I.; Sporn, Peter H. S.; Beitel, Greg J.

    2016-01-01

    Patients with severe lung disease may develop hypercapnia, elevation of the levels of CO2 in the lungs and blood, which is associated with increased risk of death, often from infection. To identify compounds that ameliorate the adverse effects of hypercapnia, we performed a focused screen of 8832 compounds using a CO2-responsive luciferase reporter in Drosophila S2* cells. We found that evoxine, a plant alkaloid, counteracts the CO2-induced transcriptional suppression of antimicrobial peptides in S2* cells. Strikingly, evoxine also inhibits hypercapnic suppression of interleukin-6 and the chemokine CCL2 expression in human THP-1 macrophages. Evoxine's effects are selective, since it does not prevent hypercapnic inhibition of phagocytosis by THP-1 cells or CO2-induced activation of AMPK in rat ATII pulmonary epithelial cells. The results suggest that hypercapnia suppresses innate immune gene expression by definable pathways that are evolutionarily conserved and demonstrate for the first time that specific CO2 effects can be targeted pharmacologically. PMID:26701099

  9. Genetic screening of Greek patients with Huntington’s disease phenocopies identifies an SCA8 expansion.

    PubMed

    Koutsis, G; Karadima, G; Pandraud, A; Sweeney, M G; Paudel, R; Houlden, H; Wood, N W; Panas, M

    2012-09-01

    Huntington’s disease (HD) is an autosomal dominant disorder characterized by a triad of chorea, psychiatric disturbance and cognitive decline. Around 1% of patients with HD-like symptoms lack the causative HD expansion and are considered HD phenocopies. Genetic diseases that can present as HD phenocopies include HD-like syndromes such as HDL1, HDL2 and HDL4 (SCA17), some spinocerebellar ataxias (SCAs) and dentatorubral-pallidoluysian atrophy (DRPLA). In this study we screened a cohort of 21 Greek patients with HD phenocopy syndromes formutations causing HDL2, SCA17, SCA1, SCA2, SCA3,SCA8, SCA12 and DRPLA. Fifteen patients (71%) had a positive family history. We identified one patient (4.8% of the total cohort) with an expansion of 81 combined CTA/CTG repeats at the SCA8 locus. This falls within what is believed to be the high-penetrance allele range. In addition to the classic HD triad, the patient had features of dystonia and oculomotor apraxia. There were no cases of HDL2, SCA17, SCA1, SCA2, SCA3, SCA12 or DRPLA. Given the controversy surrounding the SCA8 expansion, the present finding may be incidental. However, if pathogenic, it broadens the phenotype that may be associated with SCA8 expansions. The absence of any other mutations in our cohort is not surprising, given the low probability of reaching a genetic diagnosis in HD phenocopy patients.

  10. siRNA Screen Identifies Trafficking Host Factors that Modulate Alphavirus Infection

    PubMed Central

    Radoshitzky, Sheli R.; Pegoraro, Gianluca; Chī, Xiǎolì; Dǒng, Lián; Chiang, Chih-Yuan; Jozwick, Lucas; Clester, Jeremiah C.; Cooper, Christopher L.; Courier, Duane; Langan, David P.; Underwood, Knashka; Kuehl, Kathleen A.; Sun, Mei G.; Caì, Yíngyún; Yú, Shuǐqìng; Burk, Robin; Zamani, Rouzbeh; Kota, Krishna; Kuhn, Jens H.; Bavari, Sina

    2016-01-01

    Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2) expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1–PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling. PMID:27031835

  11. Phenotypic screening identifies Axl kinase as a negative regulator of an alveolar epithelial cell phenotype.

    PubMed

    Fujino, Naoya; Kubo, Hiroshi; Maciewicz, Rose A

    2017-09-01

    Loss of epithelial barrier integrity is implicated in a number of human lung diseases. However, the molecular pathways underlying this process are poorly understood. In a phenotypic screen, we identified Axl kinase as a negative regulator of epithelial phenotype and function. Furthermore, suppression of Axl activity by a small molecule kinase inhibitor or downregulation of Axl expression by small interfering RNA led to: (1) the increase in epithelial surfactant protein expression; (2) a cell morphology transition from front-rear polarity to cuboidal shape; (3) the cytoskeletal re-organization resulting in decreased cell mobility; and (4) the acquisition of epithelial junctions. Loss of Axl activity reduced activation of the Axl canonical pathway members, Akt and extracellular signal-regulated kinase-1/2 and resulted in the loss of gene expression of a unique profile of epithelial-to-mesenchymal transition transcription factors including SNAI2, HOXA5, TBX2 or TBX3. Finally, we observed that Axl was activated in hyperplasia of epithelial cells in idiopathic pulmonary fibrosis where epithelial barrier integrity was lost. These results suggest that the Axl kinase signaling pathway is associated with the loss integrity of alveolar epithelium in pathological remodeling of human lung diseases.

  12. Identifying genes of agronomic importance in maize by screening microsatellites for evidence of selection during domestication.

    PubMed

    Vigouroux, Y; McMullen, M; Hittinger, C T; Houchins, K; Schulz, L; Kresovich, S; Matsuoka, Y; Doebley, J

    2002-07-23

    Crop species experienced strong selective pressure directed at genes controlling traits of agronomic importance during their domestication and subsequent episodes of selective breeding. Consequently, these genes are expected to exhibit the signature of selection. We screened 501 maize genes for the signature of selection using microsatellites or simple sequence repeats (SSRs). We applied the Ewens-Watterson test, which can reveal deviations from a neutral-equilibrium model, as well as two nonequilibrium tests that incorporate the domestication bottleneck. We investigated two classes of SSRs: those known to be polymorphic in maize (Class I) and those previously classified as monomorphic in maize (Class II). Fifteen SSRs exhibited some evidence for selection in maize and 10 showed evidence under stringent criteria. The genes containing nonneutral SSRs are candidates for agronomically important genes. Because demographic factors can bias our tests, further independent tests of these candidates are necessary. We applied such an additional test to one candidate, which encodes a MADS box transcriptional regulator, and confirmed that this gene experienced a selective sweep during maize domestication. Genomic scans for the signature of selection offer a means of identifying new genes of agronomic importance even when gene function and the phenotype of interest are unknown.

  13. Tumor cell migration screen identifies SRPK1 as breast cancer metastasis determinant

    PubMed Central

    van Roosmalen, Wies; Le Dévédec, Sylvia E.; Golani, Ofra; Smid, Marcel; Pulyakhina, Irina; Timmermans, Annemieke M.; Look, Maxime P.; Zi, Di; Pont, Chantal; de Graauw, Marjo; Naffar-Abu-Amara, Suha; Kirsanova, Catherine; Rustici, Gabriella; Hoen, Peter A.C. ‘t; Martens, John W.M.; Foekens, John A.; Geiger, Benjamin; van de Water, Bob

    2015-01-01

    Tumor cell migration is a key process for cancer cell dissemination and metastasis that is controlled by signal-mediated cytoskeletal and cell matrix adhesion remodeling. Using a phagokinetic track assay with migratory H1299 cells, we performed an siRNA screen of almost 1,500 genes encoding kinases/phosphatases and adhesome- and migration-related proteins to identify genes that affect tumor cell migration speed and persistence. Thirty candidate genes that altered cell migration were validated in live tumor cell migration assays. Eight were associated with metastasis-free survival in breast cancer patients, with integrin β3–binding protein (ITGB3BP), MAP3K8, NIMA-related kinase (NEK2), and SHC-transforming protein 1 (SHC1) being the most predictive. Examination of genes that modulate migration indicated that SRPK1, encoding the splicing factor kinase SRSF protein kinase 1, is relevant to breast cancer outcomes, as it was highly expressed in basal breast cancer. Furthermore, high SRPK1 expression correlated with poor breast cancer disease outcome and preferential metastasis to the lungs and brain. In 2 independent murine models of breast tumor metastasis, stable shRNA-based SRPK1 knockdown suppressed metastasis to distant organs, including lung, liver, and spleen, and inhibited focal adhesion reorganization. Our study provides comprehensive information on the molecular determinants of tumor cell migration and suggests that SRPK1 has potential as a drug target for limiting breast cancer metastasis. PMID:25774502

  14. Validation of a fluorescence-based screening concept to identify ribosome assembly defects in Escherichia coli.

    PubMed

    Nikolay, Rainer; Schloemer, Renate; Schmidt, Sabine; Mueller, Silke; Heubach, Anja; Deuerling, Elke

    2014-07-01

    While the structure of mature ribosomes is analyzed in atomic detail considerably less is known about their assembly process in living cells. This is mainly due to technical and conceptual hurdles. To analyze ribosome assembly in vivo, we designed and engineered an Escherichiacoli strain--using chromosomal gene knock-in techniques--that harbors large and small ribosomal subunits labeled with the fluorescent proteins EGFP and mCherry, respectively. A thorough characterization of this reporter strain revealed that its growth properties and translation apparatus were wild-type like. Alterations in the ratio of EGFP over mCherry fluorescence are supposed to indicate ribosome assembly defects. To provide proof of principle, subunit specific assembly defects were provoked and could be identified by both manual and fully automated fluorometric in vivo assays. This is to our knowledge the first methodology that directly detects ribosome assembly defects in vivo in a high-throughput compatible format. Screening of knock-out collections and small molecule libraries will allow identification of new ribosome assembly factors and possible inhibitors. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Validation of a fluorescence-based screening concept to identify ribosome assembly defects in Escherichia coli

    PubMed Central

    Nikolay, Rainer; Schloemer, Renate; Schmidt, Sabine; Mueller, Silke; Heubach, Anja; Deuerling, Elke

    2014-01-01

    While the structure of mature ribosomes is analyzed in atomic detail considerably less is known about their assembly process in living cells. This is mainly due to technical and conceptual hurdles. To analyze ribosome assembly in vivo, we designed and engineered an Escherichiacoli strain—using chromosomal gene knock-in techniques—that harbors large and small ribosomal subunits labeled with the fluorescent proteins EGFP and mCherry, respectively. A thorough characterization of this reporter strain revealed that its growth properties and translation apparatus were wild-type like. Alterations in the ratio of EGFP over mCherry fluorescence are supposed to indicate ribosome assembly defects. To provide proof of principle, subunit specific assembly defects were provoked and could be identified by both manual and fully automated fluorometric in vivo assays. This is to our knowledge the first methodology that directly detects ribosome assembly defects in vivo in a high-throughput compatible format. Screening of knock-out collections and small molecule libraries will allow identification of new ribosome assembly factors and possible inhibitors. PMID:24792169

  16. A zebrafish genetic screen identifies neuromedin U as a regulator of sleep/wake states

    PubMed Central

    Chiu, Cindy N.; Rihel, Jason; Lee, Daniel A.; Singh, Chanpreet; Mosser, Eric A.; Chen, Shijia; Sapin, Viveca; Pham, Uyen; Engle, Jae; Niles, Brett J.; Montz, Christin J.; Chakravarthy, Sridhara; Zimmerman, Steven; Salehi-Ashtiani, Kourosh; Vidal, Marc; Schier, Alexander F.; Prober, David A.

    2016-01-01

    Summary Neuromodulation of arousal states ensures that an animal appropriately responds to its environment and engages in behaviors necessary for survival. However, the molecular and circuit properties underlying neuromodulation of arousal states such as sleep and wakefulness remain unclear. To tackle this challenge in a systematic and unbiased manner, we performed a genetic overexpression screen to identify genes that affect larval zebrafish arousal. We found that the neuropeptide neuromedin U (Nmu) promotes hyperactivity and inhibits sleep in zebrafish larvae, whereas nmu mutant animals are hypoactive. We show that Nmu-induced arousal requires Nmu receptor 2 and signaling via corticotrophin-releasing hormone (Crh) receptor 1. In contrast to previously proposed models, we find that Nmu does not promote arousal via the hypothalamic-pituitary-adrenal axis, but rather likely acts via brainstem crh-expressing neurons. These results reveal an unexpected functional and anatomical interface between the Nmu system and brainstem arousal systems that represents a novel wake-promoting pathway. PMID:26889812

  17. A new class of temporarily phenotypic enhancers identified by CRISPR/Cas9-mediated genetic screening

    PubMed Central

    Diao, Yarui; Li, Bin; Meng, Zhipeng; Jung, Inkyung; Lee, Ah Young; Dixon, Jesse; Maliskova, Lenka; Guan, Kun-liang; Shen, Yin; Ren, Bing

    2016-01-01

    With <2% of the human genome coding for proteins, a major challenge is to interpret the function of the noncoding DNA. Millions of regulatory sequences have been predicted in the human genome through analysis of DNA methylation, chromatin modification, hypersensitivity to nucleases, and transcription factor binding, but few have been shown to regulate transcription in their native contexts. We have developed a high-throughput CRISPR/Cas9-based genome-editing strategy and used it to interrogate 174 candidate regulatory sequences within the 1-Mbp POU5F1 locus in human embryonic stem cells (hESCs). We identified two classical regulatory elements, including a promoter and a proximal enhancer, that are essential for POU5F1 transcription in hESCs. Unexpectedly, we also discovered a new class of enhancers that contribute to POU5F1 transcription in an unusual way: Disruption of such sequences led to a temporary loss of POU5F1 transcription that is fully restored after a few rounds of cell division. These results demonstrate the utility of high-throughput screening for functional characterization of noncoding DNA and reveal a previously unrecognized layer of gene regulation in human cells. PMID:26813977

  18. Virtual screening and experimental validation identify novel modulators of nuclear receptor RXRα from Drugbank database.

    PubMed

    Xu, Dan; Cai, Lijun; Guo, Shangjie; Xie, Lei; Yin, Meimei; Chen, Ziwen; Zhou, Hu; Su, Ying; Zeng, Zhiping; Zhang, Xiaokun

    2017-02-15

    Retinoid X receptor alpha (RXRα), an important ligand-dependent transcription factor, plays a critical role in the development of various cancers and metabolic and neurodegenerative diseases. Therefore, RXRα represents one of the most important targets in modern drug discovery. In this study, Drugbank 2.0 with 1280 old drugs were virtually screened by Glide according to the crystal structure of ligand-binding domain (LBP) of RXRα. 15 compounds selected were tested for their binding and transcriptional activity toward RXRα by Biacore and reporter gene assay, respectively. The identified new scafford ligand of RXRα, Pitavastatin (1), was chemically optimized. Our results demonstrated that statin compounds Pitavastatin (1) and Fluvastatin (4) could bind to the LBP of RXRα (KD=13.30μM and 11.04μM, respectively) and serve as transcriptional antagonists of RXRα. On the contrary, compound (12) (domperidone) and (13) (rosiglitazone maleate) could bind to the LBP of RXRα (KD=8.80μM and 15.01μM, respectively) but serve as transcriptional agonists of RXRα.

  19. Tumor cell migration screen identifies SRPK1 as breast cancer metastasis determinant.

    PubMed

    van Roosmalen, Wies; Le Dévédec, Sylvia E; Golani, Ofra; Smid, Marcel; Pulyakhina, Irina; Timmermans, Annemieke M; Look, Maxime P; Zi, Di; Pont, Chantal; de Graauw, Marjo; Naffar-Abu-Amara, Suha; Kirsanova, Catherine; Rustici, Gabriella; Hoen, Peter A C 't; Martens, John W M; Foekens, John A; Geiger, Benjamin; van de Water, Bob

    2015-04-01

    Tumor cell migration is a key process for cancer cell dissemination and metastasis that is controlled by signal-mediated cytoskeletal and cell matrix adhesion remodeling. Using a phagokinetic track assay with migratory H1299 cells, we performed an siRNA screen of almost 1,500 genes encoding kinases/phosphatases and adhesome- and migration-related proteins to identify genes that affect tumor cell migration speed and persistence. Thirty candidate genes that altered cell migration were validated in live tumor cell migration assays. Eight were associated with metastasis-free survival in breast cancer patients, with integrin β3-binding protein (ITGB3BP), MAP3K8, NIMA-related kinase (NEK2), and SHC-transforming protein 1 (SHC1) being the most predictive. Examination of genes that modulate migration indicated that SRPK1, encoding the splicing factor kinase SRSF protein kinase 1, is relevant to breast cancer outcomes, as it was highly expressed in basal breast cancer. Furthermore, high SRPK1 expression correlated with poor breast cancer disease outcome and preferential metastasis to the lungs and brain. In 2 independent murine models of breast tumor metastasis, stable shRNA-based SRPK1 knockdown suppressed metastasis to distant organs, including lung, liver, and spleen, and inhibited focal adhesion reorganization. Our study provides comprehensive information on the molecular determinants of tumor cell migration and suggests that SRPK1 has potential as a drug target for limiting breast cancer metastasis.

  20. An unbiased expression screen for synaptogenic proteins identifies the LRRTM protein family as synaptic organizers.

    PubMed

    Linhoff, Michael W; Laurén, Juha; Cassidy, Robert M; Dobie, Frederick A; Takahashi, Hideto; Nygaard, Haakon B; Airaksinen, Matti S; Strittmatter, Stephen M; Craig, Ann Marie

    2009-03-12

    Delineating the molecular basis of synapse development is crucial for understanding brain function. Cocultures of neurons with transfected fibroblasts have demonstrated the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the coculture assay using custom-made cDNA libraries. Reisolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is incomplete. We identify LRRTM1 as a transmembrane protein that induces presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially induced clustering of LRRTMs mediates postsynaptic differentiation. We generate LRRTM1(-/-) mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the reported linkage of LRRTM1 to handedness and schizophrenia.

  1. A novel chemical screening strategy in zebrafish identifies common pathways in embryogenesis and rhabdomyosarcoma development.

    PubMed

    Le, Xiuning; Pugach, Emily K; Hettmer, Simone; Storer, Narie Y; Liu, Jianing; Wills, Airon A; DiBiase, Antony; Chen, Eleanor Y; Ignatius, Myron S; Poss, Kenneth D; Wagers, Amy J; Langenau, David M; Zon, Leonard I

    2013-06-01

    The zebrafish is a powerful genetic model that has only recently been used to dissect developmental pathways involved in oncogenesis. We hypothesized that operative pathways during embryogenesis would also be used for oncogenesis. In an effort to define RAS target genes during embryogenesis, gene expression was evaluated in Tg(hsp70-HRAS(G12V)) zebrafish embryos subjected to heat shock. dusp6 was activated by RAS, and this was used as the basis for a chemical genetic screen to identify small molecules that interfere with RAS signaling during embryogenesis. A KRAS(G12D)-induced zebrafish embryonal rhabdomyosarcoma was then used to assess the therapeutic effects of the small molecules. Two of these inhibitors, PD98059 and TPCK, had anti-tumor activity as single agents in both zebrafish embryonal rhabdomyosarcoma and a human cell line of rhabdomyosarcoma that harbored activated mutations in NRAS. PD98059 inhibited MEK1 whereas TPCK suppressed S6K1 activity; however, the combined treatment completely suppressed eIF4B phosphorylation and decreased translation initiation. Our work demonstrates that the activated pathways in RAS induction during embryogenesis are also important in oncogenesis and that inhibition of these pathways suppresses tumor growth.

  2. AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma.

    PubMed

    Chow, Ryan D; Guzman, Christopher D; Wang, Guangchuan; Schmidt, Florian; Youngblood, Mark W; Ye, Lupeng; Errami, Youssef; Dong, Matthew B; Martinez, Michael A; Zhang, Sensen; Renauer, Paul; Bilguvar, Kaya; Gunel, Murat; Sharp, Phillip A; Zhang, Feng; Platt, Randall J; Chen, Sidi

    2017-10-01

    A causative understanding of genetic factors that regulate glioblastoma pathogenesis is of central importance. Here we developed an adeno-associated virus-mediated, autochthonous genetic CRISPR screen in glioblastoma. Stereotaxic delivery of a virus library targeting genes commonly mutated in human cancers into the brains of conditional-Cas9 mice resulted in tumors that recapitulate human glioblastoma. Capture sequencing revealed diverse mutational profiles across tumors. The mutation frequencies in mice correlated with those in two independent patient cohorts. Co-mutation analysis identified co-occurring driver combinations such as B2m-Nf1, Mll3-Nf1 and Zc3h13-Rb1, which were subsequently validated using AAV minipools. Distinct from Nf1-mutant tumors, Rb1-mutant tumors are undifferentiated and aberrantly express homeobox gene clusters. The addition of Zc3h13 or Pten mutations altered the gene expression profiles of Rb1 mutants, rendering them more resistant to temozolomide. Our study provides a functional landscape of gliomagenesis suppressors in vivo.

  3. A Zebrafish Genetic Screen Identifies Neuromedin U as a Regulator of Sleep/Wake States.

    PubMed

    Chiu, Cindy N; Rihel, Jason; Lee, Daniel A; Singh, Chanpreet; Mosser, Eric A; Chen, Shijia; Sapin, Viveca; Pham, Uyen; Engle, Jae; Niles, Brett J; Montz, Christin J; Chakravarthy, Sridhara; Zimmerman, Steven; Salehi-Ashtiani, Kourosh; Vidal, Marc; Schier, Alexander F; Prober, David A

    2016-02-17

    Neuromodulation of arousal states ensures that an animal appropriately responds to its environment and engages in behaviors necessary for survival. However, the molecular and circuit properties underlying neuromodulation of arousal states such as sleep and wakefulness remain unclear. To tackle this challenge in a systematic and unbiased manner, we performed a genetic overexpression screen to identify genes that affect larval zebrafish arousal. We found that the neuropeptide neuromedin U (Nmu) promotes hyperactivity and inhibits sleep in zebrafish larvae, whereas nmu mutant animals are hypoactive. We show that Nmu-induced arousal requires Nmu receptor 2 and signaling via corticotropin releasing hormone (Crh) receptor 1. In contrast to previously proposed models, we find that Nmu does not promote arousal via the hypothalamic-pituitary-adrenal axis, but rather probably acts via brainstem crh-expressing neurons. These results reveal an unexpected functional and anatomical interface between the Nmu system and brainstem arousal systems that represents a novel wake-promoting pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Genome-Wide RNAi Screen Identifies Novel Host Proteins Required for Alphavirus Entry

    PubMed Central

    Taylor, Gwen M.; Kielian, Margaret

    2013-01-01

    The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ), a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy. PMID:24367265

  5. siRNA Screen Identifies Trafficking Host Factors that Modulate Alphavirus Infection.

    PubMed

    Radoshitzky, Sheli R; Pegoraro, Gianluca; Chī, Xi Olì; D Ng, Lián; Chiang, Chih-Yuan; Jozwick, Lucas; Clester, Jeremiah C; Cooper, Christopher L; Courier, Duane; Langan, David P; Underwood, Knashka; Kuehl, Kathleen A; Sun, Mei G; Caì, Yíngyún; Yú, Shu Qìng; Burk, Robin; Zamani, Rouzbeh; Kota, Krishna; Kuhn, Jens H; Bavari, Sina

    2016-03-01

    Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2) expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1-PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling.

  6. The validation of electronic health records in accurately identifying patients eligible for colorectal cancer screening in safety net clinics.

    PubMed

    Petrik, Amanda F; Green, Beverly B; Vollmer, William M; Le, Thuy; Bachman, Barbara; Keast, Erin; Rivelli, Jennifer; Coronado, Gloria D

    2016-12-01

    While electronic health records (EHRs) play a key role in increasing colorectal cancer (CRC) screening by identifying individuals who are overdue, important shortfalls remain. As part of the Strategies and Opportunities to STOP Colon Cancer (STOP CRC) study, we assessed the accuracy of EHR codes in identifying patients eligible for CRC screening. We selected a stratified random sample of 800 study participants from 26 participating clinics, in the Pacific Northwest region of the USA. We compared data obtained through codes in the EHR to conduct a manual chart audit. A trained chart abstractor completed the abstraction of eligible and ineligible patients. Of 520 individuals in need of CRC screening, identified via the EHR, 459 were confirmed through chart review (positive predictive value = 88%). Of 280 individuals flagged as up-to-date in their screening per EHR data, 269 were confirmed through chart review (negative predictive value = 96%). Among the 61 patients incorrectly classified as eligible, 83.6% of disagreements were due to evidence of a prior colonoscopy or referral that was not captured in recognizable fields in the EHR. Our findings highlight importance of better capture of past screening events in the EHR. While the need for better population-based data is not unique to CRC screening, it provides an important example of the use of population-based data not only for tracking care, but also for delivering interventions. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Pyrrolamide DNA Gyrase Inhibitors: Fragment-Based Nuclear Magnetic Resonance Screening To Identify Antibacterial Agents

    PubMed Central

    Green, Oluyinka; Hales, Neil; Walkup, Grant K.; Bist, Shanta; Singh, Alok; Mullen, George; Bryant, Joanna; Embrey, Kevin; Gao, Ning; Breeze, Alex; Timms, Dave; Andrews, Beth; Uria-Nickelsen, Maria; Demeritt, Julie; Loch, James T.; Hull, Ken; Blodgett, April; Illingworth, Ruth N.; Prince, Bryan; Boriack-Sjodin, P. Ann; Hauck, Sheila; MacPherson, Lawrence J.; Ni, Haihong; Sherer, Brian

    2012-01-01

    DNA gyrase is an essential enzyme in bacteria, and its inhibition results in the disruption of DNA synthesis and, subsequently, cell death. The pyrrolamides are a novel class of antibacterial agents targeting DNA gyrase. These compounds were identified by a fragment-based lead generation (FBLG) approach using nuclear magnetic resonance (NMR) screening to identify low-molecular-weight compounds that bind to the ATP pocket of DNA gyrase. A pyrrole hit with a binding constant of 1 mM formed the basis of the design and synthesis of a focused library of compounds that resulted in the rapid identification of a lead compound that inhibited DNA gyrase with a 50% inhibitory concentration (IC50) of 3 μM. The potency of the lead compound was further optimized by utilizing iterative X-ray crystallography to yield DNA gyrase inhibitors that also displayed antibacterial activity. Spontaneous mutants were isolated in Staphylococcus aureus by plating on agar plates containing pyrrolamide 4 at the MIC. The resistant variants displayed 4- to 8-fold-increased MIC values relative to the parent strain. DNA sequencing revealed two independent point mutations in the pyrrolamide binding region of the gyrB genes from these variants, supporting the hypothesis that the mode of action of these compounds was inhibition of DNA gyrase. Efficacy of a representative pyrrolamide was demonstrated against Streptococcus pneumoniae in a mouse lung infection model. These data demonstrate that the pyrrolamides are a novel class of DNA gyrase inhibitors with the potential to deliver future antibacterial agents targeting multiple clinical indications. PMID:22183167

  8. A functional screen for copper homeostasis genes identifies a pharmacologically tractable cellular system.

    PubMed

    Schlecht, Ulrich; Suresh, Sundari; Xu, Weihong; Aparicio, Ana Maria; Chu, Angela; Proctor, Michael J; Davis, Ronald W; Scharfe, Curt; St Onge, Robert P

    2014-04-05

    Copper is essential for the survival of aerobic organisms. If copper is not properly regulated in the body however, it can be extremely cytotoxic and genetic mutations that compromise copper homeostasis result in severe clinical phenotypes. Understanding how cells maintain optimal copper levels is therefore highly relevant to human health. We found that addition of copper (Cu) to culture medium leads to increased respiratory growth of yeast, a phenotype which we then systematically and quantitatively measured in 5050 homozygous diploid deletion strains. Cu's positive effect on respiratory growth was quantitatively reduced in deletion strains representing 73 different genes, the function of which identify increased iron uptake as a cause of the increase in growth rate. Conversely, these effects were enhanced in strains representing 93 genes. Many of these strains exhibited respiratory defects that were specifically rescued by supplementing the growth medium with Cu. Among the genes identified are known and direct regulators of copper homeostasis, genes required to maintain low vacuolar pH, and genes where evidence supporting a functional link with Cu has been heretofore lacking. Roughly half of the genes are conserved in man, and several of these are associated with Mendelian disorders, including the Cu-imbalance syndromes Menkes and Wilson's disease. We additionally demonstrate that pharmacological agents, including the approved drug disulfiram, can rescue Cu-deficiencies of both environmental and genetic origin. A functional screen in yeast has expanded the list of genes required for Cu-dependent fitness, revealing a complex cellular system with implications for human health. Respiratory fitness defects arising from perturbations in this system can be corrected with pharmacological agents that increase intracellular copper concentrations.

  9. A Functional Screen Identifies miRs that Induce Radioresistance in Glioblastomas

    PubMed Central

    Moskwa, Patryk; Zinn, Pascal O.; Choi, Young Eun; Shukla, Sachet A; Fendler, Wojciech; Chen, Clark C; Lu, Jun; Golub, Todd R; Hjelmeland, Anita; Chowdhury, Dipanjan

    2015-01-01

    The efficacy of radiotherapy in many tumor types is limited by normal tissue toxicity and by intrinsic or acquired radioresistance. Therefore, it is essential to understand the molecular network responsible for regulating radiosensitivity/resistance. Here, an unbiased functional screen identified four microRNAs (miR-1, miR-125a, miR-150, and miR-425) that induce radioresistance. Considering the clinical importance of radiotherapy for glioblastoma (GBM) patients, the impact of these miRNAs on GBM radioresistance was investigated. Overexpression of miR-1, miR-125a, miR-150 and/or miR-425 in GBM promotes radioresistance through upregulation of the cell cycle checkpoint response. Conversely, antagonizing with antagomiRs sensitizes GBM cells to irradiation, suggesting their potential as targets for inhibiting therapeutic resistance. Analysis of GBM datasets from TCGA revealed that these miRNAs are expressed in GBM patient specimens and correlate with Transforming Growth Factor Beta (TGF-β) signaling. Finally, it is demonstrated that expression of miR-1 and miR-125a can be induced by TGF-β and antagonized by a TGF-β receptor inhibitor. Together, these results identify and characterize a new role for miR-425, miR-1, miR-125, and miR-150 in promoting radioresistance in GBMs and provide insight into the therapeutic application of TGF-β inhibitors in radiotherapy. Systematic identification of miRs that cause radioresistance in gliomas is important for uncovering predictive markers for radiotherapy or targets for overcoming radioresistance. PMID:25256711

  10. Genome Screen to Identify Susceptibility Genes for Parkinson Disease in a Sample without parkin Mutations

    PubMed Central

    Pankratz, Nathan; Nichols, William C.; Uniacke, Sean K.; Halter, Cheryl; Rudolph, Alice; Shults, Cliff; Conneally, P. Michael; Foroud, Tatiana

    2002-01-01

    Parkinson disease (PD) is a common neurodegenerative disorder characterized by bradykinesia, resting tremor, muscular rigidity, and postural instability, as well as by a clinically significant response to treatment with levodopa. Mutations in the α-synuclein gene have been found to result in autosomal dominant PD, and mutations in the parkin gene produce autosomal recessive juvenile-onset PD. We have studied 203 sibling pairs with PD who were evaluated by a rigorous neurological assessment based on (a) inclusion criteria consisting of clinical features highly associated with autopsy-confirmed PD and (b) exclusion criteria highly associated with other, non-PD pathological diagnoses. Families with positive LOD scores for a marker in an intron of the parkin gene were prioritized for parkin-gene testing, and mutations in the parkin gene were identified in 22 families. To reduce genetic heterogeneity, these families were not included in subsequent genome-screen analysis. Thus, a total of 160 multiplex families without evidence of a parkin mutation were used in multipoint nonparametric linkage analysis to identify PD-susceptibility genes. Two models of PD affection status were considered: model I included only those individuals with a more stringent diagnosis of verified PD (96 sibling pairs from 90 families), whereas model II included all examined individuals as affected, regardless of their final diagnostic classification (170 sibling pairs from 160 families). Under model I, the highest LOD scores were observed on chromosome X (LOD score 2.1) and on chromosome 2 (LOD score 1.9). Analyses performed with all available sibling pairs (model II) found even greater evidence of linkage to chromosome X (LOD score 2.7) and to chromosome 2 (LOD score 2.5). Evidence of linkage was also found to chromosomes 4, 5, and 13 (LOD scores >1.5). Our findings are consistent with those of other linkage studies that have reported linkage to chromosomes 5 and X. PMID:12058349

  11. A functional screen for copper homeostasis genes identifies a pharmacologically tractable cellular system

    PubMed Central

    2014-01-01

    Background Copper is essential for the survival of aerobic organisms. If copper is not properly regulated in the body however, it can be extremely cytotoxic and genetic mutations that compromise copper homeostasis result in severe clinical phenotypes. Understanding how cells maintain optimal copper levels is therefore highly relevant to human health. Results We found that addition of copper (Cu) to culture medium leads to increased respiratory growth of yeast, a phenotype which we then systematically and quantitatively measured in 5050 homozygous diploid deletion strains. Cu’s positive effect on respiratory growth was quantitatively reduced in deletion strains representing 73 different genes, the function of which identify increased iron uptake as a cause of the increase in growth rate. Conversely, these effects were enhanced in strains representing 93 genes. Many of these strains exhibited respiratory defects that were specifically rescued by supplementing the growth medium with Cu. Among the genes identified are known and direct regulators of copper homeostasis, genes required to maintain low vacuolar pH, and genes where evidence supporting a functional link with Cu has been heretofore lacking. Roughly half of the genes are conserved in man, and several of these are associated with Mendelian disorders, including the Cu-imbalance syndromes Menkes and Wilson’s disease. We additionally demonstrate that pharmacological agents, including the approved drug disulfiram, can rescue Cu-deficiencies of both environmental and genetic origin. Conclusions A functional screen in yeast has expanded the list of genes required for Cu-dependent fitness, revealing a complex cellular system with implications for human health. Respiratory fitness defects arising from perturbations in this system can be corrected with pharmacological agents that increase intracellular copper concentrations. PMID:24708151

  12. Pyrrolamide DNA gyrase inhibitors: fragment-based nuclear magnetic resonance screening to identify antibacterial agents.

    PubMed

    Eakin, Ann E; Green, Oluyinka; Hales, Neil; Walkup, Grant K; Bist, Shanta; Singh, Alok; Mullen, George; Bryant, Joanna; Embrey, Kevin; Gao, Ning; Breeze, Alex; Timms, Dave; Andrews, Beth; Uria-Nickelsen, Maria; Demeritt, Julie; Loch, James T; Hull, Ken; Blodgett, April; Illingworth, Ruth N; Prince, Bryan; Boriack-Sjodin, P Ann; Hauck, Sheila; MacPherson, Lawrence J; Ni, Haihong; Sherer, Brian

    2012-03-01

    DNA gyrase is an essential enzyme in bacteria, and its inhibition results in the disruption of DNA synthesis and, subsequently, cell death. The pyrrolamides are a novel class of antibacterial agents targeting DNA gyrase. These compounds were identified by a fragment-based lead generation (FBLG) approach using nuclear magnetic resonance (NMR) screening to identify low-molecular-weight compounds that bind to the ATP pocket of DNA gyrase. A pyrrole hit with a binding constant of 1 mM formed the basis of the design and synthesis of a focused library of compounds that resulted in the rapid identification of a lead compound that inhibited DNA gyrase with a 50% inhibitory concentration (IC(50)) of 3 μM. The potency of the lead compound was further optimized by utilizing iterative X-ray crystallography to yield DNA gyrase inhibitors that also displayed antibacterial activity. Spontaneous mutants were isolated in Staphylococcus aureus by plating on agar plates containing pyrrolamide 4 at the MIC. The resistant variants displayed 4- to 8-fold-increased MIC values relative to the parent strain. DNA sequencing revealed two independent point mutations in the pyrrolamide binding region of the gyrB genes from these variants, supporting the hypothesis that the mode of action of these compounds was inhibition of DNA gyrase. Efficacy of a representative pyrrolamide was demonstrated against Streptococcus pneumoniae in a mouse lung infection model. These data demonstrate that the pyrrolamides are a novel class of DNA gyrase inhibitors with the potential to deliver future antibacterial agents targeting multiple clinical indications.

  13. Ectopic expression screen identifies genes affecting Drosophila mesoderm development including the HSPG Trol.

    PubMed

    Trisnadi, Nathanie; Stathopoulos, Angelike

    2014-12-23

    Gastrulation of the embryo involves coordinate cell movements likely supported by multiple signaling pathways, adhesion molecules, and extracellular matrix components. Fibroblast growth factors (FGFs) have a major role in Drosophila melanogaster mesoderm migration; however, few other inputs are known and the mechanism supporting cell movement is unclear. To provide insight, we performed an ectopic expression screen to identify secreted or membrane-associated molecules that act to support mesoderm migration. Twenty-four UAS insertions were identified that cause lethality when expressed in either the mesoderm (Twi-Gal4) or the ectoderm (69B-Gal4). The list was narrowed to a subset of 10 genes that were shown to exhibit loss-of-function mutant phenotypes specifically affecting mesoderm migration. These include the FGF ligand Pyramus, α-integrins, E-cadherin, Cueball, EGFR, JAK/STAT signaling components, as well as the heparan sulfate proteoglycan (HSPG) Terribly reduced optic lobes (Trol). Trol encodes the ortholog of mammalian HSPG Perlecan, a demonstrated FGF signaling cofactor. Here, we examine the role of Trol in Drosophila mesoderm migration and compare and contrast its role with that of Syndecan (Sdc), another HSPG previously implicated in this process. Embryos mutant for Trol or Sdc were obtained and analyzed. Our data support the view that both HSPGs function to support FGF-dependent processes in the early embryo as they share phenotypes with FGF mutants: Trol in terms of effects on mesoderm migration and caudal visceral mesoderm (CVM) migration and Sdc in terms of dorsal mesoderm specification. The differential roles uncovered for these two HSPGs suggest that HSPG cofactor choice may modify FGF-signaling outputs. Copyright © 2015 Trisnadi and Stathopoulos.

  14. A yeast-based genetic screening to identify human proteins that increase homologous recombination.

    PubMed

    Collavoli, Anita; Comelli, Laura; Rainaldi, Giuseppe; Galli, Alvaro

    2008-05-01

    To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR.

  15. In vitro functional screening as a means to identify new plasticizers devoid of reproductive toxicity.

    PubMed

    Boisvert, Annie; Jones, Steven; Issop, Leeyah; Erythropel, Hanno C; Papadopoulos, Vassilios; Culty, Martine

    2016-10-01

    Plasticizers are indispensable additives providing flexibility and malleability to plastics. Among them, several phthalates, including di (2-ethylhexyl) phthalate (DEHP), have emerged as endocrine disruptors, leading to their restriction in consumer products and creating a need for new, safer plasticizers. The goal of this project was to use in vitro functional screening tools to select novel non-toxic plasticizers suitable for further in vivo evaluation. A panel of novel compounds with satisfactory plasticizer properties and biodegradability were tested, along with several commercial plasticizers, such as diisononyl-cyclohexane-1,2-dicarboxylate (DINCH®). MEHP, the monoester metabolite of DEHP was also included as reference compound. Because phthalates target mainly testicular function, including androgen production and spermatogenesis, we used the mouse MA-10 Leydig and C18-4 spermatogonial cell lines as surrogates to examine cell survival, proliferation, steroidogenesis and mitochondrial integrity. The most promising compounds were further assessed on organ cultures of rat fetal and neonatal testes, corresponding to sensitive developmental windows. Dose-response studies revealed the toxicity of most maleates and fumarates, while identifying several dibenzoate and succinate plasticizers as innocuous on Leydig and germ cells. Interestingly, DINCH®, a plasticizer marketed as a safe alternative to phthalates, exerted a biphasic effect on steroid production in MA-10 and fetal Leydig cells. MEHP was the only plasticizer inducing the formation of multinucleated germ cells (MNG) in organ culture. Overall, organ cultures corroborated the cell line data, identifying one dibenzoate and one succinate as the most promising candidates. The adoption of such collaborative approaches for developing new chemicals should help prevent the development of compounds potentially harmful to human health.

  16. No evidence for intracellular magnetite in putative vertebrate magnetoreceptors identified by magnetic screening

    PubMed Central

    Edelman, Nathaniel B.; Fritz, Tanja; Nimpf, Simon; Pichler, Paul; Lauwers, Mattias; Hickman, Robert W.; Papadaki-Anastasopoulou, Artemis; Ushakova, Lyubov; Heuser, Thomas; Resch, Guenter P.; Saunders, Martin; Shaw, Jeremy A.; Keays, David A.

    2015-01-01

    The cellular basis of the magnetic sense remains an unsolved scientific mystery. One theory that aims to explain how animals detect the magnetic field is the magnetite hypothesis. It argues that intracellular crystals of the iron oxide magnetite (Fe3O4) are coupled to mechanosensitive channels that elicit neuronal activity in specialized sensory cells. Attempts to find these primary sensors have largely relied on the Prussian Blue stain that labels cells rich in ferric iron. This method has proved problematic as it has led investigators to conflate iron-rich macrophages with magnetoreceptors. An alternative approach developed by Eder et al. [Eder SH, et al. (2012) Proc Natl Acad Sci USA 109(30):12022–12027] is to identify candidate magnetoreceptive cells based on their magnetic moment. Here, we explore the utility of this method by undertaking a screen for magnetic cells in the pigeon. We report the identification of a small number of cells (1 in 476,000) with large magnetic moments (8–106 fAm2) from various tissues. The development of single-cell correlative light and electron microscopy (CLEM) coupled with electron energy loss spectroscopy (EELS) and energy-filtered transmission electron microscopy (EFTEM) permitted subcellular analysis of magnetic cells. This revealed the presence of extracellular structures composed of iron, titanium, and chromium accounting for the magnetic properties of these cells. Application of single-cell CLEM to magnetic cells from the trout failed to identify any intracellular structures consistent with biogenically derived magnetite. Our work illustrates the need for new methods to test the magnetite hypothesis of magnetosensation. PMID:25535350

  17. No evidence for intracellular magnetite in putative vertebrate magnetoreceptors identified by magnetic screening.

    PubMed

    Edelman, Nathaniel B; Fritz, Tanja; Nimpf, Simon; Pichler, Paul; Lauwers, Mattias; Hickman, Robert W; Papadaki-Anastasopoulou, Artemis; Ushakova, Lyubov; Heuser, Thomas; Resch, Guenter P; Saunders, Martin; Shaw, Jeremy A; Keays, David A

    2015-01-06

    The cellular basis of the magnetic sense remains an unsolved scientific mystery. One theory that aims to explain how animals detect the magnetic field is the magnetite hypothesis. It argues that intracellular crystals of the iron oxide magnetite (Fe3O4) are coupled to mechanosensitive channels that elicit neuronal activity in specialized sensory cells. Attempts to find these primary sensors have largely relied on the Prussian Blue stain that labels cells rich in ferric iron. This method has proved problematic as it has led investigators to conflate iron-rich macrophages with magnetoreceptors. An alternative approach developed by Eder et al. [Eder SH, et al. (2012) Proc Natl Acad Sci USA 109(30):12022-12027] is to identify candidate magnetoreceptive cells based on their magnetic moment. Here, we explore the utility of this method by undertaking a screen for magnetic cells in the pigeon. We report the identification of a small number of cells (1 in 476,000) with large magnetic moments (8-106 fAm(2)) from various tissues. The development of single-cell correlative light and electron microscopy (CLEM) coupled with electron energy loss spectroscopy (EELS) and energy-filtered transmission electron microscopy (EFTEM) permitted subcellular analysis of magnetic cells. This revealed the presence of extracellular structures composed of iron, titanium, and chromium accounting for the magnetic properties of these cells. Application of single-cell CLEM to magnetic cells from the trout failed to identify any intracellular structures consistent with biogenically derived magnetite. Our work illustrates the need for new methods to test the magnetite hypothesis of magnetosensation.

  18. Signalome-wide RNAi screen identifies GBA1 as a positive mediator of autophagic cell death

    PubMed Central

    Dasari, Santosh K; Bialik, Shani; Levin-Zaidman, Smadar; Levin-Salomon, Vered; Merrill, Alfred H; Futerman, Anthony H; Kimchi, Adi

    2017-01-01

    Activating alternative cell death pathways, including autophagic cell death, is a promising direction to overcome the apoptosis resistance observed in various cancers. Yet, whether autophagy acts as a death mechanism by over consumption of intracellular components is still controversial and remains undefined at the ultrastructural and the mechanistic levels. Here we identified conditions under which resveratrol-treated A549 lung cancer cells die by a mechanism that fulfills the previous definition of autophagic cell death. The cells displayed a strong and sustained induction of autophagic flux, cell death was prevented by knocking down autophagic genes and death occurred in the absence of apoptotic or necroptotic pathway activation. Detailed ultrastructural characterization revealed additional critical events, including a continuous increase over time in the number of autophagic vacuoles, in particular autolysosomes, occupying most of the cytoplasm at terminal stages. This was followed by loss of organelles, disruption of intracellular membranes including the swelling of perinuclear space and, occasionally, a unique type of nuclear shedding. A signalome-wide shRNA-based viability screen was applied to identify positive mediators of this type of autophagic cell death. One top hit was GBA1, the Gaucher disease-associated gene, which encodes glucocerebrosidase, an enzyme that metabolizes glucosylceramide to ceramide and glucose. Interestingly, glucocerebrosidase expression levels and activity were elevated, concomitantly with increased intracellular ceramide levels, both of which correlated in time with the appearance of the unique death characteristics. Transfection with siGBA1 attenuated the increase in glucocerebrosidase activity and the intracellular ceramide levels. Most importantly, GBA1 knockdown prevented the strong increase in LC3 lipidation, and many of the ultrastructural changes characteristic of this type of autophagic cell death, including a significant

  19. Hearing Screening Follow-Up: Completing the Process to Identify Hearing Health Needs

    ERIC Educational Resources Information Center

    Eiserman, William; Shisler, Lenore; Hoffman, Jeff

    2015-01-01

    Hearing is at the heart of language development and school readiness; increasing numbers of Early Head Start programs have come to rely on otoacoustic emissions (OAE) technology to screen all infants and toddlers for hearing loss. Successful identification of hearing health needs is dependent not only on an appropriate screening method, but also…

  20. Identifying Barriers to Colonoscopy Screening for Nonadherent African American Participants in a Patient Navigation Intervention

    ERIC Educational Resources Information Center

    Sly, Jamilia R.; Edwards, Tiffany; Shelton, Rachel C.; Jandorf, Lina

    2013-01-01

    African Americans have a higher rate of colorectal cancer (CRC) mortality than other racial/ethnic groups. This disparity is alarming given that CRC is largely preventable through the use of endoscopy (screening colonoscopy or sigmoidoscopy), yet rates of CRC screening among African Americans is suboptimal. Only 48.9% of African Americans are…

  1. Identifying Barriers to Colonoscopy Screening for Nonadherent African American Participants in a Patient Navigation Intervention

    ERIC Educational Resources Information Center

    Sly, Jamilia R.; Edwards, Tiffany; Shelton, Rachel C.; Jandorf, Lina

    2013-01-01

    African Americans have a higher rate of colorectal cancer (CRC) mortality than other racial/ethnic groups. This disparity is alarming given that CRC is largely preventable through the use of endoscopy (screening colonoscopy or sigmoidoscopy), yet rates of CRC screening among African Americans is suboptimal. Only 48.9% of African Americans are…

  2. Hearing Screening Follow-Up: Completing the Process to Identify Hearing Health Needs

    ERIC Educational Resources Information Center

    Eiserman, William; Shisler, Lenore; Hoffman, Jeff

    2015-01-01

    Hearing is at the heart of language development and school readiness; increasing numbers of Early Head Start programs have come to rely on otoacoustic emissions (OAE) technology to screen all infants and toddlers for hearing loss. Successful identification of hearing health needs is dependent not only on an appropriate screening method, but also…

  3. Phenotypic Screening Identifies Modulators of Amyloid Precursor Protein Processing in Human Stem Cell Models of Alzheimer's Disease.

    PubMed

    Brownjohn, Philip W; Smith, James; Portelius, Erik; Serneels, Lutgarde; Kvartsberg, Hlin; De Strooper, Bart; Blennow, Kaj; Zetterberg, Henrik; Livesey, Frederick J

    2017-03-06

    Human stem cell models have the potential to provide platforms for phenotypic screens to identify candidate treatments and cellular pathways involved in the pathogenesis of neurodegenerative disorders. Amyloid precursor protein (APP) processing and the accumulation of APP-derived amyloid β (Aβ) peptides are key processes in Alzheimer's disease (AD). We designed a phenotypic small-molecule screen to identify modulators of APP processing in trisomy 21/Down syndrome neurons, a complex genetic model of AD. We identified the avermectins, commonly used as anthelmintics, as compounds that increase the relative production of short Aβ peptides at the expense of longer, potentially more toxic peptides. Further studies demonstrated that this effect is not due to an interaction with the core γ-secretase responsible for Aβ production. This study demonstrates the feasibility of phenotypic drug screening in human stem cell models of Alzheimer-type dementia, and points to possibilities for indirectly modulating APP processing, independently of γ-secretase modulation.

  4. Efficacy of ACL injury risk screening methods in identifying high-risk landing patterns during a sport-specific task.

    PubMed

    Fox, A S; Bonacci, J; McLean, S G; Saunders, N

    2016-06-12

    Screening methods sensitive to movement strategies that increase anterior cruciate ligament (ACL) loads are likely to be effective in identifying athletes at-risk of ACL injury. Current ACL injury risk screening methods are yet to be evaluated for their ability to identify athletes' who exhibit high-risk lower limb mechanics during sport-specific maneuvers associated with ACL injury occurrences. The purpose of this study was to examine the efficacy of two ACL injury risk screening methods in identifying high-risk lower limb mechanics during a sport-specific landing task. Thirty-two female athletes were screened using the Landing Error Scoring System (LESS) and Tuck Jump Assessment. Participants' also completed a sport-specific landing task, during which three-dimensional kinematic and kinetic data were collected. One-dimensional statistical parametric mapping was used to examine the relationships between screening method scores, and the three-dimensional hip and knee joint rotation and moment data from the sport-specific landing. Higher LESS scores were associated with reduced knee flexion from 30 to 57 ms after initial contact (P = 0.003) during the sport-specific landing; however, no additional relationships were found. These findings suggest the LESS and Tuck Jump Assessment may have minimal applicability in identifying athletes' who exhibit high-risk landing postures in the sport-specific task examined.

  5. Combined rational design and a high throughput screening platform for identifying chemical inhibitors of a Ras-activating enzyme.

    PubMed

    Evelyn, Chris R; Biesiada, Jacek; Duan, Xin; Tang, Hong; Shang, Xun; Papoian, Ruben; Seibel, William L; Nelson, Sandra; Meller, Jaroslaw; Zheng, Yi

    2015-05-15

    The Ras family small GTPases regulate multiple cellular processes, including cell growth, survival, movement, and gene expression, and are intimately involved in cancer pathogenesis. Activation of these small GTPases is catalyzed by a special class of enzymes, termed guanine nucleotide exchange factors (GEFs). Herein, we developed a small molecule screening platform for identifying lead hits targeting a Ras GEF enzyme, SOS1. We employed an ensemble structure-based virtual screening approach in combination with a multiple tier high throughput experimental screen utilizing two complementary fluorescent guanine nucleotide exchange assays to identify small molecule inhibitors of GEF catalytic activity toward Ras. From a library of 350,000 compounds, we selected a set of 418 candidate compounds predicted to disrupt the GEF-Ras interaction, of which dual wavelength GDP dissociation and GTP-loading experimental screening identified two chemically distinct small molecule inhibitors. Subsequent biochemical validations indicate that they are capable of dose-dependently inhibiting GEF catalytic activity, binding to SOS1 with micromolar affinity, and disrupting GEF-Ras interaction. Mutagenesis studies in conjunction with structure-activity relationship studies mapped both compounds to different sites in the catalytic pocket, and both inhibited Ras signaling in cells. The unique screening platform established here for targeting Ras GEF enzymes could be broadly useful for identifying lead inhibitors for a variety of small GTPase-activating GEF reactions.

  6. Combined Rational Design and a High Throughput Screening Platform for Identifying Chemical Inhibitors of a Ras-activating Enzyme*

    PubMed Central

    Evelyn, Chris R.; Biesiada, Jacek; Duan, Xin; Tang, Hong; Shang, Xun; Papoian, Ruben; Seibel, William L.; Nelson, Sandra; Meller, Jaroslaw; Zheng, Yi

    2015-01-01

    The Ras family small GTPases regulate multiple cellular processes, including cell growth, survival, movement, and gene expression, and are intimately involved in cancer pathogenesis. Activation of these small GTPases is catalyzed by a special class of enzymes, termed guanine nucleotide exchange factors (GEFs). Herein, we developed a small molecule screening platform for identifying lead hits targeting a Ras GEF enzyme, SOS1. We employed an ensemble structure-based virtual screening approach in combination with a multiple tier high throughput experimental screen utilizing two complementary fluorescent guanine nucleotide exchange assays to identify small molecule inhibitors of GEF catalytic activity toward Ras. From a library of 350,000 compounds, we selected a set of 418 candidate compounds predicted to disrupt the GEF-Ras interaction, of which dual wavelength GDP dissociation and GTP-loading experimental screening identified two chemically distinct small molecule inhibitors. Subsequent biochemical validations indicate that they are capable of dose-dependently inhibiting GEF catalytic activity, binding to SOS1 with micromolar affinity, and disrupting GEF-Ras interaction. Mutagenesis studies in conjunction with structure-activity relationship studies mapped both compounds to different sites in the catalytic pocket, and both inhibited Ras signaling in cells. The unique screening platform established here for targeting Ras GEF enzymes could be broadly useful for identifying lead inhibitors for a variety of small GTPase-activating GEF reactions. PMID:25825487

  7. Structure- and ligand-based virtual screening identifies new scaffolds for inhibitors of the oncoprotein MDM2.

    PubMed

    Houston, Douglas R; Yen, Li-Hsuan; Pettit, Simon; Walkinshaw, Malcolm D

    2015-01-01

    A major challenge in the field of ligand discovery is to identify chemically useful fragments that can be developed into inhibitors of specific protein-protein interactions. Low molecular weight fragments (with molecular weight less than 250 Da) are likely to bind weakly to a protein's surface. Here we use a new virtual screening procedure which uses a combination of similarity searching and docking to identify chemically tractable scaffolds that bind to the p53-interaction site of MDM2. The binding has been verified using capillary electrophoresis which has proven to be an excellent screening method for such small, weakly binding ligands.

  8. High Throughput Screening Identifies Two Classes of Antibiotics as Radioprotectors: Tetracyclines and Fluoroquinolones

    PubMed Central

    Kim, Kwanghee; Pollard, Julianne M.; Norris, Andrew J.; McDonald, J. Tyson; Sun, Yingli; Micewicz, Ewa; Pettijohn, Kelly; Damoiseaux, Robert; Iwamoto, Keisuke S.; Sayre, James W.; Price, Brendan D.; Gatti, Richard A.; McBride, William H.

    2009-01-01

    Purpose Discovery of agents that protect or mitigate normal tissue from radiation injury during radiotherapy, accidents, or terrorist attacks, is of importance. Specifically, bone marrow insufficiency, with possible infection due to immune suppression, can occur after total body irradiation (TBI) or regional irradiation and is a major component of the acute radiation syndrome. The purpose of this study was to identify novel radioprotectors and mitigators of the hematopoietic system. Experimental Design High throughput screening of small molecule libraries was performed using viability of a murine lymphocyte line as a read out with further validation in human lymphoblastoid cells. The selected compounds were then tested for their ability to counter TBI lethality in mice. Results All of two major classes of antibiotics, tetracyclines and fluoroquinolones, which share a common planar ring moiety, were radioprotective. Furthermore, tetracycline protected murine hematopoietic stem/progenitor cell populations from radiation damage and allowed 87.5% of mice to survive when given before and 35% when given 24 h after lethal TBI. Interestingly, tetracycline did not alter the radiosensitivity of Lewis lung cancer cells. Tetracycline and ciprofloxacine also protected human lymphoblastoid cells, reducing radiation-induced DNA double strand breaks by 33% and 21%, respectively. The effects of these agents on radiation lethality are not due to the classical mechanism of free radical scavenging but potentially through activation of the histone acetyl transferase Tip60 and altered chromatin structure. Conclusions Tetracyclines and fluoroquinolones can be robust radioprotectors and mitigators of the hematopoietic system with potential utility in anti-cancer radiotherapy and radiation emergencies. PMID:19920105

  9. High Throughput Screening against the Peroxidase Cascade of African Trypanosomes Identifies Antiparasitic Compounds That Inactivate Tryparedoxin*

    PubMed Central

    Fueller, Florian; Jehle, Britta; Putzker, Kerstin; Lewis, Joe D.; Krauth-Siegel, R. Luise

    2012-01-01

    In African trypanosomes, the detoxification of broad spectrum hydroperoxides relies on a unique cascade composed of trypanothione (T(SH)2), trypanothione reductase, tryparedoxin (Tpx), and nonselenium glutathione peroxidase-type enzymes. All three proteins are essential for Trypanosoma brucei. Here, we subjected the complete system to a high throughput screening approach with nearly 80,000 chemicals. Twelve compounds inhibited the peroxidase system. All but one carried chloroalkyl substituents. The detailed kinetic analysis showed that two compounds weakly inhibited trypanothione reductase, but none of them specifically interacted with the peroxidase. They proved to be time-dependent inhibitors of Tpx-modifying Cys-40, the first cysteine of its active site WCPPC motif. Importantly, gel shift assays verified Tpx as a target in the intact parasites. T(SH)2, present in the in vitro assays and in the cells in high molar excess, did not interfere with Tpx inactivation. The compounds inhibited the proliferation of bloodstream T. brucei with EC50 values down to <1 μm and exerted up to 83-fold lower toxicity toward HeLa cells. Irreversible inhibitors are traditionally regarded as unfavorable. However, a large number of antimicrobials and anticancer therapeutics acts covalently with their target protein. The compounds identified here also interacted with recombinant human thioredoxin, a distant relative of Tpx. This finding might even be exploited for thioredoxin-based anticancer drug development approaches reported recently. The fact that the T(SH)2/Tpx couple occupies a central position within the trypanosomal thiol metabolism and delivers electrons also for the synthesis of DNA precursors renders the parasite-specific oxidoreductase an attractive drug target molecule. PMID:22275351

  10. Drug screening identifies niclosamide as an inhibitor of breast cancer stem-like cells.

    PubMed

    Wang, Yu-Chi; Chao, Tai-Kuang; Chang, Cheng-Chang; Yo, Yi-Te; Yu, Mu-Hsien; Lai, Hung-Cheng

    2013-01-01

    The primary cause of death from breast cancer is the progressive growth of tumors and resistance to conventional therapies. It is currently believed that recurrent cancer is repopulated according to a recently proposed cancer stem cell hypothesis. New therapeutic strategies that specifically target cancer stem-like cells may represent a new avenue of cancer therapy. We aimed to discover novel compounds that target breast cancer stem-like cells. We used a dye-exclusion method to isolate side population (SP) cancer cells and, subsequently, subjected these SP cells to a sphere formation assay to generate SP spheres (SPS) from breast cancer cell lines. Surface markers, stemness genes, and tumorigenicity were used to test stem properties. We performed a high-throughput drug screening using these SPS. The effects of candidate compounds were assessed in vitro and in vivo. We successfully generated breast cancer SPS with stem-like properties. These SPS were enriched for CD44(high) (2.8-fold) and CD24(low) (4-fold) cells. OCT4 and ABCG2 were overexpressed in SPS. Moreover, SPS grew tumors at a density of 10(3), whereas an equivalent number of parental cells did not initiate tumor formation. A clinically approved drug, niclosamide, was identified from the LOPAC chemical library of 1,258 compounds. Niclosamide downregulated stem pathways, inhibited the formation of spheroids, and induced apoptosis in breast cancer SPS. Animal studies also confirmed this therapeutic effect. The results of this proof-of-principle study may facilitate the development of new breast cancer therapies in the near future. The extension of niclosamide clinical trials is warranted.

  11. A systems-wide screen identifies substrates of the SCFβTrCP ubiquitin ligase.

    PubMed

    Low, Teck Yew; Peng, Mao; Magliozzi, Roberto; Mohammed, Shabaz; Guardavaccaro, Daniele; Heck, Albert J R

    2014-12-16

    Cellular proteins are degraded by the ubiquitin-proteasome system (UPS) in a precise and timely fashion. Such precision is conferred by the high substrate specificity of ubiquitin ligases. Identification of substrates of ubiquitin ligases is crucial not only to unravel the molecular mechanisms by which the UPS controls protein degradation but also for drug discovery purposes because many established UPS substrates are implicated in disease. We developed a combined bioinformatics and affinity purification-mass spectrometry (AP-MS) workflow for the system-wide identification of substrates of SCF(βTrCP), a member of the SCF family of ubiquitin ligases. These ubiquitin ligases are characterized by a multisubunit architecture typically consisting of the invariable subunits Rbx1, Cul1, and Skp1 and one of 69 F-box proteins. The F-box protein of this member of the family is βTrCP. SCF(βTrCP) binds, through the WD40 repeats of βTrCP, to the DpSGXX(X)pS diphosphorylated motif in its substrates. We recovered 27 previously reported SCF(βTrCP) substrates, of which 22 were verified by two independent statistical protocols, thereby confirming the reliability of this approach. In addition to known substrates, we identified 221 proteins that contained the DpSGXX(X)pS motif and also interacted specifically with the WD40 repeats of βTrCP. Thus, with SCF(βTrCP), as the example, we showed that integration of structural information, AP-MS, and degron motif mining constitutes an effective method to screen for substrates of ubiquitin ligases.

  12. Mutagenesis and behavioral screening for altered circadian activity identifies the mouse mutant, Wheels.

    PubMed

    Pickard, G E; Sollars, P J; Rinchik, E M; Nolan, P M; Bucan, M

    1995-12-24

    The molecular processes underlying the generation of circadian behavior in mammals are virtually unknown. To identify genes that regulate or alter circadian activity rhythms, a mouse mutagenesis program was initiated in conjunction with behavioral screening for alterations in circadian period (tau), a fundamental property of the biological clock. Male mice of the inbred BALB/c strain, treated with the potent mutagen N-ethyl-N-nitrosourea were mated with wild-type hybrids. Wheel-running activity of approximately 300 male progeny was monitored for 6-10 weeks under constant dark (DD) conditions. The tau DD of a single mouse (#187) was longer than the population mean by more than three standard deviations (24.20 vs. 23.32 +/- 0.02 h; mean +/- S.E.M.; n = 277). In addition, mouse #187 exhibited other abnormal phenotypes, including hyperactive bi-directional circling/spinning activity and an abnormal response to light. Heterozygous progeny of the founder mouse, generated from outcrossings with wild-type C57BL/6J mice, displayed lengthened tau DD although approximately 20% of the animals showed no wheel-running activity despite being quite active. Under light:dark conditions, all animals displaying circling behavior that ran in the activity wheels exhibited robust wheel-running activity at lights-ON and these animals also showed enhanced wheel-running activity in constant light conditions. The genetic dissection of the complex behavior associated with this mutation was facilitated by the previously described genetic mapping of the mutant locus causing circling behavior, designated Wheels (Whl), to the subcentromeric portion of mouse chromosome 4. In this report, the same locus is shown to be responsible for the abnormal responses to light and presumably for the altered circadian behavior. Characterization of the gene altered in the novel Whl mutation will contribute to understanding the molecular elements involved in mammalian circadian regulation.

  13. High throughput screening against the peroxidase cascade of African trypanosomes identifies antiparasitic compounds that inactivate tryparedoxin.

    PubMed

    Fueller, Florian; Jehle, Britta; Putzker, Kerstin; Lewis, Joe D; Krauth-Siegel, R Luise

    2012-03-16

    In African trypanosomes, the detoxification of broad spectrum hydroperoxides relies on a unique cascade composed of trypanothione (T(SH)(2)), trypanothione reductase, tryparedoxin (Tpx), and nonselenium glutathione peroxidase-type enzymes. All three proteins are essential for Trypanosoma brucei. Here, we subjected the complete system to a high throughput screening approach with nearly 80,000 chemicals. Twelve compounds inhibited the peroxidase system. All but one carried chloroalkyl substituents. The detailed kinetic analysis showed that two compounds weakly inhibited trypanothione reductase, but none of them specifically interacted with the peroxidase. They proved to be time-dependent inhibitors of Tpx-modifying Cys-40, the first cysteine of its active site WCPPC motif. Importantly, gel shift assays verified Tpx as a target in the intact parasites. T(SH)(2), present in the in vitro assays and in the cells in high molar excess, did not interfere with Tpx inactivation. The compounds inhibited the proliferation of bloodstream T. brucei with EC(50) values down to <1 μM and exerted up to 83-fold lower toxicity toward HeLa cells. Irreversible inhibitors are traditionally regarded as unfavorable. However, a large number of antimicrobials and anticancer therapeutics acts covalently with their target protein. The compounds identified here also interacted with recombinant human thioredoxin, a distant relative of Tpx. This finding might even be exploited for thioredoxin-based anticancer drug development approaches reported recently. The fact that the T(SH)(2)/Tpx couple occupies a central position within the trypanosomal thiol metabolism and delivers electrons also for the synthesis of DNA precursors renders the parasite-specific oxidoreductase an attractive drug target molecule.

  14. High throughput Screening to Identify Natural Human Monoamine Oxidase B Inhibitors

    PubMed Central

    Mazzio, E; Deiab, S; Park, K; Soliman, KFA

    2012-01-01

    Age-related increase in monoamine oxidase B (MAO-B) may contribute to CNS neurodegenerative diseases. Moreover, MAO-B inhibitors are used in the treatment of idiopathic Parkinson disease as preliminary monotherapy or adjunct therapy with L-dopa. To date, meager natural sources of MAO-B inhibitors have been identified, and the relative strength, potency and rank of many plants relative to standard drugs such as Selegiline (L-deprenyl, Eldepryl) are not known. In this work, we developed and utilized a high throughput enzyme microarray format to screen and evaluate 905 natural product extracts (0.025–.7 mg/ml) to inhibit human MAO-B derived from BTI-TN-5B1-4 cells infected with recombinant baculovirus. The protein sequence of purified enzyme was confirmed using 1D gel electrophoresis-matrix assisted laser desorption ionization-time-of-flight-tandem mass spectroscopy, and enzyme activity was confirmed by [1] substrate conversion (3-mM benzylamine) to H202 and [2] benzaldehyde. Of the 905 natural extracts tested, the lowest IC50s [<0.07 mg/ml] were obtained with extracts of Amur Corktree (Phellodendron amurense), Bakuchi Seed(Cyamopsis psoralioides), Licorice Root (Glycyrrhiza glabra/uralensis), Babchi (Psoralea corylifolia seed). The data also show, albeit to a lesser extent, inhibitory properties of herbs originating from the mint family (Lamiaceae) and Turmeric, Comfrey, Bringraj, Skullcap, Kava-kava, Wild Indigo, Gentian and Green Tea. In conclusion, the data reflect relative potency information by rank of commonly used herbs and plants that contain human MAO-B inhibitory properties in their natural form. PMID:22887993

  15. A high throughput pharmaceutical screen identifies compounds with specific toxicity against BRCA2-deficient tumors

    PubMed Central

    Evers, Bastiaan; Schut, Eva; van der Burg, Eline; Braumuller, Tanya M.; Egan, David A.; Holstege, Henne; Edser, Pauline; Adams, David J.; Wade-Martins, Richard; Bouwman, Peter; Jonkers, Jos

    2009-01-01

    Purpose: Hereditary breast cancer is partly explained by germline mutations in BRCA1 and BRCA2. While patients carry heterozygous mutations, their tumors have typically lost the remaining wild-type allele. Selectively targeting BRCA-deficiency may therefore constitute an important therapeutic approach. Clinical trials applying this principle are underway, but it is unknown whether the compounds tested are optimal. It is therefore important to identify alternative compounds that specifically target BRCA-deficiency and to test new combination therapies to establish optimal treatment strategies. Experimental Design: We performed a high-throughput pharmaceutical screen on BRCA2-deficient mouse mammary tumor cells and isogenic controls with restored BRCA2 function. Subsequently, we validated positive hits in vitro and in vivo using mice carrying BRCA2-deficient mammary tumors. Results: Three alkylators – chlorambucil, melphalan and nimustine – displayed strong and specific toxicity against BRCA2-deficient cells. In vivo, these showed heterogeneous but generally strong BRCA2-deficient antitumor activity, with melphalan and nimustine outperforming cisplatin and the poly-(ADP-ribose)-polymerase (PARP) inhibitor olaparib (AZD2281) in this small study. In vitro drug combination experiments showed synergistic interactions between the alkylators and olaparib. Tumor intervention studies combining nimustine and olaparib resulted in recurrence-free survival exceeding 330 days in 3 out of 5 animals tested. Conclusions: We generated and validated a platform for identification of compounds with specific activity against BRCA2-deficient cells that translates well to the preclinical setting. Our data call for the re-evaluation of alkylators – especially melphalan and nimustine – alone or in combination with PARP inhibitors for the treatment of breast cancers with a defective BRCA pathway. PMID:20008842

  16. Large-Scale Screening of a Targeted Enterococcus faecalis Mutant Library Identifies Envelope Fitness Factors

    PubMed Central

    Rigottier-Gois, Lionel; Alberti, Adriana; Houel, Armel; Taly, Jean-François; Palcy, Philippe; Manson, Janet; Pinto, Daniela; Matos, Renata C.; Carrilero, Laura; Montero, Natalia; Tariq, Muhammad; Karsens, Harma; Repp, Christian; Kropec, Andrea; Budin-Verneuil, Aurélie; Benachour, Abdellah; Sauvageot, Nicolas; Bizzini, Alain; Gilmore, Michael S.; Bessières, Philippe; Kok, Jan; Huebner, Johannes; Lopes, Fatima; Gonzalez-Zorn, Bruno; Hartke, Axel; Serror, Pascale

    2011-01-01

    Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% Gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence). For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i) resistance to oxidative stress, ii) antibiotic resistance, iii) resistance to opsonophagocytosis, iv) adherence to the human colon carcinoma Caco-2 epithelial cells and v) virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence. PMID:22194979

  17. Genome-wide RNAi screening identifies protein damage as a regulator of osmoprotective gene expression

    PubMed Central

    Lamitina, Todd; Huang, Chunyi George; Strange, Kevin

    2006-01-01

    The detection, stabilization, and repair of stress-induced damage are essential requirements for cellular life. All cells respond to osmotic stress-induced water loss with increased expression of genes that mediate accumulation of organic osmolytes, solutes that function as chemical chaperones and restore osmotic homeostasis. The signals and signaling mechanisms that regulate osmoprotective gene expression in animal cells are poorly understood. Here, we show that gpdh-1 and gpdh-2, genes that mediate the accumulation of the organic osmolyte glycerol, are essential for survival of the nematode Caenorhabditis elegans during osmotic stress. Expression of GFP driven by the gpdh-1 promoter (Pgpdh-1::GFP) is detected only during hypertonic stress but is not induced by other stressors. Using Pgpdh-1::GFP expression as a phenotype, we screened ≈16,000 genes by RNAi feeding and identified 122 that cause constitutive activation of gpdh-1 expression and glycerol accumulation. Many of these genes function to regulate protein translation and cotranslational protein folding and to target and degrade denatured proteins, suggesting that the accumulation of misfolded proteins functions as a signal to activate osmoprotective gene expression and organic osmolyte accumulation in animal cells. Consistent with this hypothesis, 73% of these protein-homeostasis genes have been shown to slow age-dependent protein aggregation in C. elegans. Because diverse environmental stressors and numerous disease states result in protein misfolding, mechanisms must exist that discriminate between osmotically induced and other forms of stress-induced protein damage. Our findings provide a foundation for understanding how these damage-selectivity mechanisms function. PMID:16880390

  18. The challenge of small lung nodules identified in CT screening: can biomarkers assist diagnosis?

    PubMed

    Veronesi, Giulia; Bianchi, Fabrizio; Infante, Maurizio; Alloisio, Marco

    2016-01-01

    Various biomarkers have been developed as noninvasive tests to indicate the presence of lung cancer in asymptomatic persons, and in particular to provide evidence as to whether indeterminate lung nodules detected by screening are malignant. We performed an overview of the range of biomarkers reported in the literature and described those that can complement low-dose computed tomography screening. Several have promising sensitivity and specificity. However to our knowledge, only three techniques have reached the prospective screening phase (phase 4) of the five-phase biomarker development process. Two miRNA signatures (the miR-Test for serum and the miRNA signature classifier test for plasma) are being assessed in prospective screening trials, as is the EarlyCDT-Lung test based on autoantibodies. All will need to undergo prospective studies to determine their ability to improve outcomes before they can become an established adjunct to lung cancer control strategies.

  19. Evaluation of a Community Pharmacy-Based Screening Questionnaire to Identify Patients at Risk for Drug Therapy Problems.

    PubMed

    Pammett, Robert T; Blackburn, David; Taylor, Jeff; Mansell, Kerry; Kwan, Debbie; Papoushek, Christine; Jorgenson, Derek

    2015-09-01

    To determine if a short screening questionnaire can identify patients at risk for drug therapy problems (DTPs) in a community pharmacy setting. Self-administered questionnaire. Three community pharmacies in Saskatoon, Canada. Forty-nine adults who were picking up a refill prescription for a medication that had remained stable over the past 6 months (i.e., no changes to drug, dose, or regimen) during 4 consecutive weeks at each of the three pharmacies between November 2013 and February 2014. All patients completed a self-administered screening questionnaire and underwent a blinded comprehensive medication assessment with a clinical pharmacist. Agreement between the screening questionnaire responses and responses based on information from the medication assessment were assessed with Cohen's κ coefficient. The DTPs identified during the medication assessments were categorized in one of the eight standard DTP categories: unnecessary drug therapy, inappropriate drug, subtherapeutic dose, supratherapeutic dose, drug therapy required, adverse drug reaction, noncompliance, and other or unsure. The DTPs were also assigned a severity-mild, moderate, or severe-using adapted Schneider criteria. The number and severity of DTPs identified were compared among patients categorized as high versus low risk for DTPs as determined by the questionnaire responses. Of the 49 patients who completed the study, 18 (37%) were high risk and 31 (63%) low risk. The agreement between risk categorization based on the screening questionnaire and medication assessment was very good (κ = 0.91, p<0.01). Also, patients identified as high risk on the screening questionnaire had a mean of 3.7 (p<0.01) more DTPs than low-risk patients. Seventeen (94%) of the 18 high-risk patients had at least one moderate or severe DTP compared with 15 (48%) of the 31 low-risk patients. The screening questionnaire was a reliable method for identifying patients in community pharmacies who have a large number of DTPs

  20. Automated NMR fragment based screening identified a novel interface blocker to the LARG/RhoA complex.

    PubMed

    Gao, Jia; Ma, Rongsheng; Wang, Wei; Wang, Na; Sasaki, Ryan; Snyderman, David; Wu, Jihui; Ruan, Ke

    2014-01-01

    The small GTPase cycles between the inactive GDP form and the activated GTP form, catalyzed by the upstream guanine exchange factors. The modulation of such process by small molecules has been proven to be a fruitful route for therapeutic intervention to prevent the over-activation of the small GTPase. The fragment based approach emerging in the past decade has demonstrated its paramount potential in the discovery of inhibitors targeting such novel and challenging protein-protein interactions. The details regarding the procedure of NMR fragment screening from scratch have been rarely disclosed comprehensively, thus restricts its wider applications. To achieve a consistent screening applicable to a number of targets, we developed a highly automated protocol to cover every aspect of NMR fragment screening as possible, including the construction of small but diverse libray, determination of the aqueous solubility by NMR, grouping compounds with mutual dispersity to a cocktail, and the automated processing and visualization of the ligand based screening spectra. We exemplified our streamlined screening in RhoA alone and the complex of the small GTPase RhoA and its upstream guanine exchange factor LARG. Two hits were confirmed from the primary screening in cocktail and secondary screening over individual hits for LARG/RhoA complex, while one of them was also identified from the screening for RhoA alone. HSQC titration of the two hits over RhoA and LARG alone, respectively, identified one compound binding to RhoA.GDP at a 0.11 mM affinity, and perturbed the residues at the switch II region of RhoA. This hit blocked the formation of the LARG/RhoA complex, validated by the native gel electrophoresis, and the titration of RhoA to ¹⁵N labeled LARG in the absence and presence the compound, respectively. It therefore provides us a starting point toward a more potent inhibitor to RhoA activation catalyzed by LARG.

  1. Automated NMR Fragment Based Screening Identified a Novel Interface Blocker to the LARG/RhoA Complex

    PubMed Central

    Gao, Jia; Ma, Rongsheng; Wang, Wei; Wang, Na; Sasaki, Ryan; Snyderman, David; Wu, Jihui; Ruan, Ke

    2014-01-01

    The small GTPase cycles between the inactive GDP form and the activated GTP form, catalyzed by the upstream guanine exchange factors. The modulation of such process by small molecules has been proven to be a fruitful route for therapeutic intervention to prevent the over-activation of the small GTPase. The fragment based approach emerging in the past decade has demonstrated its paramount potential in the discovery of inhibitors targeting such novel and challenging protein-protein interactions. The details regarding the procedure of NMR fragment screening from scratch have been rarely disclosed comprehensively, thus restricts its wider applications. To achieve a consistent screening applicable to a number of targets, we developed a highly automated protocol to cover every aspect of NMR fragment screening as possible, including the construction of small but diverse libray, determination of the aqueous solubility by NMR, grouping compounds with mutual dispersity to a cocktail, and the automated processing and visualization of the ligand based screening spectra. We exemplified our streamlined screening in RhoA alone and the complex of the small GTPase RhoA and its upstream guanine exchange factor LARG. Two hits were confirmed from the primary screening in cocktail and secondary screening over individual hits for LARG/RhoA complex, while one of them was also identified from the screening for RhoA alone. HSQC titration of the two hits over RhoA and LARG alone, respectively, identified one compound binding to RhoA.GDP at a 0.11 mM affinity, and perturbed the residues at the switch II region of RhoA. This hit blocked the formation of the LARG/RhoA complex, validated by the native gel electrophoresis, and the titration of RhoA to 15N labeled LARG in the absence and presence the compound, respectively. It therefore provides us a starting point toward a more potent inhibitor to RhoA activation catalyzed by LARG. PMID:24505392

  2. Wide screening of phage-displayed libraries identifies immune targets in planta.

    PubMed

    Rioja, Cristina; Van Wees, Saskia C; Charlton, Keith A; Pieterse, Corné M J; Lorenzo, Oscar; García-Sánchez, Susana

    2013-01-01

    Microbe-Associated Molecular Patterns and virulence effectors are recognized by plants as a first step to mount a defence response against potential pathogens. This recognition involves a large family of extracellular membrane receptors and other immune proteins located in different sub-cellular compartments. We have used phage-display technology to express and select for Arabidopsis proteins able to bind bacterial pathogens. To rapidly identify microbe-bound phage, we developed a monitoring method based on microarrays. This combined strategy allowed for a genome-wide screening of plant proteins involved in pathogen perception. Two phage libraries for high-throughput selection were constructed from cDNA of plants infected with Pseudomonas aeruginosa PA14, or from combined samples of the virulent isolate DC3000 of Pseudomonas syringae pv. tomato and its avirulent variant avrRpt2. These three pathosystems represent different degrees in the specificity of plant-microbe interactions. Libraries cover up to 2 × 10(7) different plant transcripts that can be displayed as functional proteins on the surface of T7 bacteriophage. A number of these were selected in a bio-panning assay for binding to Pseudomonas cells. Among the selected clones we isolated the ethylene response factor ATERF-1, which was able to bind the three bacterial strains in competition assays. ATERF-1 was rapidly exported from the nucleus upon infiltration of either alive or heat-killed Pseudomonas. Moreover, aterf-1 mutants exhibited enhanced susceptibility to infection. These findings suggest that ATERF-1 contains a microbe-recognition domain with a role in plant defence. To identify other putative pathogen-binding proteins on a genome-wide scale, the copy number of selected-vs.-total clones was compared by hybridizing phage cDNAs with Arabidopsis microarrays. Microarray analysis revealed a set of 472 candidates with significant fold change. Within this set defence-related genes, including well

  3. Wide Screening of Phage-Displayed Libraries Identifies Immune Targets in Planta

    PubMed Central

    Rioja, Cristina; Van Wees, Saskia C.; Charlton, Keith A.; Pieterse, Corné M. J.; Lorenzo, Oscar; García-Sánchez, Susana

    2013-01-01

    Microbe-Associated Molecular Patterns and virulence effectors are recognized by plants as a first step to mount a defence response against potential pathogens. This recognition involves a large family of extracellular membrane receptors and other immune proteins located in different sub-cellular compartments. We have used phage-display technology to express and select for Arabidopsis proteins able to bind bacterial pathogens. To rapidly identify microbe-bound phage, we developed a monitoring method based on microarrays. This combined strategy allowed for a genome-wide screening of plant proteins involved in pathogen perception. Two phage libraries for high-throughput selection were constructed from cDNA of plants infected with Pseudomonas aeruginosa PA14, or from combined samples of the virulent isolate DC3000 of Pseudomonas syringae pv. tomato and its avirulent variant avrRpt2. These three pathosystems represent different degrees in the specificity of plant-microbe interactions. Libraries cover up to 2×107 different plant transcripts that can be displayed as functional proteins on the surface of T7 bacteriophage. A number of these were selected in a bio-panning assay for binding to Pseudomonas cells. Among the selected clones we isolated the ethylene response factor ATERF-1, which was able to bind the three bacterial strains in competition assays. ATERF-1 was rapidly exported from the nucleus upon infiltration of either alive or heat-killed Pseudomonas. Moreover, aterf-1 mutants exhibited enhanced susceptibility to infection. These findings suggest that ATERF-1 contains a microbe-recognition domain with a role in plant defence. To identify other putative pathogen-binding proteins on a genome-wide scale, the copy number of selected-vs.-total clones was compared by hybridizing phage cDNAs with Arabidopsis microarrays. Microarray analysis revealed a set of 472 candidates with significant fold change. Within this set defence-related genes, including well

  4. A Synthetic Lethal Screen Identifies a Role for Lin-44/Wnt in C. elegans Embryogenesis

    PubMed Central

    Hartin, Samantha N.; Hudson, Martin L.; Yingling, Curtis; Ackley, Brian D.

    2015-01-01

    Background The C. elegans proteins PTP-3/LAR-RPTP and SDN-1/Syndecan are conserved cell adhesion molecules. Loss-of-function (LOF) mutations in either ptp-3 or sdn-1 result in low penetrance embryonic developmental defects. Work from other systems has shown that syndecans can function as ligands for LAR receptors in vivo. We used double mutant analysis to test whether ptp-3 and sdn-1 function in a linear genetic pathway during C. elegans embryogenesis. Results We found animals with LOF in both sdn-1 and ptp-3 exhibited a highly penetrant synthetic lethality (SynLet), with only a small percentage of animals surviving to adulthood. Analysis of the survivors demonstrated that these animals had a synergistic increase in the penetrance of embryonic developmental defects. Together, these data strongly suggested PTP-3 and SDN-1 function in parallel during embryogenesis. We subsequently used RNAi to knockdown ~3,600 genes predicted to encode secreted and/or transmembrane molecules to identify genes that interacted with ptp-3 or sdn-1. We found that the Wnt ligand, lin-44, was SynLet with sdn-1, but not ptp-3. We used 4-dimensional time-lapse analysis to characterize the interaction between lin-44 and sdn-1. We found evidence that loss of lin-44 caused defects in the polarization and migration of endodermal precursors during gastrulation, a previously undescribed role for lin-44 that is strongly enhanced by the loss of sdn-1. Conclusions PTP-3 and SDN-1 function in compensatory pathways during C. elegans embryonic and larval development, as simultaneous loss of both genes has dire consequences for organismal survival. The Wnt ligand lin-44 contributes to the early stages of gastrulation in parallel to sdn-1, but in a genetic pathway with ptp-3. Overall, the SynLet phenotype provides a robust platform to identify ptp-3 and sdn-1 interacting genes, as well as other genes that function in development, yet might be missed in traditional forward genetic screens. PMID:25938228

  5. A Synthetic Lethal Screen Identifies a Role for Lin-44/Wnt in C. elegans Embryogenesis.

    PubMed

    Hartin, Samantha N; Hudson, Martin L; Yingling, Curtis; Ackley, Brian D

    2015-01-01

    The C. elegans proteins PTP-3/LAR-RPTP and SDN-1/Syndecan are conserved cell adhesion molecules. Loss-of-function (LOF) mutations in either ptp-3 or sdn-1 result in low penetrance embryonic developmental defects. Work from other systems has shown that syndecans can function as ligands for LAR receptors in vivo. We used double mutant analysis to test whether ptp-3 and sdn-1 function in a linear genetic pathway during C. elegans embryogenesis. We found animals with LOF in both sdn-1 and ptp-3 exhibited a highly penetrant synthetic lethality (SynLet), with only a small percentage of animals surviving to adulthood. Analysis of the survivors demonstrated that these animals had a synergistic increase in the penetrance of embryonic developmental defects. Together, these data strongly suggested PTP-3 and SDN-1 function in parallel during embryogenesis. We subsequently used RNAi to knockdown ~3,600 genes predicted to encode secreted and/or transmembrane molecules to identify genes that interacted with ptp-3 or sdn-1. We found that the Wnt ligand, lin-44, was SynLet with sdn-1, but not ptp-3. We used 4-dimensional time-lapse analysis to characterize the interaction between lin-44 and sdn-1. We found evidence that loss of lin-44 caused defects in the polarization and migration of endodermal precursors during gastrulation, a previously undescribed role for lin-44 that is strongly enhanced by the loss of sdn-1. PTP-3 and SDN-1 function in compensatory pathways during C. elegans embryonic and larval development, as simultaneous loss of both genes has dire consequences for organismal survival. The Wnt ligand lin-44 contributes to the early stages of gastrulation in parallel to sdn-1, but in a genetic pathway with ptp-3. Overall, the SynLet phenotype provides a robust platform to identify ptp-3 and sdn-1 interacting genes, as well as other genes that function in development, yet might be missed in traditional forward genetic screens.

  6. Efforts to identify at-risk children for blood lead screening in pediatric clinics--Clark County, Nevada.

    PubMed

    Burns, Mackenzie S; Shah, Lopa H; Marquez, Erika R; Denton, Scott L; Neyland, Beverly A; Vereschzagin, Diana; Gremse, David A; Gerstenberger, Shawn L

    2012-11-01

    Childhood lead poisoning continues to be a public health problem; however, lead screening rates remain low in many areas. Our objective is to increase screening in pediatric clinics, while testing a questionnaire for its predictability of elevated blood lead levels (BLLs). Participants were approached at pediatric clinics in Las Vegas, Nevada. A brief questionnaire assessed the child's potential exposure to lead and a blood sample was collected from each child. Of 564 children tested, 35 had detectable BLLs. Two questions from the questionnaire demonstrated significant differences in proportions (Fisher's exact test: P < .05) of affirmative/negative responses, for the 35 participants with detectable BLLs. The questionnaire failed to identify reliable associations between detectable BLLs and affirmative responses, limiting its use as an in-office tool. More research is recommended to identify and alleviate barriers to childhood lead screening in the clinical setting and to develop more applicable risk assessment tools.

  7. Single Measure and Gated Screening Approaches for Identifying Students At-Risk for Academic Problems: Implications for Sensitivity and Specificity

    ERIC Educational Resources Information Center

    Van Norman, Ethan R.; Nelson, Peter M.; Klingbeil, David A.

    2017-01-01

    Educators need recommendations to improve screening practices without limiting students' instructional opportunities. Repurposing previous years' state test scores has shown promise in identifying at-risk students within multitiered systems of support. However, researchers have not directly compared the diagnostic accuracy of previous years' state…

  8. Random small interfering RNA library screen identifies siRNAs that induce human erythroleukemia cell differentiation.

    PubMed

    Fan, Cuiqing; Xiong, Yuan; Zhu, Ning; Lu, Yabin; Zhang, Jiewen; Wang, Song; Liang, Zicai; Shen, Yan; Chen, Meihong

    2011-03-01

    Cancers are characterized by poor differentiation. Differentiation therapy is a strategy to alleviate malignant phenotypes by inducing cancer cell differentiation. Here we carried out a combinatorial high-throughput screen with a random siRNA library on human erythroleukemia K-562 cell differentiation. Two siRNAs screened from the library were validated to be able to induce erythroid differentiation to varying degrees, determined by CD235 and globin up-regulation, GATA-2 down-regulation, and cell growth inhibition. The screen we performed here is the first trial of screening cancer differentiation-inducing agents from a random siRNA library, demonstrating that a random siRNA library can be considered as a new resource in efforts to seek new therapeutic agents for cancers. As a random siRNA library has a broad coverage for the entire genome, including known/unknown genes and protein coding/non-coding sequences, screening using a random siRNA library can be expected to greatly augment the repertoire of therapeutic siRNAs for cancers.

  9. Time-Resolved Human Kinome RNAi Screen Identifies a Network Regulating Mitotic-Events as Early Regulators of Cell Proliferation

    PubMed Central

    Bechtel, Stephanie; Bender, Christian; Keklikoglou, Ioanna; Schmidt, Christian; Irsigler, Anja; Ernst, Ute; Sahin, Özgür; Wiemann, Stefan; Tschulena, Ulrich

    2011-01-01

    Analysis of biological processes is frequently performed with the help of phenotypic assays where data is mostly acquired in single end-point analysis. Alternative phenotypic profiling techniques are desired where time-series information is essential to the biological question, for instance to differentiate early and late regulators of cell proliferation in loss-of-function studies. So far there is no study addressing this question despite of high unmet interests, mostly due to the limitation of conventional end-point assaying technologies. We present the first human kinome screen with a real-time cell analysis system (RTCA) to capture dynamic RNAi phenotypes, employing time-resolved monitoring of cell proliferation via electrical impedance. RTCA allowed us to investigate the dynamics of phenotypes of cell proliferation instead of using conventional end-point analysis. By introducing data transformation with first-order derivative, i.e. the cell-index growth rate, we demonstrate this system suitable for high-throughput screenings (HTS). The screen validated previously identified inhibitor genes and, additionally, identified activators of cell proliferation. With the information of time kinetics available, we could establish a network of mitotic-event related genes to be among the first displaying inhibiting effects after RNAi knockdown. The time-resolved screen captured kinetics of cell proliferation caused by RNAi targeting human kinome, serving as a resource for researchers. Our work establishes RTCA technology as a novel robust tool with biological and pharmacological relevance amenable for high-throughput screening. PMID:21765947

  10. Identifying infants at risk for neonatal abstinence syndrome: a retrospective cohort comparison study of 3 screening approaches.

    PubMed

    Murphy-Oikonen, Jodie; Montelpare, William J; Southon, Sarah; Bertoldo, Larry; Persichino, Nancy

    2010-01-01

    The main objective of this study was to analyze the consistency in using a standardized newborn toxicology screening protocol to identify infants at risk of developing neonatal abstinence syndrome (NAS). A retrospective cohort comparison design was approved by the institutional review board at the regional hospital and used to gather data from the infants' medical records during the study period. The data were collected for a period of 1 year from a regional hospital serving 100,000 patients per annum. Data were based on expectant mothers who delivered between March 2006 and March 2007. Data of maternal self-reported substance use, and urine toxicology results and meconium results were obtained through retrospective chart review of infants exhibiting signs of NAS as noted by nurses on the Finnegan Scoring Tool. In the absence of accurate prenatal screening, this study lends positively to support the use of toxicology screening protocols at birth to adequately assess and treat infants exposed to illicit substances. Toxicology screening in not intrusive and despite emotional discomfort experienced by mothers of the infants tested, the benefits of attaining accurate information regarding substance exposure is critical for the well-being of the infant. The use of a toxicology screening protocol at birth appears beneficial in determining the need for identifying infants with NAS. Early detection of substance exposure in newborns leads to timely assessment for NAS and subsequent treatment to reduce symptoms in newborns.

  11. Development and validation of a screening procedure to identify speech-language delay in toddlers with cleft palate.

    PubMed

    Jørgensen, Line Dahl; Willadsen, Elisabeth

    2017-05-10

    The purpose of this study was to develop and validate a clinically useful speech-language screening procedure for young children with cleft palate ± cleft lip (CP) to identify those in need of speech-language intervention. Twenty-two children with CP were assigned to a +/- need for intervention conditions based on assessment of consonant inventory using a real-time listening procedure in combination with parent-reported expressive vocabulary. These measures allowed evaluation of early speech-language skills found to correlate significantly with later speech-language performance in longitudinal studies of children with CP. The external validity of this screening procedure was evaluated by comparing the +/- need for intervention assignment determined by the screening procedure to experienced speech-language pathologist (SLP)s' clinical judgement of whether or not a child needed early intervention. The results of real-time listening assessment showed good-excellent inter-rater agreement on different consonant inventory measures. Furthermore, there was almost perfect agreement between the children selected for intervention with the screening procedure and the clinical judgement of experienced SLPs indicate that the screening procedure is a valid way of identifying children with CP who need early intervention.

  12. Developing a brief depression screen and identifying associations with comorbid physical and psychological illness in Australian Gulf War veterans.

    PubMed

    McKenzie, Dean P; Sim, Malcolm R; Clarke, David M; Forbes, Andrew B; Ikin, Jillian F; Kelsall, Helen L

    2015-12-01

    Major depression occurs frequently in veterans, and is associated with comorbid psychological and physical disorders and poorer quality of life. Depression can be difficult to detect in primary care, while lengthy assessment instruments can deter use. Our study aimed to develop a brief depression screen that could be used by veterans and caregivers, and then to compare the association between the brief screen and comorbidities and quality of life with that of a longer instrument. Our dataset comprised 1204 male Royal Australian Navy veterans of the 1990/91 Gulf War. Depressive symptoms were assessed using the General Health Questionnaire (GHQ-12), health-related quality of life by the Short-Form Health Survey (SF-12), major depression and comorbid psychiatric diagnoses such as posttraumatic disorder (PTSD) using Diagnostic and Statistical Manual (DSM-IV) criteria. Comorbid physical illnesses including musculoskeletal disorders, chronic fatigue and diabetes were examined. A brief depression screen of three key self-reported symptoms was identified. Veterans with major depression present according to the screen were over four times more likely to have multisymptom illness or PTSD, and almost twice as likely to have musculoskeletal disorders. Having depression according to the brief screen and having at least one other physical or psychological condition was associated with poorer quality of life. Similar results were obtained for a longer screen based on all GHQ-12 items. A 3 item depression screen performed as well as a 12 item one in identifying major depression, comorbid physical and psychological illness and poorer quality of life in veterans. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  13. A simplified screening test for identifying people with low vision in developing countries.

    PubMed Central

    Keeffe, J. E.; Lovie-Kitchin, J. E.; Maclean, H.; Taylor, H. R.

    1996-01-01

    Simple but effective tests have been produced for screening subjects with low vision in developing countries. These tests of distance and near vision, based on the E test, were evaluated and validated in trials with people aged 4-90 years, and have been field tested in the health, education and rehabilitation services in 32 developing countries. Their sensitivity and specificity as screening tools for low vision have been calculated; sensitivity of 85% and specificity of 96% for the distance vision test, and sensitivity of 100% and specificity of 84% for the near vision test. The content and format of the tests have been demonstrated to be appropriate for developing countries, and their effectiveness for screening for low vision has been confirmed. PMID:9002333

  14. Niche-based screening identifies small-molecule inhibitors of leukemia stem cells.

    PubMed

    Hartwell, Kimberly A; Miller, Peter G; Mukherjee, Siddhartha; Kahn, Alissa R; Stewart, Alison L; Logan, David J; Negri, Joseph M; Duvet, Mildred; Järås, Marcus; Puram, Rishi; Dancik, Vlado; Al-Shahrour, Fatima; Kindler, Thomas; Tothova, Zuzana; Chattopadhyay, Shrikanta; Hasaka, Thomas; Narayan, Rajiv; Dai, Mingji; Huang, Christina; Shterental, Sebastian; Chu, Lisa P; Haydu, J Erika; Shieh, Jae Hung; Steensma, David P; Munoz, Benito; Bittker, Joshua A; Shamji, Alykhan F; Clemons, Paul A; Tolliday, Nicola J; Carpenter, Anne E; Gilliland, D Gary; Stern, Andrew M; Moore, Malcolm A S; Scadden, David T; Schreiber, Stuart L; Ebert, Benjamin L; Golub, Todd R

    2013-12-01

    Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those compounds that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on target, via inhibition of HMG-CoA reductase. These results illustrate the power of merging physiologically relevant models with high-throughput screening.

  15. A Chemical Screen to Identify Novel Inhibitors of Fin Regeneration in Zebrafish

    PubMed Central

    Oppedal, Douglas

    2010-01-01

    Abstract We performed a chemical screen to look for novel inhibitors of zebrafish caudal fin regeneration. In a pilot screen, 520 compounds were tested. Two compounds, budesonide and AGN192403, abrogated fin regeneration. One compound in particular, AGN192403, targets the imidazoline receptor, a pathway not previously linked to fin regeneration. In addition to inhibiting regeneration of the adult fin, AGN192403 also blocked regeneration of the larval fin fold. Finally, the inhibitory effect of AGN192403 on fin regeneration persisted after removal of the drug. These studies demonstrate that chemical screening is feasible in adult zebrafish and that it is a reasonable strategy to use for exploring the biology of regeneration. PMID:20384483

  16. Use of a single alcohol screening question to identify other drug use.

    PubMed

    Smith, Peter C; Cheng, Debbie M; Allensworth-Davies, Donald; Winter, Michael R; Saitz, Richard

    2014-06-01

    People who consume unhealthy amounts of alcohol are more likely to use illicit drugs. We tested the ability of a screening test for unhealthy alcohol use to simultaneously detect drug use. Adult English speaking patients (n=286) were enrolled from a primary care waiting room. They were asked the screening question for unhealthy alcohol use "How many times in the past year have you had X or more drinks in a day?", where X is 5 for men and 4 for women, and a response of one or more is considered positive. A standard diagnostic interview was used to determine current (past year) drug use or a drug use disorder (abuse or dependence). Oral fluid testing was also used to detect recent use of common drugs of abuse. The single screening question for unhealthy alcohol use was 67.6% sensitive (95% confidence interval [CI], 50.2-82.0%) and 64.7% specific (95% CI, 58.4-70.6%) for the detection of a drug use disorder. It was similarly insensitive for drug use detected by oral fluid testing and/or self-report. Although a patient with a drug use disorder has twice the odds of screening positive for unhealthy alcohol use compared to one without a drug use disorder, suggesting patients who screen positive for alcohol should be asked about drug use, a single screening question for unhealthy alcohol use was not sensitive or specific for the detection of other drug use or drug use disorders in a sample of primary care patients. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  17. High-Throughput Screening of Myometrial Calcium-Mobilization to Identify Modulators of Uterine Contractility

    PubMed Central

    Herington, Jennifer L.; Swale, Daniel R.; Brown, Naoko; Shelton, Elaine L.; Choi, Hyehun; Williams, Charles H.; Hong, Charles C.; Paria, Bibhash C.; Denton, Jerod S.; Reese, Jeff

    2015-01-01

    The uterine myometrium (UT-myo) is a therapeutic target for preterm labor, labor induction, and postpartum hemorrhage. Stimulation of intracellular Ca2+-release in UT-myo cells by oxytocin is a final pathway controlling myometrial contractions. The goal of this study was to develop a dual-addition assay for high-throughput screening of small molecular compounds, which could regulate Ca2+-mobilization in UT-myo cells, and hence, myometrial contractions. Primary murine UT-myo cells in 384-well plates were loaded with a Ca2+-sensitive fluorescent probe, and then screened for inducers of Ca2+-mobilization and inhibitors of oxytocin-induced Ca2+-mobilization. The assay exhibited robust screening statistics (Z´ = 0.73), DMSO-tolerance, and was validated for high-throughput screening against 2,727 small molecules from the Spectrum, NIH Clinical I and II collections of well-annotated compounds. The screen revealed a hit-rate of 1.80% for agonist and 1.39% for antagonist compounds. Concentration-dependent responses of hit-compounds demonstrated an EC50 less than 10μM for 21 hit-antagonist compounds, compared to only 7 hit-agonist compounds. Subsequent studies focused on hit-antagonist compounds. Based on the percent inhibition and functional annotation analyses, we selected 4 confirmed hit-antagonist compounds (benzbromarone, dipyridamole, fenoterol hydrobromide and nisoldipine) for further analysis. Using an ex vivo isometric contractility assay, each compound significantly inhibited uterine contractility, at different potencies (IC50). Overall, these results demonstrate for the first time that high-throughput small-molecules screening of myometrial Ca2+-mobilization is an ideal primary approach for discovering modulators of uterine contractility. PMID:26600013

  18. Use of a single alcohol screening question to identify other drug use

    PubMed Central

    Smith, Peter C; Cheng, Debbie M; Allensworth-Davies, Donald; Winter, Michael R; Saitz, Richard

    2014-01-01

    Background People who consume unhealthy amounts of alcohol are more likely to use illicit drugs. We tested the ability of a screening test for unhealthy alcohol use to simultaneously detect drug use. Methods Adult English speaking patients (n=286) were enrolled from a primary care waiting room. They were asked the screening question for unhealthy alcohol use “How many times in the past year have you had X or more drinks in a day?”, where X is 5 for men and 4 for women, and a response of one or more is considered positive. A standard diagnostic interview was used to determine current (past year) drug use or a drug use disorder (abuse or dependence). Oral fluid testing was also used to detect recent use of common drugs of abuse. Results The single screening question for unhealthy alcohol use was 67.6% sensitive (95% confidence interval [CI], 50.2%- 82.0%) and 64.7% specific (95% CI, 58.4%- 70.6%) for the detection of a drug use disorder. It was similarly insensitive for drug use detected by oral fluid testing and/or self-report. Conclusions Although a patient with a drug use disorder has twice the odds of screening positive for unhealthy alcohol use compared to one without a drug use disorder, suggesting patients who screen positive for alcohol should be asked about drug use, a single screening question for unhealthy alcohol use was not sensitive or specific for the detection of other drug use or drug use disorders in a sample of primary care patients. PMID:24768061

  19. Screening to Identify and Eradicate Helicobacter pylori Infection in Teenagers in Japan.

    PubMed

    Akamatsu, Taiji; Okamura, Takuma; Iwaya, Yugo; Suga, Tomoaki

    2015-09-01

    The purpose of this study was to elucidate the prevalence and effect of Helicobacter pylori infection in Japanese teenagers. The study subjects were students ages 16 to 17 from one high school studied between 2007 and 2013. Students who tested positive on this screening examination underwent esophagogastroduodenoscopy and biopsy samples to determine their H pylori status using culture and histology. Cure of H pylori infections was determined by urea breath test. The low rate of prevalence of H pylori infection in present Japanese teenagers makes it possible and cost effective to perform examinations and carry out treatment of this infection in nationwide health screenings of high school students.

  20. Image-based siRNA screen to identify kinases regulating Weibel-Palade body size control using electroporation.

    PubMed

    Ketteler, Robin; Freeman, Jamie; Ferraro, Francesco; Bata, Nicole; Cutler, Dan F; Kriston-Vizi, Janos

    2017-03-01

    High-content screening of kinase inhibitors is important in order to identify biogenesis and function mechanisms of subcellular organelles. Here, we present a human kinome siRNA high-content screen on primary human umbilical vein endothelial cells, that were transfected by electroporation. The data descriptor contains a confocal fluorescence, microscopic image dataset. We also describe an open source, automated image analysis workflow that can be reused to perform high-content analysis of other organelles. This dataset is suitable for analysis of morphological parameters that are linked to human umbilical vein endothelial cell (HUVEC) biology.

  1. Image-based siRNA screen to identify kinases regulating Weibel-Palade body size control using electroporation

    PubMed Central

    Ketteler, Robin; Freeman, Jamie; Ferraro, Francesco; Bata, Nicole; Cutler, Dan F.; Kriston-Vizi, Janos

    2017-01-01

    High-content screening of kinase inhibitors is important in order to identify biogenesis and function mechanisms of subcellular organelles. Here, we present a human kinome siRNA high-content screen on primary human umbilical vein endothelial cells, that were transfected by electroporation. The data descriptor contains a confocal fluorescence, microscopic image dataset. We also describe an open source, automated image analysis workflow that can be reused to perform high-content analysis of other organelles. This dataset is suitable for analysis of morphological parameters that are linked to human umbilical vein endothelial cell (HUVEC) biology. PMID:28248923

  2. Identifiability of the Joint Distribution of Age and Tumor Size at Detection in the Presence of Screening

    PubMed Central

    Hanin, Leonid; Yakovlev, Andrei

    2007-01-01

    In recent years, a stochastic model of cancer development and detection allowing for arbitrary screening schedules has been developed and applied to analysis of screening trials and population-based cancer incidence and mortality data. The model is entirely mechanistic, builds on a minimal set of biologically plausible assumptions, and yields the joint distribution of tumor size and age of a patient at the time of diagnosis. Whether or not parameters of the model can be estimated from data generated by cohort studies depends on model identifiability. The present paper provides a proof of this important property of the model. PMID:17303192

  3. Large-Scale Computational Screening Identifies First in Class Multitarget Inhibitor of EGFR Kinase and BRD4

    PubMed Central

    Allen, Bryce K.; Mehta, Saurabh; Ember, Stewart W. J.; Schonbrunn, Ernst; Ayad, Nagi; Schürer, Stephan C.

    2015-01-01

    Inhibition of cancer-promoting kinases is an established therapeutic strategy for the treatment of many cancers, although resistance to kinase inhibitors is common. One way to overcome resistance is to target orthogonal cancer-promoting pathways. Bromo and Extra-Terminal (BET) domain proteins, which belong to the family of epigenetic readers, have recently emerged as promising therapeutic targets in multiple cancers. The development of multitarget drugs that inhibit kinase and BET proteins therefore may be a promising strategy to overcome tumor resistance and prolong therapeutic efficacy in the clinic. We developed a general computational screening approach to identify novel dual kinase/bromodomain inhibitors from millions of commercially available small molecules. Our method integrated machine learning using big datasets of kinase inhibitors and structure-based drug design. Here we describe the computational methodology, including validation and characterization of our models and their application and integration into a scalable virtual screening pipeline. We screened over 6 million commercially available compounds and selected 24 for testing in BRD4 and EGFR biochemical assays. We identified several novel BRD4 inhibitors, among them a first in class dual EGFR-BRD4 inhibitor. Our studies suggest that this computational screening approach may be broadly applicable for identifying dual kinase/BET inhibitors with potential for treating various cancers. PMID:26596901

  4. High-content phenotypic screening and triaging strategy to identify small molecules driving oligodendrocyte progenitor cell differentiation.

    PubMed

    Peppard, Jane V; Rugg, Catherine A; Smicker, Matthew A; Powers, Elaine; Harnish, Erica; Prisco, Joy; Cirovic, Dragan; Wright, Paul S; August, Paul R; Chandross, Karen J

    2015-03-01

    Multiple Sclerosis is a demyelinating disease of the CNS and the primary cause of neurological disability in young adults. Loss of myelinating oligodendrocytes leads to neuronal dysfunction and death and is an important contributing factor to this disease. Endogenous oligodendrocyte precursor cells (OPCs), which on differentiation are responsible for replacing myelin, are present in the adult CNS. As such, therapeutic agents that can stimulate OPCs to differentiate and remyelinate demyelinated axons under pathologic conditions may improve neuronal function and clinical outcome. We describe the details of an automated, cell-based, morphometric-based, high-content screen that is used to identify small molecules eliciting the differentiation of OPCs after 3 days. Primary screening was performed using rat CG-4 cells maintained in culture conditions that normally support a progenitor cell-like state. From a library of 73,000 diverse small molecules within the Sanofi collection, 342 compounds were identified that increased OPC morphological complexity as an indicator of oligodendrocyte maturation. Subsequent to the primary high-content screen, a suite of cellular assays was established that identified 22 nontoxic compounds that selectively stimulated primary rat OPCs but not C2C12 muscle cell differentiation. This rigorous triaging yielded several chemical series for further expansion and bio- or cheminformatics studies, and their compelling biological activity merits further investigation. © 2014 Society for Laboratory Automation and Screening.

  5. High-Throughput Screening Identifies Idarubicin as a Preferential Inhibitor of Smooth Muscle versus Endothelial Cell Proliferation

    PubMed Central

    Wang, Bowen; Guo, Song; Roenneburg, Drew; Ananiev, Gene E.; Hoffmann, F. Michael; Kent, K. Craig

    2014-01-01

    Intimal hyperplasia is the cause of the recurrent occlusive vascular disease (restenosis). Drugs currently used to treat restenosis effectively inhibit smooth muscle cell (SMC) proliferation, but also inhibit the growth of the protective luminal endothelial cell (EC) lining, leading to thrombosis. To identify compounds that selectively inhibit SMC versus EC proliferation, we have developed a high-throughput screening (HTS) format using human cells and have employed this to screen a multiple compound collection (NIH Clinical Collection). We developed an automated, accurate proliferation assay in 96-well plates using human aortic SMCs and ECs. Using this HTS format we screened a 447-drug NIH Clinical Library. We identified 11 compounds that inhibited SMC proliferation greater than 50%, among which idarubicin exhibited a unique feature of preferentially inhibiting SMC versus EC proliferation. Concentration-response analysis revealed this differential effect most evident over an ∼10 nM-5 µM window. In vivo testing of idarubicin in a rat carotid injury model at 14 days revealed an 80% reduction of intimal hyperplasia and a 45% increase of lumen size with no significant effect on re-endothelialization. Taken together, we have established a HTS assay of human vascular cell proliferation, and identified idarubicin as a selective inhibitor of SMC versus EC proliferation both in vitro and in vivo. Screening of larger and more diverse compound libraries may lead to the discovery of next-generation therapeutics that can inhibit intima hyperplasia without impairing re-endothelialization. PMID:24586708

  6. Identifying Adolescents at Risk through Voluntary School-Based Mental Health Screening

    ERIC Educational Resources Information Center

    Husky, Mathilde M.; Kaplan, Adam; McGuire, Leslie; Flynn, Laurie; Chrostowski, Christine; Olfson, Mark

    2011-01-01

    This study compares referrals for mental health services among high school students randomized to two means of referral to mental health services: referral via systematic identification through a brief mental health screening procedure (n = 365) or referral via the usual process of identification by school personnel, parents, or students…

  7. Identifying Adolescents at Risk through Voluntary School-Based Mental Health Screening

    ERIC Educational Resources Information Center

    Husky, Mathilde M.; Kaplan, Adam; McGuire, Leslie; Flynn, Laurie; Chrostowski, Christine; Olfson, Mark

    2011-01-01

    This study compares referrals for mental health services among high school students randomized to two means of referral to mental health services: referral via systematic identification through a brief mental health screening procedure (n = 365) or referral via the usual process of identification by school personnel, parents, or students…

  8. Effectiveness of the Revised Denver Developmental Screening Test in Identifying Children at Risk for Learning Problems.

    ERIC Educational Resources Information Center

    Diamond, Karen E.

    1990-01-01

    Findings from a 5-year follow-up study of 78 kindergartners suggest that while the Revised Denver Developmental Screening Test (RDDST) accurately predicts academic achievement and standardized test performance, it consistently misclassifies as normal the performance of a significant number of children who require special help in their early…

  9. Using in Vitro High Throughput Screening Assays to Identify Potential Endocrine-Disrupting Chemicals

    EPA Science Inventory

    Over the past 20 years, an increased focus on detecting environmental chemicals posing a risk of adverse effects due to endocrine disruption has driven the creation of the U.S. EPA Endocrine Disruptor Screening Program (EDSP). Thousands of chemicals are subject to the EDSP, whic...

  10. Using in Vitro High Throughput Screening Assays to Identify Potential Endocrine-Disrupting Chemicals

    EPA Science Inventory

    Over the past 20 years, an increased focus on detecting environmental chemicals posing a risk of adverse effects due to endocrine disruption has driven the creation of the U.S. EPA Endocrine Disruptor Screening Program (EDSP). Thousands of chemicals are subject to the EDSP, whic...

  11. Use of cell-based screening to identify small-molecule compounds that modulate claudin-4 expression.

    PubMed

    Watari, Akihiro; Hashegawa, Maki; Muangman, Thanchanok; Yagi, Kiyohito; Kondoh, Masuo

    2015-06-01

    Claudins constitute a family of at least 27 proteins with four transmembrane domains, and play a pivotal role in maintaining tight-junctions seals in diverse epithelial tissues. The expression of claudin-4 often changes in intestinal tissues of inflammatory bowel disease and various human cancers. Therefore, claudin-4 is a promising target for treatment of these diseases. In our previous study, we established a reporter cell line to monitor claudin-4 expression on the basis of a functional claudin-4 promoter. Using this cell line, we have performed a cell-based screen of a library containing 2642 biologically active small-molecule compounds to identify modulators of claudin-4 expression. The screen identified 24 potential modulators of the claudin-4 promoter activity. Fourteen of these compounds (12 of them novel) induced endogenous claudin-4 expression. The identified compounds might serve as lead compounds targeting aberrant gene expression in inflammatory bowel disease.

  12. Evaluation of an inpatient fall risk screening tool to identify the most critical fall risk factors in inpatients.

    PubMed

    Hou, Wen-Hsuan; Kang, Chun-Mei; Ho, Mu-Hsing; Kuo, Jessie Ming-Chuan; Chen, Hsiao-Lien; Chang, Wen-Yin

    2017-03-01

    To evaluate the accuracy of the inpatient fall risk screening tool and to identify the most critical fall risk factors in inpatients. Variations exist in several screening tools applied in acute care hospitals for examining risk factors for falls and identifying high-risk inpatients. Secondary data analysis. A subset of inpatient data for the period from June 2011-June 2014 was extracted from the nursing information system and adverse event reporting system of an 818-bed teaching medical centre in Taipei. Data were analysed using descriptive statistics, receiver operating characteristic curve analysis and logistic regression analysis. During the study period, 205 fallers and 37,232 nonfallers were identified. The results revealed that the inpatient fall risk screening tool (cut-off point of ≥3) had a low sensitivity level (60%), satisfactory specificity (87%), a positive predictive value of 2·0% and a negative predictive value of 99%. The receiver operating characteristic curve analysis revealed an area under the curve of 0·805 (sensitivity, 71·8%; specificity, 78%). To increase the sensitivity values, the Youden index suggests at least 1·5 points to be the most suitable cut-off point for the inpatient fall risk screening tool. Multivariate logistic regression analysis revealed a considerably increased fall risk in patients with impaired balance and impaired elimination. The fall risk factor was also significantly associated with days of hospital stay and with admission to surgical wards. The findings can raise awareness about the two most critical risk factors for falls among future clinical nurses and other healthcare professionals and thus facilitate the development of fall prevention interventions. This study highlights the needs for redefining the cut-off points of the inpatient fall risk screening tool to effectively identify inpatients at a high risk of falls. Furthermore, inpatients with impaired balance and impaired elimination should be closely

  13. Bioluminescence-Based High-Throughput Screen Identifies Pharmacological Agents That Target Neurotransmitter Signaling in Small Cell Lung Carcinoma

    PubMed Central

    Improgo, Ma. Reina D.; Johnson, Christopher W.; Tapper, Andrew R.; Gardner, Paul D.

    2011-01-01

    Background Frontline treatment of small cell lung carcinoma (SCLC) relies heavily on chemotherapeutic agents and radiation therapy. Though SCLC patients respond well to initial cycles of chemotherapy, they eventually develop resistance. Identification of novel therapies against SCLC is therefore imperative. Methods and Findings We have designed a bioluminescence-based cell viability assay for high-throughput screening of anti-SCLC agents. The assay was first validated via standard pharmacological agents and RNA interference using two human SCLC cell lines. We then utilized the assay in a high-throughput screen using the LOPAC1280 compound library. The screening identified several drugs that target classic cancer signaling pathways as well as neuroendocrine markers in SCLC. In particular, perturbation of dopaminergic and serotonergic signaling inhibits SCLC cell viability. Conclusions The convergence of our pharmacological data with key SCLC pathway components reiterates the importance of neurotransmitter signaling in SCLC etiology and points to possible leads for drug development. PMID:21931655

  14. Parameter screening: the use of a dummy parameter to identify non-influential parameters in a global sensitivity analysis

    NASA Astrophysics Data System (ADS)

    Khorashadi Zadeh, Farkhondeh; Nossent, Jiri; van Griensven, Ann; Bauwens, Willy

    2017-04-01

    Parameter estimation is a major concern in hydrological modeling, which may limit the use of complex simulators with a large number of parameters. To support the selection of parameters to include in or exclude from the calibration process, Global Sensitivity Analysis (GSA) is widely applied in modeling practices. Based on the results of GSA, the influential and the non-influential parameters are identified (i.e. parameters screening). Nevertheless, the choice of the screening threshold below which parameters are considered non-influential is a critical issue, which has recently received more attention in GSA literature. In theory, the sensitivity index of a non-influential parameter has a value of zero. However, since numerical approximations, rather than analytical solutions, are utilized in GSA methods to calculate the sensitivity indices, small but non-zero indices may be obtained for the indices of non-influential parameters. In order to assess the threshold that identifies non-influential parameters in GSA methods, we propose to calculate the sensitivity index of a "dummy parameter". This dummy parameter has no influence on the model output, but will have a non-zero sensitivity index, representing the error due to the numerical approximation. Hence, the parameters whose indices are above the sensitivity index of the dummy parameter can be classified as influential, whereas the parameters whose indices are below this index are within the range of the numerical error and should be considered as non-influential. To demonstrated the effectiveness of the proposed "dummy parameter approach", 26 parameters of a Soil and Water Assessment Tool (SWAT) model are selected to be analyzed and screened, using the variance-based Sobol' and moment-independent PAWN methods. The sensitivity index of the dummy parameter is calculated from sampled data, without changing the model equations. Moreover, the calculation does not even require additional model evaluations for the Sobol

  15. Screening of whole genome sequences identified high-impact variants for stallion fertility.

    PubMed

    Schrimpf, Rahel; Gottschalk, Maren; Metzger, Julia; Martinsson, Gunilla; Sieme, Harald; Distl, Ottmar

    2016-04-14

    Stallion fertility is an economically important trait due to the increase of artificial insemination in horses. The availability of whole genome sequence data facilitates identification of rare high-impact variants contributing to stallion fertility. The aim of our study was to genotype rare high-impact variants retrieved from next-generation sequencing (NGS)-data of 11 horses in order to unravel harmful genetic variants in large samples of stallions. Gene ontology (GO) terms and search results from public databases were used to obtain a comprehensive list of human und mice genes predicted to participate in the regulation of male reproduction. The corresponding equine orthologous genes were searched in whole genome sequence data of seven stallions and four mares and filtered for high-impact genetic variants using SnpEFF, SIFT and Polyphen 2 software. All genetic variants with the missing homozygous mutant genotype were genotyped on 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. Mixed linear model analysis was employed for an association analysis with de-regressed estimated breeding values of the paternal component of the pregnancy rate per estrus (EBV-PAT). We screened next generation sequenced data of whole genomes from 11 horses for equine genetic variants in 1194 human and mice genes involved in male fertility and linked through common gene ontology (GO) with male reproductive processes. Variants were filtered for high-impact on protein structure and validated through SIFT and Polyphen 2. Only those genetic variants were followed up when the homozygote mutant genotype was missing in the detection sample comprising 11 horses. After this filtering process, 17 single nucleotide polymorphism (SNPs) were left. These SNPs were genotyped in 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. An association analysis in 216 Hanoverian stallions revealed a significant association of the splice-site disruption variant

  16. IspE inhibitors identified by a combination of in silico and in vitro high-throughput screening.

    PubMed

    Tidten-Luksch, Naomi; Grimaldi, Raffaella; Torrie, Leah S; Frearson, Julie A; Hunter, William N; Brenk, Ruth

    2012-01-01

    CDP-ME kinase (IspE) contributes to the non-mevalonate or deoxy-xylulose phosphate (DOXP) pathway for isoprenoid precursor biosynthesis found in many species of bacteria and apicomplexan parasites. IspE has been shown to be essential by genetic methods and since it is absent from humans it constitutes a promising target for antimicrobial drug development. Using in silico screening directed against the substrate binding site and in vitro high-throughput screening directed against both, the substrate and co-factor binding sites, non-substrate-like IspE inhibitors have been discovered and structure-activity relationships were derived. The best inhibitors in each series have high ligand efficiencies and favourable physico-chemical properties rendering them promising starting points for drug discovery. Putative binding modes of the ligands were suggested which are consistent with established structure-activity relationships. The applied screening methods were complementary in discovering hit compounds, and a comparison of both approaches highlights their strengths and weaknesses. It is noteworthy that compounds identified by virtual screening methods provided the controls for the biochemical screens.

  17. Telephone screening to identify potential dementia cases in a population-based sample of older adults.

    PubMed

    Gatz, Margaret; Reynolds, Chandra A; John, Richard; Johansson, Boo; Mortimer, James A; Pedersen, Nancy L

    2002-09-01

    This study examined the utility of the TELE, a telephone assessment for dementia, in a sample of 269 individuals that was not selected on the basis of previous dementia diagnosis. Thus, the conditions of the study reflect the actual situation in which a screening instrument might be employed. Scores on TELE were compared to dementia diagnoses. Using the best cutoff score, sensitivity was .86 and specificity was .90. Longitudinal follow-up established that false positives primarily included those who subsequently developed dementia. Telephone screening for dementia has both clinical and research applications. One recommendation based on our experience is that longitudinal studies should include a telephone interview component for anyone who drops out of the study, to enable characterizing the cognitive status of dropouts.

  18. Combination of Biological Screening in a Cellular Model of Viral Latency and Virtual Screening Identifies Novel Compounds That Reactivate HIV-1

    PubMed Central

    Gallastegui, Edurne; Marshall, Brett; Vidal, David; Sanchez-Duffhues, Gonzalo; Collado, Juan A.; Alvarez-Fernández, Carmen; Luque, Neus; Terme, Jean-Michel; Gatell, Josep M.; Sánchez-Palomino, Sonsoles; Muñoz, Eduardo; Mestres, Jordi; Verdin, Eric

    2012-01-01

    Although highly active antiretroviral therapy (HAART) has converted HIV into a chronic disease, a reservoir of HIV latently infected resting T cells prevents the eradication of the virus from patients. To achieve eradication, HAART must be combined with drugs that reactivate the dormant viruses. We examined this problem in an established model of HIV postintegration latency by screening a library of small molecules. Initially, we identified eight molecules that reactivated latent HIV. Using them as templates, additional hits were identified by means of similarity-based virtual screening. One of those hits, 8-methoxy-6-methylquinolin-4-ol (MMQO), proved to be useful to reactivate HIV-1 in different cellular models, especially in combination with other known reactivating agents, without causing T-cell activation and with lower toxicity than that of the initial hits. Interestingly, we have established that MMQO produces Jun N-terminal protein kinase (JNK) activation and enhances the T-cell receptor (TCR)/CD3 stimulation of HIV-1 reactivation from latency but inhibits CD3-induced interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α) gene transcription. Moreover, MMQO prevents TCR-induced cell cycle progression and proliferation in primary T cells. The present study documents that the combination of biological screening in a cellular model of viral latency with virtual screening is useful for the identification of novel agents able to reactivate HIV-1. Moreover, we set the bases for a hypothetical therapy to reactivate latent HIV by combining MMQO with physiological or pharmacological TCR/CD3 stimulation. PMID:22258251

  19. A screening approach for identifying environmental justice issues in environmental impact statements

    SciTech Connect

    Schexnayder, S.S.

    1995-12-01

    Executive Order 12898 and the accompanying memorandum addressed to Federal agency heads, both issued on February 11, 1994, require NEPA processes to incorporate environmental justice. The NEPA processes affected are: (1) public involvement formats, (2) analyses of potential impacts. The Executive Order clearly indicates that research strategies and mitigation measure should be developed with the input of the populations mentioned in the Executive Order, i.e., minority and low-income populations. However, an enhanced public involvement process may not occur because the NEPA activity may have been underway before the Executive Order was issued or because the agency chooses not to change traditional public participation mechanisms. It is also possible that enhanced mechanisms may not effectively elicit involvement. In either case, analysis that considers environmental justice must proceed. These analyses could be highly data-intensive--requiring new or modified methodological approaches-- and time-intensive, particularly if the process elements of the executive order are interpreted broadly, Federal agencies and NEPA project managers already have expressed concern about the potential cost of conducting exhaustive environmental justice related analyses where they may not be warranted. Also, the time and resources required to conduct a full environmental justice analysis is counter to recent trends to streamline the NEPA process. In light of this, a process to screen for indicators of the potential for environmental justice issues has been developed. The method incorporates separate screens for human health impacts, socioeconomic impacts, and social structural impacts. Positive results of any screen indicates the need for full-scale, environmental-justice-related analysis of that category of impact. The screen is intended as a useful tool in implementing environmental justice in environmental impact statements.

  20. Drosophila glucome screening identifies Ck1alpha as a regulator of mammalian glucose metabolism

    PubMed Central

    Ugrankar, Rupali; Berglund, Eric; Akdemir, Fatih; Tran, Christopher; Kim, Min Soo; Noh, Jungsik; Schneider, Rebekka; Ebert, Benjamin; Graff, Jonathan M.

    2015-01-01

    Circulating carbohydrates are an essential energy source, perturbations in which are pathognomonic of various diseases, diabetes being the most prevalent. Yet many of the genes underlying diabetes and its characteristic hyperglycaemia remain elusive. Here we use physiological and genetic interrogations in D. melanogaster to uncover the ‘glucome', the complete set of genes involved in glucose regulation in flies. Partial genomic screens of ∼1,000 genes yield ∼160 hyperglycaemia ‘flyabetes' candidates that we classify using fat body- and muscle-specific knockdown and biochemical assays. The results highlight the minor glucose fraction as a physiological indicator of metabolism in Drosophila. The hits uncovered in our screen may have conserved functions in mammalian glucose homeostasis, as heterozygous and homozygous mutants of Ck1alpha in the murine adipose lineage, develop diabetes. Our findings demonstrate that glucose has a role in fly biology and that genetic screenings carried out in flies may increase our understanding of mammalian pathophysiology. PMID:25994086

  1. High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells

    PubMed Central

    Gerby, Bastien; Veiga, Diogo F.T.; Krosl, Jana; Nourreddine, Sami; Ouellette, Julianne; Haman, André; Lavoie, Geneviève; Fares, Iman; Tremblay, Mathieu; Litalien, Véronique; Ottoni, Elizabeth; Geoffrion, Dominique; Maddox, Paul S.; Chagraoui, Jalila; Hébert, Josée; Sauvageau, Guy; Kwok, Benjamin H.; Roux, Philippe P.

    2016-01-01

    Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non–cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy. PMID:27797342

  2. High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells.

    PubMed

    Gerby, Bastien; Veiga, Diogo F T; Krosl, Jana; Nourreddine, Sami; Ouellette, Julianne; Haman, André; Lavoie, Geneviève; Fares, Iman; Tremblay, Mathieu; Litalien, Véronique; Ottoni, Elizabeth; Kosic, Milena; Geoffrion, Dominique; Ryan, Joël; Maddox, Paul S; Chagraoui, Jalila; Marinier, Anne; Hébert, Josée; Sauvageau, Guy; Kwok, Benjamin H; Roux, Philippe P; Hoang, Trang

    2016-12-01

    Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non-cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy.

  3. Organ-targeted high-throughput in vivo biologics screen identifies materials for RNA delivery.

    PubMed

    Chang, Tsung-Yao; Shi, Peng; Steinmeyer, Joseph D; Chatnuntawech, Itthi; Tillberg, Paul; Love, Kevin T; Eimon, Peter M; Anderson, Daniel G; Yanik, Mehmet Fatih

    2014-10-01

    Therapies based on biologics involving delivery of proteins, DNA, and RNA are currently among the most promising approaches. However, although large combinatorial libraries of biologics and delivery vehicles can be readily synthesized, there are currently no means to rapidly characterize them in vivo using animal models. Here, we demonstrate high-throughput in vivo screening of biologics and delivery vehicles by automated delivery into target tissues of small vertebrates with developed organs. Individual zebrafish larvae are automatically oriented and immobilized within hydrogel droplets in an array format using a microfluidic system, and delivery vehicles are automatically microinjected to target organs with high repeatability and precision. We screened a library of lipid-like delivery vehicles for their ability to facilitate the expression of protein-encoding RNAs in the central nervous system. We discovered delivery vehicles that are effective in both larval zebrafish and rats. Our results showed that the in vivo zebrafish model can be significantly more predictive of both false positives and false negatives in mammals than in vitro mammalian cell culture assays. Our screening results also suggest certain structure-activity relationships, which can potentially be applied to design novel delivery vehicles.

  4. Organ-targeted high-throughput in vivo biologics screen identifies materials for RNA delivery

    PubMed Central

    Chang, Tsung-Yao; Shi, Peng; Steinmeyer, Joseph D.; Chatnuntawech, Itthi; Tillberg, Paul; Love, Kevin T.; Eimon, Peter M.; Anderson, Daniel G.; Yanik, Mehmet Fatih

    2014-01-01

    Therapies based on biologics involving delivery of proteins, DNA, and RNA are currently among the most promising approaches. However, although large combinatorial libraries of biologics and delivery vehicles can be readily synthesized, there are currently no means to rapidly characterize them in vivo using animal models. Here, we demonstrate high-throughput in vivo screening of biologics and delivery vehicles by automated delivery into target tissues of small vertebrates with developed organs. Individual zebrafish larvae are automatically oriented and immobilized within hydrogel droplets in an array format using a microfluidic system, and delivery vehicles are automatically microinjected to target organs with nearly perfect repeatability and precision. We screened a library of lipid-like delivery vehicles for their ability to facilitate the expression of protein-encoding RNAs in the central nervous system. We discovered delivery vehicles that are effective in both larval zebrafish and rats. Our results showed that the in vivo zebrafish model can be significantly more predictive of both false positives and false negatives in mammals than in vitro mammalian cell culture assays. Our screening results also suggest certain structure-activity relationship, which can potentially be applied to design novel delivery vehicles. PMID:25184623

  5. Development of a Novel Virtual Screening Cascade Protocol to Identify Potential Trypanothione Reductase Inhibitors

    PubMed Central

    2009-01-01

    The implementation of a novel sequential computational approach that can be used effectively for virtual screening and identification of prospective ligands that bind to trypanothione reductase (TryR) is reported. The multistep strategy combines a ligand-based virtual screening for building an enriched library of small molecules with a docking protocol (AutoDock, X-Score) for screening against the TryR target. Compounds were ranked by an exhaustive conformational consensus scoring approach that employs a rank-by-rank strategy by combining both scoring functions. Analysis of the predicted ligand−protein interactions highlights the role of bulky quaternary amine moieties for binding affinity. The scaffold hopping (SHOP) process derived from this computational approach allowed the identification of several chemotypes, not previously reported as antiprotozoal agents, which includes dibenzothiepine, dibenzooxathiepine, dibenzodithiepine, and polycyclic cationic structures like thiaazatetracyclo-nonadeca-hexaen-3-ium. Assays measuring the inhibiting effect of these compounds on T. cruzi and T. brucei TryR confirm their potential for further rational optimization. PMID:19296695

  6. A Systematic Approach to Identify Biased Agonists of the Apelin Receptor through High-Throughput Screening.

    PubMed

    McAnally, Danielle; Siddiquee, Khandaker; Sharir, Haleli; Qi, Feng; Phatak, Sharangdhar; Li, Jian-Liang; Berg, Eric; Fishman, Jordan; Smith, Layton

    2017-03-01

    Biased agonists are defined by their ability to selectively activate distinct signaling pathways of a receptor, and they hold enormous promise for the development of novel drugs that specifically elicit only the desired therapeutic response and avoid potential adverse effects. Unfortunately, most high-throughput screening (HTS) assays are designed to detect signaling of G protein-coupled receptors (GPCRs) downstream of either G protein or β-arrestin-mediated signaling but not both. A comprehensive drug discovery program seeking biased agonists must employ assays that report on the activity of each compound at multiple discrete pathways, particularly for HTS campaigns. Here, we report a systematic approach to the identification of biased agonists of human apelin receptor (APJ). We synthesized 448 modified versions of apelin and screened them against a cascade of cell-based assays, including intracellular cAMP and β-arrestin recruitment to APJ, simultaneously. The screen yielded potent and highly selective APJ agonists. Representative hits displaying preferential signaling via either G-protein or β-arrestin were subjected to a battery of confirmation assays. These biased agonists will be useful as tools to probe the function and pharmacology of APJ and provide proof of concept of our systematic approach to the discovery of biased ligands. This approach is likely universally applicable to the search for biased agonists of GPCRs.

  7. Novel Natural Inhibitors of CYP1A2 Identified by in Silico and in Vitro Screening

    PubMed Central

    Zhu, Ruixin; Hu, Liwei; Li, Haiyun; Su, Juan; Cao, Zhiwei; Zhang, Weidong

    2011-01-01

    Inhibition of cytochrome P450 (CYP) is a major cause of herb–drug interactions. The CYP1A2 enzyme plays a major role in the metabolism of drugs in humans. Its broad substrate specificity, as well as its inhibition by a vast array of structurally diverse herbal active ingredients, has indicated the possibility of metabolic herb–drug interactions. Therefore nowadays searching inhibitors for CYP1A2 from herbal medicines are drawing much more attention by biological, chemical and pharmological scientists. In our work, a pharmacophore model as well as the docking technology is proposed to screen inhibitors from herbal ingredients data. Firstly different pharmaphore models were constructed and then validated and modified by 202 herbal ingredients. Secondly the best pharmaphore model was chosen to virtually screen the herbal data (a curated database of 989 herbal compounds). Then the hits (147 herbal compounds) were continued to be filtered by a docking process, and were tested in vitro successively. Finally, five of eighteen candidate compounds (272, 284, 300, 616 and 817) were found to have inhibition of CYP1A2 activity. The model developed in our study is efficient for in silico screening of large herbal databases in the identification of CYP1A2 inhibitors. It will play an important role to prevent the risk of herb–drug interactions at an early stage of the drug development process. PMID:21686183

  8. Niche-based screening identifies small-molecule inhibitors of leukemia stem cells

    PubMed Central

    Mukherjee, Siddhartha; Kahn, Alissa R; Stewart, Alison L; Logan, David J; Negri, Joseph M; Duvet, Mildred; Järås, Marcus; Puram, Rishi; Dancik, Vlado; Al-Shahrour, Fatima; Kindler, Thomas; Tothova, Zuzana; Chattopadhyay, Shrikanta; Hasaka, Thomas; Narayan, Rajiv; Dai, Mingji; Huang, Christina; Shterental, Sebastian; Chu, Lisa P; Haydu, J Erika; Shieh, Jae Hung; Steensma, David P; Munoz, Benito; Bittker, Joshua A; Shamji, Alykhan F; Clemons, Paul A; Tolliday, Nicola J; Carpenter, Anne E; Gilliland, D Gary; Stern, Andrew M; Moore, Malcolm A S; Scadden, David T; Schreiber, Stuart L; Ebert, Benjamin L; Golub, Todd R

    2014-01-01

    Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone-marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on-target, via inhibition of HMGCoA reductase. These results illustrate the power of merging physiologically-relevant models with high-throughput screening. PMID:24161946

  9. Loss-of-function screen in rhabdomyosarcoma identifies CRKL-YES as a critical signal for tumor growth.

    PubMed

    Yeung, C L; Ngo, V N; Grohar, P J; Arnaldez, F I; Asante, A; Wan, X; Khan, J; Hewitt, S M; Khanna, C; Staudt, L M; Helman, L J

    2013-11-21

    To identify novel signaling pathways necessary for rhabdomyosarcoma (RMS) survival, we performed a loss-of-function screen using an inducible small hairpin RNA (shRNA) library in an alveolar and an embryonal RMS cell line. This screen identified CRKL expression as necessary for growth of alveolar RMS and embryonal RMS both in vitro and in vivo. We also found that CRKL was uniformly highly expressed in both RMS cell lines and tumor tissue. As CRKL is a member of the CRK adapter protein family that contains an SH2 and two SH3 domains and is involved in signal transduction from multiple tyrosine kinase receptors, we evaluated CRKL interaction with multiple tyrosine kinase receptor signaling pathways in RMS cells. While we saw no interaction of CRKL with IGFIR, MET or PI3KAKT/mTOR pathways, we determined that CRKL signaling was associated with SRC family kinase (SFK) signaling, specifically with YES kinase. Inhibition of SFK signaling with dasatinib or another SFK inhibitor, sarcatinib, suppressed RMS cell growth in vitro and in vivo. These data identify CRKL as a novel critical component of RMS growth. This study also demonstrates the use of functional screening to identify a potentially novel therapeutic target and treatment approach for these highly aggressive pediatric cancers.

  10. Screening with an NMNAT2-MSD platform identifies small molecules that modulate NMNAT2 levels in cortical neurons

    PubMed Central

    Ali, Yousuf O.; Bradley, Gillian; Lu, Hui-Chen

    2017-01-01

    Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) is a key neuronal maintenance factor and provides potent neuroprotection in numerous preclinical models of neurological disorders. NMNAT2 is significantly reduced in Alzheimer’s, Huntington’s, Parkinson’s diseases. Here we developed a Meso Scale Discovery (MSD)-based screening platform to quantify endogenous NMNAT2 in cortical neurons. The high sensitivity and large dynamic range of this NMNAT2-MSD platform allowed us to screen the Sigma LOPAC library consisting of 1280 compounds. This library had a 2.89% hit rate, with 24 NMNAT2 positive and 13 negative modulators identified. Western analysis was conducted to validate and determine the dose-dependency of identified modulators. Caffeine, one identified NMNAT2 positive-modulator, when systemically administered restored NMNAT2 expression in rTg4510 tauopathy mice to normal levels. We confirmed in a cell culture model that four selected positive-modulators exerted NMNAT2-specific neuroprotection against vincristine-induced cell death while four selected NMNAT2 negative modulators reduced neuronal viability in an NMNAT2-dependent manner. Many of the identified NMNAT2 positive modulators are predicted to increase cAMP concentration, suggesting that neuronal NMNAT2 levels are tightly regulated by cAMP signaling. Taken together, our findings indicate that the NMNAT2-MSD platform provides a sensitive phenotypic screen to detect NMNAT2 in neurons. PMID:28266613

  11. On the Design of Broad Based Screening Assays to Identify Potential Pharmacological Chaperones of Protein Misfolding Diseases†

    PubMed Central

    Naik, Subhashchandra; Zhang, Na; Gao, Phillip; Fisher, Mark T.

    2013-01-01

    Correcting aberrant folds that develop during protein folding disease states is now an active research endeavor that is attracting increasing attention from both academic and industrial circles. One particular approach focuses on developing or identifying small molecule correctors or pharmacological chaperones that specifically stabilize the native fold. Unfortunately, the limited screening platforms available to rapidly identify or validate potential drug candidates are usually inadequate or slow because the folding disease proteins in question are often transiently folded and/or aggregation-prone, complicating and/or interfering with the assay outcomes. In this review, we outline and discuss the numerous platform options currently being employed to identify small molecule therapeutics for folding diseases. Finally, we describe a new stability screening approach that is broad based and is easily applicable toward a very large number of both common and rare protein folding diseases. The label free screening method described herein couples the promiscuity of the GroEL binding to transient aggregation-prone hydrophobic folds with surface plasmon resonance enabling one to rapidly identify potential small molecule pharmacological chaperones. PMID:23339304

  12. Novel small molecule modulators of plant growth and development identified by high-content screening with plant pollen.

    PubMed

    Chuprov-Netochin, Roman; Neskorodov, Yaroslav; Marusich, Elena; Mishutkina, Yana; Volynchuk, Polina; Leonov, Sergey; Skryabin, Konstantin; Ivashenko, Andrey; Palme, Klaus; Touraev, Alisher

    2016-09-06

    Small synthetic molecules provide valuable tools to agricultural biotechnology to circumvent the need for genetic engineering and provide unique benefits to modulate plant growth and development. We developed a method to explore molecular mechanisms of plant growth by high-throughput phenotypic screening of haploid populations of pollen cells. These cells rapidly germinate to develop pollen tubes. Compounds acting as growth inhibitors or stimulators of pollen tube growth are identified in a screen lasting not longer than 8 h high-lighting the potential broad applicability of this assay to prioritize chemicals for future mechanism focused investigations in plants. We identified 65 chemical compounds that influenced pollen development. We demonstrated the usefulness of the identified compounds as promotors or inhibitors of tobacco and Arabidopsis thaliana seed growth. When 7 days old seedlings were grown in the presence of these chemicals twenty two of these compounds caused a reduction in Arabidopsis root length in the range from 4.76 to 49.20 % when compared to controls grown in the absence of the chemicals. Two of the chemicals sharing structural homology with thiazolidines stimulated root growth and increased root length by 129.23 and 119.09 %, respectively. The pollen tube growth stimulating compound (S-02) belongs to benzazepin-type chemicals and increased Arabidopsis root length by 126.24 %. In this study we demonstrate the usefulness of plant pollen tube based assay for screening small chemical compound libraries for new biologically active compounds. The pollen tubes represent an ultra-rapid screening tool with which even large compound libraries can be analyzed in very short time intervals. The broadly applicable high-throughput protocol is suitable for automated phenotypic screening of germinating pollen resulting in combination with seed germination assays in identification of plant growth inhibitors and stimulators.

  13. Cell-Based High-Throughput Screening Identifies Rifapentine as an Inhibitor of Amyloid and Biofilm Formation in Escherichia coli.

    PubMed

    Maher, Marie C; Lim, Ji Youn; Gunawan, Cheston; Cegelski, Lynette

    2015-10-09

    Escherichia coli assemble functional amyloid fibers termed curli that contribute to bacterial adhesion, biofilm formation, and host pathogenesis. We developed a cell-based high-throughput screen to identify inhibitors of curli-mediated adhesion in the laboratory strain MC4100 and curli-associated biofilm formation in the uropathogenic E. coli clinical isolate UTI89. Inhibitors of biofilm formation can operate through many mechanisms, and such inhibitors could hold therapeutic value in preventing and treating urinary tract infections. The curli-specific screen allows the identification of compounds that inhibit either curli expression, curli biogenesis, or adhesion by normally produced curli. In screening the NIH Clinical Collection of 446 compounds, we identified rifapentine as a potent inhibitor in both of these screens. Rifapentine is an antibiotic used to treat tuberculosis that targets RNA polymerase, but prevents curli-dependent adhesion and biofilm formation in E. coli at concentrations below those that affect viability. Rifapentine inhibits curli production and prevents biofilm formation on plastic, on agar, and at the air-liquid interface by inhibiting curli gene transcription. Comparisons with a cephalosporin antibiotic further revealed that curli production is not affected by standard antibiotic treatment and cell killing pressure. Thus, we reveal a new role independent of killing activity for rifapentine as an inhibitor of curli and curli-mediated biofilm formation.

  14. Development of a High-Throughput Screening Assay to Identify Inhibitors of the Lipid Kinase PIP5K1C.

    PubMed

    Wright, Brittany D; Simpson, Catherine; Stashko, Michael; Kireev, Dmitri; Hull-Ryde, Emily A; Zylka, Mark J; Janzen, William P

    2015-06-01

    Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) regulate a variety of cellular processes, including signaling through G protein-coupled receptors (GPCRs), endocytosis, exocytosis, and cell migration. These lipid kinases synthesize phosphatidylinositol 4,5-bisphosphate (PIP2) from phosphatidylinositol 4-phosphate [PI(4)P]. Because small-molecule inhibitors of these lipid kinases did not exist, molecular and genetic approaches were predominantly used to study PIP5K1 regulation of these cellular processes. Moreover, standard radioisotope-based lipid kinase assays cannot be easily adapted for high-throughput screening. Here, we report a novel, high-throughput, microfluidic mobility shift assay to identify inhibitors of PIP5K1C. This assay uses fluorescently labeled phosphatidylinositol 4-phosphate as the substrate and recombinant human PIP5K1C. Our assay exhibited high reproducibility, had a calculated adenosine triphosphate Michaelis constant (Km) of 15 µM, performed with z' values >0.7, and was used to screen a kinase-focused library of ~4700 compounds. From this screen, we identified several potent inhibitors of PIP5K1C, including UNC3230, a compound that we recently found can reduce nociceptive sensitization in animal models of chronic pain. This novel assay will allow continued drug discovery efforts for PIP5K1C and can be adapted easily to screen additional lipid kinases.

  15. Development of a high-throughput screening assay to identify inhibitors of the lipid kinase PIP5K1C

    PubMed Central

    Wright, Brittany D.; Simpson, Catherine; Stashko, Michael; Kireev, Dmitri; Hull-Ryde, Emily A.; Zylka, Mark J.; Janzen, William P.

    2015-01-01

    Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) regulate a variety of cellular processes including signaling through G protein-coupled receptors (GPCRs), endocytosis, exocytosis, and cell migration. These lipid kinases synthesize phosphatidylinositol 4,5-bisphosphate (PIP2) from phosphatidylinositol 4-phosphate [PI(4)P]. Since small molecule inhibitors of these lipid kinases did not exist, molecular and genetic approaches were predominantly used to study PIP5K1 regulation of these cellular processes. Moreover, standard radioisotope-based lipid kinase assays cannot be easily adapted for high-throughput screening. Here, we report a novel high-throughput microfluidic mobility shift assay to identify inhibitors of PIP5K1C. This assay utilizes fluorescently labeled phosphatidylinositol 4-phosphate as the substrate and recombinant human PIP5K1C. Our assay exhibited high reproducibility, had a calculated ATP Km of 15 µM, performed with z’ values >0.7, and was used to screen a kinase-focused library of ~4,700 compounds. From this screen, we identified several potent inhibitors of PIP5K1C, including UNC3230, a compound that we recently found can reduce nociceptive sensitization in animal models of chronic pain. This novel assay will allow continued drug discovery efforts for PIP5K1C and can be easily adapted to screen additional lipid kinases. PMID:25534829

  16. High-throughput screening platform identifies small molecules that prevent sequestration of Plasmodium falciparum-infected erythrocytes.

    PubMed

    Gullingsrud, Justin; Milman, Neta; Saveria, Tracy; Chesnokov, Olga; Williamson, Kathryn; Srivastava, Anand; Gamain, Benoit; Duffy, Patrick E; Oleinikov, Andrew V

    2015-04-01

    We developed a 2-step approach to screen molecules that prevent and/or reverse Plasmodium falciparum-infected erythrocyte (IE) binding to host receptors. IE adhesion and sequestration in vasculature causes severe malaria, and therefore antiadhesion therapy might be useful as adjunctive treatment. IE adhesion is mediated by the polymorphic family (approximately 60 members) of P. falciparum EMP1 (PfEMP1) multidomain proteins. We constructed sets of PfEMP1 domains that bind ICAM-1, CSA, or CD36, receptors that commonly support IE binding. Combinations of domain-coated beads were assayed by Bio-Plex technology as a high-throughput molecular platform to screen antiadhesion molecules (antibodies and small molecules). Molecules identified as so-called hits in the screen (first step) then could be assayed individually for inhibition of binding of live IE to receptors (second step). In proof-of-principle studies, the antiadhesion activity of several antibodies was concordant in Bio-Plex and live IE assays. Using this 2-step approach, we identified several molecules in a small molecule library of 10 000 compounds that could inhibit and reverse binding of IEs to ICAM-1 and CSA receptors. This 2-step screening approach should be efficient for identification of antiadhesion drug candidates for falciparum malaria. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  17. High-Throughput Screening Platform Identifies Small Molecules That Prevent Sequestration of Plasmodium falciparum–Infected Erythrocytes

    PubMed Central

    Gullingsrud, Justin; Milman, Neta; Saveria, Tracy; Chesnokov, Olga; Williamson, Kathryn; Srivastava, Anand; Gamain, Benoit; Duffy, Patrick E.; Oleinikov, Andrew V.

    2015-01-01

    Background. We developed a 2-step approach to screen molecules that prevent and/or reverse Plasmodium falciparum–infected erythrocyte (IE) binding to host receptors. IE adhesion and sequestration in vasculature causes severe malaria, and therefore antiadhesion therapy might be useful as adjunctive treatment. IE adhesion is mediated by the polymorphic family (approximately 60 members) of P. falciparum EMP1 (PfEMP1) multidomain proteins. Methods. We constructed sets of PfEMP1 domains that bind ICAM-1, CSA, or CD36, receptors that commonly support IE binding. Combinations of domain-coated beads were assayed by Bio-Plex technology as a high-throughput molecular platform to screen antiadhesion molecules (antibodies and small molecules). Molecules identified as so-called hits in the screen (first step) then could be assayed individually for inhibition of binding of live IE to receptors (second step). Results. In proof-of-principle studies, the antiadhesion activity of several antibodies was concordant in Bio-Plex and live IE assays. Using this 2-step approach, we identified several molecules in a small molecule library of 10 000 compounds that could inhibit and reverse binding of IEs to ICAM-1 and CSA receptors. Conclusion. This 2-step screening approach should be efficient for identification of antiadhesion drug candidates for falciparum malaria. PMID:25355939

  18. Novel plant immune-priming compounds identified via high-throughput chemical screening target salicylic acid glucosyltransferases in Arabidopsis.

    PubMed

    Noutoshi, Yoshiteru; Okazaki, Masateru; Kida, Tatsuya; Nishina, Yuta; Morishita, Yoshihiko; Ogawa, Takumi; Suzuki, Hideyuki; Shibata, Daisuke; Jikumaru, Yusuke; Hanada, Atsushi; Kamiya, Yuji; Shirasu, Ken

    2012-09-01

    Plant activators are compounds, such as analogs of the defense hormone salicylic acid (SA), that protect plants from pathogens by activating the plant immune system. Although some plant activators have been widely used in agriculture, the molecular mechanisms of immune induction are largely unknown. Using a newly established high-throughput screening procedure that screens for compounds that specifically potentiate pathogen-activated cell death in Arabidopsis thaliana cultured suspension cells, we identified five compounds that prime the immune response. These compounds enhanced disease resistance against pathogenic Pseudomonas bacteria in Arabidopsis plants. Pretreatments increased the accumulation of endogenous SA, but reduced its metabolite, SA-O-β-d-glucoside. Inducing compounds inhibited two SA glucosyltransferases (SAGTs) in vitro. Double knockout plants that lack both SAGTs consistently exhibited enhanced disease resistance. Our results demonstrate that manipulation of the active free SA pool via SA-inactivating enzymes can be a useful strategy for fortifying plant disease resistance and may identify useful crop protectants.

  19. Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries

    PubMed Central

    Inglese, James; Auld, Douglas S.; Jadhav, Ajit; Johnson, Ronald L.; Simeonov, Anton; Yasgar, Adam; Zheng, Wei; Austin, Christopher P.

    2006-01-01

    High-throughput screening (HTS) of chemical compounds to identify modulators of molecular targets is a mainstay of pharmaceutical development. Increasingly, HTS is being used to identify chemical probes of gene, pathway, and cell functions, with the ultimate goal of comprehensively delineating relationships between chemical structures and biological activities. Achieving this goal will require methodologies that efficiently generate pharmacological data from the primary screen and reliably profile the range of biological activities associated with large chemical libraries. Traditional HTS, which tests compounds at a single concentration, is not suited to this task, because HTS is burdened by frequent false positives and false negatives and requires extensive follow-up testing. We have developed a paradigm, quantitative HTS (qHTS), tested with the enzyme pyruvate kinase, to generate concentration–response curves for >60,000 compounds in a single experiment. We show that this method is precise, refractory to variations in sample preparation, and identifies compounds with a wide range of activities. Concentration–response curves were classified to rapidly identify pyruvate kinase activators and inhibitors with a variety of potencies and efficacies and elucidate structure–activity relationships directly from the primary screen. Comparison of qHTS with traditional single-concentration HTS revealed a high prevalence of false negatives in the single-point screen. This study demonstrates the feasibility of qHTS for accurately profi